New Insight into the Substrate Selectivity of Bovine Milk γ-glutamyl Transferase via Structural and Molecular Dynamics Predictions.

Bovine milk γ-glutamyltransferase (BoGGT) can produce γ-glutamyl peptides using L-glutamine as a donor substrate, and the transpeptidase activity is highly dependent on both γ-glutamyl donors and acceptors. To explore the molecular mechanism behind the donor and acceptor substrate preferences for BoGGT, molecular docking and molecular dynamic simulations were performed with L-glutamine and L-γ-glutamyl-p-nitroanilide (γ-GpNA) as donors. Ser450 is a crucial residue for the interactions between BoGGT and donors. BoGGT forms more hydrogen bonds with L-glutamine than γ-GpNA, promoting the binding affinity between BoGGT and L-glutamine. Gly379, Ile399, and Asn400 are crucial residues for the interactions between the BoGGT intermediate and acceptors. The BoGGT intermediate forms more hydrogen bonds with Val-Gly than L-methionine and L-leucine, which can promote the transfer of the γ-glutamyl group from the intermediate to Val-Gly. This study reveals the critical residues responsible for the interactions of donors and acceptors with the BoGGT and provides a new understanding of the substrate selectivity and catalytic mechanism of GGT.

Synthesis, inhibition properties against xanthine oxidase and molecular docking studies of dimethyl N-benzyl-1H-1,2,3-triazole-4,5-dicarboxylate and (N-benzyl-1H-1,2,3-triazole-4,5-diyl)dimethanol derivatives.

This study focused on synthesis various dimethyl N-benzyl-1H-1,2,3-triazole-4,5-dicarboxylate and (N-benzyl-1H-1,2,3-triazole-4,5-diyl)dimethanol derivatives under the conditions of green chemistry without the use of solvent and catalysts. Their inhibition properties were also investigated on xanthine oxidase (XO) activity. All dimethanol and dicarboxylate derivatives exhibited significant inhibition activities with IC50 values ranging from 0.71 to 2.25 µM. Especially, (1-(3-bromobenzyl)-1H-1,2,3-triazole-4,5-diyl)dimethanol (5c) and dimethyl 1-(4-chlorobenzyl)-1H-1,2,3-triazole-4,5-dicarboxylate (6 g) compounds were found to be the most promising derivatives on the XO enzyme inhibition with IC50 values 0.71 and 0.73 µM, respectively. Moreover, the double docking procedure was to evaluate compound modes of inhibition and their interactions with the protein (XO) at atomic level. Surprisingly, the docking results showed a good correlation with IC50 [correlation coefficient (R2 = 0.7455)]. Also, the docking results exhibited that the 5c, 6f and 6 g have lowest docking scores -4.790, -4.755, and -4.730, respectively. These data were in agreement with the IC50 values. These results give promising beginning stages to assist in the improvement of novel and powerful inhibitor against XO.

Amide-based xanthine oxidase inhibitors bearing an N-(1-alkyl-3-cyano-1H-indol-5-yl) moiety: Design, synthesis and structure-activity relationship investigation.

Our previous work identified a promising isonicotinamide based xanthine oxidase (XO) inhibitor, N-(3-cyano-4-((2-cyanobenzyl)oxy)phenyl)isonicotinamide (1), and concluded that amide is an effective linker in exploring the XO inhibitor chemical space that is completely different from the five-membered ring framework of febuxostat and topiroxostat. Indole, an endogenous bioactive substance and a popular drug construction fragment, was involved in the structural optimization campaign of the present effort. After the installation of some functional groups, N-(1-alkyl-3-cyano-1H-indol-5-yl) was generated and employed to mend the missing H-bond interaction between the 3'-cyano of 1 and Asn768 residue of XO by shortening their distance. In this context, eight kinds of heterocyclic aromatic amide chemotypes were rationally designed and synthesized to investigate the structure-activity relationship (SAR) of amide-based XO inhibitors. The optimized compound a6 (IC50 = 0.018 µM) exhibits 17.2-fold improved potency than the initial compound 1 (IC50 = 0.31 µM). Its potency is comparable to that of topiroxostat (IC50 = 0.013 µM). Molecular docking and molecular dynamics studies proved the existence of the stable H-bond between the cyano group and the Asn768 residue. Moreover, oral administration of a6 (11.8 mg/kg) could effectively reduce serum uric acid levels in an acute hyperuricemia rat model. Liver microsomal stability assay illustrated that compound a6 possesses well metabolic stability in rat liver microsomes. However, the in vivo potency of a6 was much lower than that of topiroxostat, which may be explained by the poor absorption found in the parallel artificial membrane permeability assay (PAMPA). In addition, 6a has non-cytotoxicity against normal cell lines MCF10A and 16HBE. Taken together, this work culminated in the identification of compound 6a as an excellent lead for further exploration of amide-based XO inhibitors.

