Intramammary challenge of lipopolysaccharide stimulates secretion of lingual antimicrobial peptide into milk of dairy cows

Lingual antimicrobial peptide (LAP) belongs to the β-defensin family in cattle and is found in bovine milk. However, it is unclear whether LAP is involved in the early immune response to mammary infection. The aim of the study was to investigate the changes of LAP concentration in milk after intramammary challenge with lipopolysaccharide (LPS), the gram-negative bacteria cell membrane component, in dairy cows. Milk was collected before and after LPS or phosphate-buffered saline (control) challenge every hour for 12 h on d 0 and twice daily from d 1 to 7. Somatic cell count (SCC), LAP concentration, and lactoperoxidase (LPO) activity in the milk were measured. Somatic cell count started to increase at 2 h postchallenge and remained high until d 5 (694 ± 187 × 10³ to >1,000 ± 0 × 10³ cells/mL at d 0; >1,000 ± 0 × 10³ cells/mL at d 1 to 3; 684 ± 194 × 10³ to 829 ± 108 × 10³ cells/mL at d 4; 527 ± 197 × 10³ to 656 ± 145 × 10³ cells/mL at d 5). Somatic cell count increased in the control cows, although the levels were lower compared with those in the LPS challenge group. The LAP concentration in milk increased significantly at 2 h post-LPS-challenge and was maintained at high levels until d 2 (8.6 ± 0.6 to 17.5 ± 2.3 nM). In the control cow infused with phosphate-buffered saline, there was no increase of LAP concentration in milk (5.1 ± 0.6 to 7.2 ± 0.8 nM). Increase of LPO activity in the milk was observed at 6 h after LPS challenge and continued until d 3 (4.7 ± 0.3 to 9.4 ± 1.1 U). No increase of LPO activity was observed in the milk of control cows. The increase and subsequent decrease in LAP concentration after LPS challenge occurred earlier than those of LPO activity. In multiparous cows with LPS infusion, there was a significantly negative relationship between the days leading to the basal levels in LAP concentration and LPO activity (r = −0.75). These results suggest that LPS induces secretion of LAP into milk within hours and that LPO may have a synergistic antimicrobial function with LAP in mammary glands of dairy cows.     

Associations among milk quality indicators in raw bulk milk

The objective of this study was to determine characteristics and associations among bulk milk quality indicators from a cohort of dairies that used modern milk harvest, storage, and shipment systems and participated in an intensive program of milk quality monitoring. Bulk milk somatic cell count (SCC), total bacteria count (TBC), coliform count (CC), and laboratory pasteurization count (LPC) were monitored between July 2006 and July 2007. Bulk milk samples were collected 3 times daily (n = 3 farms), twice daily (n = 6 farms), once daily (n = 4 farms), or once every other day (n = 3 farms). Most farms (n = 11) had direct loading of milk into tankers on trucks, but 5 farms had stationary bulk tanks. The average herd size was 924 cows (range = 200 to 2,700), and daily milk produced per herd was 35,220 kg (range = 7,500 to 105,000 kg). Thresholds for increased bacterial counts were defined according to the 75th percentile and were >8,000 cfu/mL for TBC, >160 cfu/mL for CC, and ≥310 cfu/mL for LPC. Means values were 12,500 (n = 7,241 measurements), 242 (n = 7,275 measurements), and 226 cfu/mL (n = 7,220 measurements) for TBC, CC, and LPC, respectively. Increased TBC was 6.3 times more likely for bulk milk loads with increased CC compared with loads containing fewer coliforms. Increased TBC was 1.3 times more likely for bulk milk with increased LPC. The odds of increased TBC increased by 2.4% for every 10,000-cells/mL increase in SCC in the same milk load. The odds of increased CC increased by 4.3% for every 10,000-cells/mL increase in SCC. The odds of increased CC increased by 1% for every 0.1°C increase in the milk temperature upon arrival at the dairy plant (or at pickup for farms with bulk tank). Laboratory pasteurization count was poorly associated with other milk quality indicators. Seasonal effects on bacterial counts and milk temperature varied substantially among farms. Results of this study can be used to aid the interpretation and analysis of indicators of milk quality intensively produced by dairy processors’ laboratories.     

Effects of feeding three types of corn-milling coproducts on milk production and ruminal fermentation of lactating Holstein cattle

Two experiments were conducted to determine the effects of feeding 3 corn-milling coproducts on intake, milk production, ruminal fermentation, and digestibility of lactating Holstein cows. In experiment 1, three corn-milling coproducts were fed at 15% of the diet dry matter (DM) to 28 Holstein cows averaging (±SD) 625 ± 81 kg of body weight and 116 ± 33 d in milk to determine effects on DM intake and milk production. In experiment 2, the same rations were fed to 4 ruminally fistulated, multiparous Holstein cows averaging 677 ± 41 kg of body weight and 144 ± 5 d in milk to determine the effects on ruminal fermentation and digestibility. In both experiments, cows and treatments were assigned randomly in 4 × 4 Latin squares over four 21-d periods. Treatments were formulated by replacing portions of forage and concentrate feeds with 15% coproduct and included 1) 0% coproduct (control), 2) dried distillers grains plus solubles (DDGS), 3) dehydrated corn germ meal (germ), and 4) high-protein dried distillers grains (HPDDG). Feed intake was recorded daily, and milk samples were collected on d 19 to 21 of each period for analysis of major components. Rumen fluid was collected at 10 time points over 24 h post feeding on d 21 of experiment 2. In