Urea detection in milk by urease-assisted pH-sensitive carbon dots.

Excessive urea in milk will lead to serious health problems. To detect whether the urea concentration in milk exceeds the standard and ensure the quality of milk, it is necessary to develop detection technology for urea in milk. But it is difficult to detect urea in milk conveniently and accurately by traditional methods. To measure the concentration of urea in milk, stable green light carbon dots (CDs) were synthesized by a one-step method as a fluorescent probe. Then, 3, 5-diaminobenzoic acid was used as the precursor for CD synthesis. Experimental results showed that CDs can generate strong fluorescence when excited by light (350-450 nm). The fluorescence peak wavelength is 490 nm, and the optimum excitation wavelength is 390 nm. The fluorescence intensity of CDs has a significant change with variations of pH (pH of 6-9), and the higher the pH, the lower the fluorescence intensity. Additionally, urea can be hydrolyzed by urease to produce ammonia and carbon dioxide. Ammonia is ionized in water to produce OH-, which increases the pH of the solution. After adding standard urea to milk, urease and CDs are added. The fluorescence intensity of CDs in the mixed solution decreases as the concentration of standard added urea increases. Thus the concentration of urea in milk can be calculated. The experimental results show that the CD method for detecting urea in milk has advantages of high sensitivity and wide measurement range. The linear interval is 25-500 mg/L, R2 is 0.998, and the limit of detection is 6.27 mg/L. The concentration of urea in the milk used in the experiment is 265.46 mg/L. CDs are easy to fabricate, and the advantages of the method are simple operation, no pretreatment, safety, and low cost. A new method for the detection of urea in milk was established, to the best of our knowledge, and this method can aid in food quality control.

Inhibition effects of selected thiophene-2-sulfonamides on lactoperoxidase.

Lactoperoxidase (LPO, E.C.1.11.1.7) is a natural antibacterial agent which is secreted from salivary, mammary, and other mucosal glands. It is one of the crucial enzymes in biological systems, so protection of LPO activity is extremely important for the immune system. Within the scope of this study; in vitro effects of some thiophene-2-sulfonamide derivatives (1a-7a) on bovine milk LPO enzymatic activity were investigated. LPO was purified from the Sepharose-4B-L-tyrosine-5-amino-2-methylbenzenesulfonamide column prepared using affinity chromatography technique with a yield of 169.66 EU/mg specific activity in 452.44 times. As a result, 5-(2-thienylthio) thiophene-2-sulfonamide demonstrated the strongest inhibition impact among these compounds. This molecule has shown a competitive inhibition and it was determined that the IC50 value was 3.4 nM and the Ki value was 2 ± 0.6 nM.

Fatty acid biosynthesis and transcriptional regulation of Stearoyl-CoA Desaturase 1 (SCD1) in buffalo milk.

BACKGROUND: Buffalo milk is considered as a highly nutritious food owing to its higher contents of fatty acids (FA) and rich nutrient profile. Higher fat contents of buffalo milk make it suitable for processing to develop various healthy and nutritious products. Moreover, buffalo milk contains more unsaturated FAs (UFA) such as oleic and linolenic acid, which are important from the human health point of view owing to their desirable physiological effects. However, inadequate information is available about the chemical composition and mechanism of FA synthesis in buffalo milk. In this study, we hypothesized that expression of SCD1 gene could alter the biosynthesis of FA in epithelial cells of mammary gland and subsequently affect the FA contents in buffalo milk. We investigated the transcriptional and biological role of Stearoyl-CoA Desaturase 1 (SCD1) in the buffalo mammary epithelial cells (BMECs) during FA and triacylglycerol (TAG) synthesis. RESULTS: Results revealed that unsaturated fatty acid contents were much higher in concentration in buffalo milk as compared to Holstein cow. Significant increase in the expression level of FAS, ACACA, SREBP1, PPARG, GPAT, and AGPAT genes was observed in response to altered expression of SCD1 in buffalo milk. Moreover, change in SCD1 gene in BMECs also mediated the expression of genes related to FA biosynthesis subsequently leading to alter the FA composition. Overexpression of SCD1 significantly increased the expression of genes associated with FA and TAG synthesis leading to enhance FA and unsaturated FA contents in BMECs. However, down-regulation of SCD1 exhibited opposite consequences. CONCLUSION: Our study provides mechanistic insights on transcriptional regulation of SCD1 to alter FA and TAG synthesis through directly or indirectly mediating biosynthesis and metabolic pathways in BMECs. We provide preliminary findings regarding engineering of FA contents in buffalo milk through SCD1 signaling.

Genome-wide identification of Diacylglycerol Acyltransferases (DGAT) family genes influencing Milk production in Buffalo.

BACKGROUND: The diacylglycerol acyltransferases (DGAT) are a vital group of enzymes in catalyzing triacylglycerol biosynthesis. DGAT genes like DGAT1 and DGAT2, have been identified as two functional candidate genes affecting milk production traits, especially for fat content in milk. Buffalo milk is famous for its excellent quality, which is rich in fat and protein content. Therefore, this study aimed to characterize DGAT family genes in buffalo and to find candidate markers or DGAT genes influencing lactation performance. RESULTS: We performed a genome-wide study and identified eight DGAT genes in buffalo. All the DGAT genes classified into two distinct clades (DGAT1 and DGAT2 subfamily) based on their phylogenetic relationships and structural features. Chromosome localization displayed eight buffalo DGAT genes distributed on five chromosomes. Collinearity analysis revealed that the DGAT family genes were extensive homologous between buffalo and cattle. Afterward, we discovered genetic variants loci within the genomic regions that DGAT genes located in buffalo. Seven haplotype blocks were constructed and were associated with buffalo milk production traits. Single marker association analyses revealed four most significant single nucleotide polymorphisms (SNPs) mainly affecting milk protein percentage or milk fat yield in buffalo. Genes functional analysis indicated that these DGAT family genes could influence lactation performance in the mammal through regulating lipid metabolism. CONCLUSION: In the present study, we performed a comprehensive analysis for the DGAT family genes in buffalo, which including identification, structural characterization, phylogenetic classification, chromosomal distribution, collinearity analysis, association analysis, and functional analysis. These findings provide useful information for an in-depth study to determine the role of DGAT family gens play in the regulation of milk production and milk quality improvement in buffalo.

Mix-and-read method for assessment of milk pasteurization using a smartphone or a common digital camera.

Alkaline phosphatase (ALP) is the most widely used marker of the adequacy of milk pasteurization since it is inactivated at temperatures slightly higher than those required for elimination of pathogens. The cutoff level is 350 mU/L. The approved colorimetric, fluorometric, and chemiluminometric methods require specialized readers with photomultipliers as detectors, and the samples are usually analyzed one-by-one. We developed a low-cost mix-and-read method that exploited a smartphone or a common digital camera as detectors for the chemiluminometric determination of ALP in milk. As samples, we used pasteurized cow and sheep milk spiked with ALP, as well as mixtures of pasteurized and raw (non-pasteurized) milk. Chemiluminescence images acquired by the smartphone or the digital camera were analyzed by the ImageJ software. The limits of detection (LODs), for images captured by the smartphone, were 4.4 mU/L and 11.1 mU/L for cow milk and sheep milk, respectively, while with the digital camera, the respective LODs were 6.2 mU/L and 6.7 mU/L, respectively. The coefficients of variation (CVs) at the cutoff level of 350 mU/L were 8% and 8.5% for the cow and sheep milk, respectively. For images by the digital camera, the CVs were 5.8% and 5% for cow and sheep milk, respectively. The performance of the method is similar to methods that use a microtiter plate and a luminometer for chemiluminescence measurements. Sample pretreatment is not necessary. The microtiter well format combined with detection by a smartphone enables the analysis of multiple samples simultaneously. It is anticipated that the method will prove useful for the rapid assessment of milk pasteurization efficiency in dairy industries, especially in remote areas where expensive instruments are not available. Graphical abstract.

Short communication: Lactose utilization of Streptococcus thermophilus and correlations with ß-galactosidase and urease.

The ability to use lactose is critical for the application of Streptococcus thermophilus in fermented dairy products. Most studies have evaluated the use of lactose of S. thermophilus by measuring lactose utilization, but its correlation with ß-galactosidase and urease has rarely been investigated. In this study, 10 strains of S. thermophilus isolated from fermented yak milk exhibited a diversity of ß-galactosidase and urease activities, growth, and acid production in de Man, Rogosa, and Sharpe-lactose. Among the strains, 15G5 possessed the highest ß-galactosidase activity and showed the highest cell growth, lactic acid production, and titratable acidity during fermentation. In contrast, 7G10, with the weakest ß-galactosidase activity, produced the lowest lactic acid content and change in titratable acidity. Further investigation indicated that ß-galactosidase activity of S. thermophilus showed significant positive correlations with the growth of cell densities, the production of lactic acid, and titratable acidity, and urease activity of S. thermophilus showed a significant correlation with the use of lactose and the production of lactic acid and acetaldehyde. These findings suggest that the differences of ß-galactosidase and urease activities are essential for the performance in the lactose metabolism, growth, and acid production of S. thermophilus, providing new insights into strain selection and application.

Stability of fat globules in UHT milk during proteolysis by the AprX protease from Pseudomonas fluorescens and by plasmin.

Fat separation is a limiting factor for the shelf life of UHT milk. It may be promoted by the proteolysis of fat surface-adsorbed proteins (FSAP) by proteases that remain active after UHT treatment. The aim of this research was to explore the relationship between the proteolysis of FSAP and fat destabilization. In this study, we developed a full-fat UHT milk-based model system and added either the major bacterial protease AprX from Pseudomonas fluorescens or the major native milk protease plasmin at high levels to induce fast destabilization of the milk fat globules. We monitored changes in physical properties and FSAP composition, and structural changes in fat globules, over 24 h. Our results showed that AprX-induced sedimentation as a result of the flocculation of fat globules, and plasmin induced cream to float as a result of the coalescence of fat globules. This study confirmed that AprX and plasmin can both lead to fat destabilization in full-fat UHT milk, and it provides insights in the underlying mechanisms.

Lactoperoxidase, an antimicrobial enzyme, is inhibited by some indazoles.

Lactoperoxidase (LPO) has bactericidal and bacteriostatic activity on various microorganisms and it creates a natural antimicrobial defense system. So, LPO is one of the essential enzyme in biological systems and the protection of the LPO activity is extremely important for the immune system. Because of these features, the protection of the activity of the LPO has vital importance for the health of the organisms. Also, LPO is used in various sectors from cosmetics industry to agriculture industry due to its broad antimicrobial properties. Therefore, the identification of inhibitors and activators of the LPO is becoming increasingly important. In present study we aimed to investigate the inhibitory effects of some indazoles [1H-indazole (1a), 4-Bromo-1H-indazole (2a), 6-Bromo-1H-indazole (3a), 7-Bromo-1H-indazole (4a), 4-chloro-1H-indazole (5a), 6-chloro-1H-indazole (6a), 7-chloro-1H-indazole (7a), 4-fluoro-1H-indazole (8a), 6-fluoro-1H-indazole (9a), 7-fluoro-1H-indazole (10a)] on bovine milk LPO. Indazole derivatives are heterocyclic organic molecules with a wide range of biological activity. For this aim, bovine milk LPO was purified using Sepharose-4B-l-tyrosine-5-amino-2-methyl benzenesulfonamide affinity chromatography method. Then, the potential inhibitory effects of indazoles on LPO activity were investigated. Ki values were calculated for each indazole molecule. Ki values were ranging from 4.10 to 252.78 µM for 1a to10a. All of the indazole molecules we studied showed strong inhibitory effect on LPO activity. Also we determined inhibition types of the indazoles to clarify the mechanisms of inhibition.

Intramammary antibiotics with complementary acupuncture decreases milk serum N-acetyl-beta-D-glucosaminidase concentrations in dairy cattle with subclinical mastitis.

The objective of this research is to determine whether intramammary antibiotics with complementary acupuncture can reduce bovine mammary inflammation due to subclinical mastitis. Lactating cows were selected based on milk with a somatic cell count (SCC) greater than 500,000 cells/ml. Pre- and post-treatment milk samples were collected to determine SCC, aerobic bacterial content, milk ion conductivity, total protein, lactate dehydrogenase (LDH) and N-acetyl-beta-D-glucosaminidase (NAGase) concentrations. Milk serum was prepared from milk samples by double centrifugation. Concentrations of LDH and NAGase were determined using commercial enzyme-linked immunosorbent assays. Cows being treated with intramammary antibiotics were separated by random assignment to the acupuncture group (n = 10) and a no-acupuncture (control) group (n = 9). Both the acupuncture and control group were restrained for 30 min in a head catch 12 hr apart for a total of four times. For front quarters affected by subclinical mastitis, the acupuncture points used were spleen (SP) 12, SP 17, SP 18, SP 21, stomach (ST) 18 and conception vessel (CV) 12. For rear quarters affected by subclinical mastitis, the acupuncture points used were bladder (BL) 30, BL 30-1, BL 49, kidney (KI) 10, conception vessel (CV) 2 and CV 3. All parameters were compared using a Student t test. Significance was defined as p < .05. Compared to control cows, complementary acupuncture treatment reduced NAGase enzymatic activity in quarters of cows with subclinical mastitis. The reduction in NAGase suggests that complementary acupuncture treatment may be associated with healing of the damaged mammary epithelial cells, which are the primary source of NAGase activity in milk serum.

Selenium biofortified alfalfa hay fed in low quantities improves selenium status and glutathione peroxidase activity in transition dairy cows and their calves.

The hypothesis of the study was that feeding a relatively low amount of Se biofortified alfalfa hay during the dry period and early lactation would improve selenium status and glutathione peroxidase activity in dairy cows and their calves. Ten Jersey and 8 Holstein primiparous dairy cows were supplemented with Se biofortified (TRT; n = 9) or non-biofortified (CTR; n = 9) alfalfa hay at a rate of 1 kg/100 kg of BW mixed with the TMR from 40 d prior parturition to 2 weeks post-partum. Se concentration in whole blood, liver, milk, and colostrum, the transfer of Se to calves, and the glutathione peroxidase (GPx) activity were assessed. TRT had 2-fold larger (P < 0.05) Se in blood v. CTR that resulted in larger Se in liver and colostrum but not milk and larger GPx activity in plasma and erythrocytes but not in milk. Compared to CTR, calves from TRT had larger Se in blood but only a numerical (P = 0.09) larger GPx activity in plasma. A positive correlation was detected between Se in the blood and GPx activity in erythrocytes and plasma in cows. Our results demonstrated that feeding pregnant primiparous dairy cows with a relatively low amount of Se-biofortified alfalfa hay is an effective way to increase Se in the blood and liver, leading to greater antioxidant activity via GPx. The same treatment was effective in improving Se concentration in calves but had a modest effect on their GPx activity. Feeding Se biofortified hay increased Se concentration in colostrum but not in milk.

Advances in Analysis of Milk Proteases Activity at Surfaces and in a Volume by Acoustic Methods.

This review is focused on the application of surface and volume-sensitive acoustic methods for the detection of milk proteases such as trypsin and plasmin. While trypsin is an important protein of human milk, plasmin is a protease that plays an important role in the quality of bovine, sheep and goat milks. The increased activity of plasmin can cause an extensive cleavage of ß-casein and, thus, affect the milk gelation and taste. The basic principles of surface-sensitive acoustic methods, as well as high-resolution ultrasonic spectroscopy (HR-US), are presented. The current state-of-the-art examples of the application of acoustic sensors for protease detection in real time are discussed. The application of the HR-US method for studying the kinetics of the enzyme reaction is demonstrated. The sensitivity of the acoustics biosensors and HR-US methods for protease detection are compared.

Proteolytic Volatile Profile and Electrophoretic Analysis of Casein Composition in Milk and Cheese Derived from Mironutrient-Fed Cows.

The aim of the study was to evaluate the proteolytic process in Caciocavallo cheese obtained from Friesian cows fed zinc, selenium, and iodine supplementation. Thirty-six Friesian cows, balanced for parity, milk production, and days in milk, were randomly assigned to four groups. The control group (CG) was fed with a conventional feeding strategy, while the three remaining groups received a diet enriched with three different trace elements, respectively zinc (ZG), selenium (SG), and iodine (IG). At the end of the experimental period, samples of milk were collected and used to produce Caciocavallo cheese from each experimental group. Cheese samples were then analyzed after 7 and 120 days from the cheese making in order to obtain information on chemical composition and extent of the proteolytic process, evaluated through the electrophoretic analysis of caseins and the determination of volatiles profile. Both milk and cheese samples were richer in the amount of the microelement respectively used for the integration of the cattle's diet. The zymographic approach was helpful in evaluating, in milk, the proteolytic function performed by endogenous metalloenzymes specifically able to degrade gelatin and casein; this evaluation did not highlight significant differences among the analyzed samples. In cheese, the electrophoretic analysis in reducing and denaturing condition showed the marked ability of ß-casein to resist the proteolytic action during ripening, whereas the dietary selenium supplementation was shown to perform a protective action against the degradation of S1 and S2 isoforms of α-casein. The analysis of the volatile profile evidenced the presence of compounds associated with proteolysis of phenylalanine and leucine. This approach showed that selenium was able to negatively influence the biochemical processes that lead to the formation of 3-methyl butanol, although the identification of the specific mechanism needs further investigation.

A genome-wide association study reveals specific transferases as candidate loci for bovine milk oligosaccharides synthesis.

BACKGROUND: Human milk oligosaccharides (OS) play a key role in brain and gut microbiota development of the neonate, but the underlying biosynthetic steps of OS in the mammary gland are still largely unknown. As bovine milk contains OS with somewhat similar structures and functionalities there is increased interest in further understanding the genetic basis underlying the OS content of milk for eventual extraction and generation of value-added ingredients for infant formulas and nutraceuticals. The present study is the first to report on genetic parameter estimation as well as on a genome wide association study (GWAS) from the largest bovine milk OS dataset analyzed to date. RESULTS: In total 15 different bovine milk OS were monitored. Heritabilities ranged from 0 to 0.68 in Danish Holstein and from 0 to 0.92 in Danish Jersey. The GWAS identified in total 1770 SNPs (FDR < 0.10) for five different OS in Danish Holstein and 6913 SNPs (FDR < 0.10) for 11 OS in Danish Jersey. In Danish Holstein, a major overlapping QTL was identified on BTA1 for LNH and LNT explaining 24% of the variation in these OS. The most significant SNPs were associated with B3GNT5, a gene encoding a glycosyltransferase involved in glycan synthesis. In Danish Jersey, a very strong QTL was detected for the OS with composition 2 Hex 1 HexNAc (isomer 1) on BTA11. The most significant SNP had -log10(P-value) of 52.88 (BOVINEHD1100030300) and was assigned to ABO, a gene encoding ABO blood group glycosyltransferases. This SNP has been reported to be a missense mutation and explains 56% of the OS variation. Other candidate genes of interest identified for milk OS were ALG3, B3GALNT2, LOC520336, PIGV, MAN1C1, ST6GALNAC6, GLT6D1, GALNT14, GALNT17, COLGALT2, LFNG and SIGLEC. CONCLUSION: To our knowledge, this is the first study documenting a solid breeding potential for bovine milk OS and a strong indication of specific candidate genes related to OS synthesis underlying this genetic influence. This new information has the potential to guide breeding strategies to achieve production of milk with higher diversity and concentration of OS and ultimately facilitate large-scale extraction of bovine milk OS.

Synthesis of 8-hydroxyquinoline glycoconjugates and preliminary assay of their ß1,4-GalT inhibitory and anti-cancer properties.

8-Hydroxyquinoline scaffold is a privileged structure used in designing a new active agents with therapeutic potential. Its connections with the sugar unit is formed to improve the pharmacokinetic properties. The broad spectrum of activity of quinoline derivatives, especially glycoconjugates, is often associated with the ability to chelate metal ions or with the ability to intercalate into DNA. Simple and effective methods of synthesis glycoconjugates of 8-hydroxyquinoline and 8-hydroxyquinaldine derivatives, containing an O-glycosidic bond or a 1,2,3-triazole linker in their structure, have been developed. The obtained glycoconjugates were tested for their ability to inhibit ß-1,4-Galactosyltransferase, as well as inhibit cancer cell proliferation. It was found that used glycoconjugation strategy influenced both improvement of activity and improvement of the bioavailability of 8-HQ derivatives. Their activity depends on type of attached sugar, presence of protecting groups in sugar moiety and presence of a linker between sugar and quinolone aglycone.

Suitability of somatic cell count, electrical conductivity, and lactate dehydrogenase activity in foremilk before versus after alveolar milk ejection for mastitis detection.

Mastitis is responsible for substantial economic loss and significant animal welfare concerns for the dairy industry. Sensors that measure electrical conductivity (EC) and enzyme concentrations of lactate dehydrogenase (LDH) are presently used for automatic detection of mastitis. However, EC is not sensitive enough to detect mastitis, and the ability of LDH activity to identify mastitis caused by different pathogens is a potential option that needs to be investigated. This study was conducted to test the following hypotheses: (a) strict foremilk before milk ejection is more informative in detecting mastitis, in general, than foremilk removed after cows were stimulated for milk ejection; and (b) the value of LDH activity as a mastitis indicator depends on the type of pathogen associated with the infection. Milk samples (before afternoon milking) from 48 Holstein-Friesian cows at the University of Sydney's dairy farm (Camden, New South Wales, Australia) with EC > 7.5 mS/cm in any of the 4 quarters were collected over a period of 2 mo. Quarter milk samples (n = 343) from 48 cows were collected manually in the automatic milking rotary in 3 steps: foremilk before (strict foremilk) and after milk ejection, followed by an aseptic sample for bacteriological culture. The EC (mS), LDH (U/L), SCC (cells/mL), and milk protein and fat content (%) of foremilk in both sampling times were compared and used as predictors for gram-positive and gram-negative mastitis. Quarter (n = 515) observations from 44 cows were analyzed using a logistic mixed or linear mixed model, with cow and quarter nested within cow as random effects. Milk from both sampling times was also assessed by producing a receiver operating characteristic (ROC) curve and calculating the area under the curve (AUC) to determine ability to detect mastitis. Overall, EC and LDH were greater and milk protein (%) was lower in strict foremilk than in milk fractions obtained after milk ejection. Data from strict foremilk samples had slightly higher AUC values (0.98 to 0.99 vs. 0.97 to 0.98, respectively) than did the after-ejection milk samples. Although gram-negative coliform mastitis had significantly higher LDH activity than did gram-positive mastitis (6.19 vs. 5.34 log10 U/L), the robustness of this result is questionable due to limited sample size. We concluded that milk samples taken before ejection can influence major mastitis indicators, suggesting that automatic milking system sensors could be modified to monitor milk before ejection for more efficient mastitis detection.

Evaluation of 3 esterase tests for the diagnosis of subclinical mastitis at dry-off and freshening in dairy cattle.

Subclinical mastitis (SCM) and intramammary infection (IMI) increase esterase activity in the glandular secretions of dairy cattle. Our objective was to evaluate the clinical performance of 3 commercially available esterase tests for diagnosing SCM and IMI. Foremilk samples were collected from 380 quarters (96 cows) at dry-off and from 329 quarters (83 cows) within 4 to 7 d after calving. Quarter somatic cell count (SCC) was measured using the reference method (DeLaval cell counter; De Laval International AB, Tumba, Sweden) with SCM defined as SCC >200,000 cells/mL. Bacterial culture of foremilk samples was used to diagnose IMI based on the growth of ≥100 cfu/mL. The SCC was estimated using 3 PortaSCC tests (PortaCheck, Moorestown, NJ) from the measured esterase activity and the California Mastitis Test (CMT). Clinical performance was evaluated using logistic regression to determine the area under the receiver operating characteristic curve (AUC) and identify test sensitivity (Se) and specificity (Sp) at the optimal cut-point for diagnosing SCM and IMI. Test agreement was also evaluated using the kappa coefficient (κ) and weighted κ. The PortaSCC color test was the best-performing PortaSCC test for diagnosing SCM at dry-off (AUC = 0.90, Se = 0.91, Sp = 0.81, κ = 0.71) and at freshening (AUC = 0.86, Se = 0.74, Sp = 0.95, κ = 0.72), at an optimal cut-point of ≥250,000 cells/mL but required 45 min to produce a result. For comparison, the CMT required 2 min to produce a result and a CMT score of trace or higher was superior to the PortaSCC color test for diagnosing SCM at dry-off (AUC = 0.95, Se = 0.95, Sp = 0.86, κ = 0.81) and freshening (AUC = 0.88, Se = 0.79, Sp = 0.95, κ = 0.76). The PortaSCC quick test was the best-performing PortaSCC test for diagnosing IMI at dry-off (AUC = 0.81, Se = 0.81, Sp = 0.78 κ = 0.40) and required 5 min to produce a result, whereas the PortaSCC color test was the best performing PortaSCC test for diagnosing IMI at freshening (AUC = 0.80, Se = 0.75, Sp = 0.79 κ = 0.38). For comparison, the CMT was inferior to the PortaSCC quick test for diagnosing IMI at dry-off (AUC = 0.73, Se = 0.76, Sp = 0.60, κ = 0.20) but was equivalent to the PortaSCC color test at freshening (AUC = 0.79, Se = 0.58, Sp = 0.93, κ = 0.50). The PortaSCC color and quick tests and CMT were considered good tests for diagnosing SCM and IMI because clinically useful tests typically have an AUC >0.80 and κ >0.6. Based on the test sensitivity, cost, and analysis time, there does not appear to be a persuasive reason to select the PortaSCC tests over the traditional CMT for diagnosing SCM and IMI.

Characterization of the physical, microbiological, and chemical properties of sonicated raw bovine milk.

Innovative processing technologies, such as ultrasonication, can change the properties of milk, allowing for the improvement or development of dairy foods. Yet taking bench-scale equipment to pilot plant scale has been challenging. Raw milk, standardized to 3% fat and warmed to inlet temperatures of 42 or 54°C, was exposed to continuous, high-intensity, low-frequency ultrasonication (16/20 kHz, 1.36 kW/pass) at flow rates of 0.15, 0.30, and 0.45 L/min that resulted in resident times within the reaction cell of 6, 3, and 2 min per pass, respectively. Multiple passes (3, 5, and 7, respectively) were required to obtain a total exposure time of 14 to 18 min. Evaluation of fat droplet sizes, enzyme coagulation properties, and microstructure of milk and milk gels, as well as determining compositional and lipid properties, were conducted to determine the potential of the ultrasound system to effectively modify milk. Laser scanning particle sizing and confocal microscopy showed that the largest droplets (2.26 ± 0.13 µm) found in raw milk were selectively reduced in size with a concomitant increase in the number of submicron droplets (0.37 ± 0.06 µm), which occurred sooner when exposed to shorter bursts of ultrasonication (0.45 L/min flow rates) and at an inlet temperature of 54°C. Ultrasound processing with milk entering at 42°C resulted in faster gelling times and firmer curds at 30 min; however, extended processing at inlet temperature of 54°C reduced curd firmness and lengthened coagulation time. This showed that ultrasonication altered protein-protein and protein-lipid interactions, thus the strength of the enzyme-set curds. Scanning electron microscopy revealed a denser curd matrix with less continuous and more irregular shaped and clustered strands, whereas transmission electron microscopy showed submicron lipid droplets embedded within the protein strands of the curd matrix. Processing at inlet temperature of 54°C with flow rates of 0.30 and 0.45 L/min also reduced the total aerobic bacterial count by more than 1 log cfu/mL, and the number of psychrophiles below the limit of detection (10 cfu/mL) for this study. Ultrasonication exposures of 14 to 18 min had minimal effect on the milk composition, fatty acid profiles, and lipid heat capacity and enthalpy. The findings show that this continuous ultrasound system, which is conducive to commercial scale-up, modifies the physical and functional properties of milk under the parameters used in this study and has potential use in dairy processing.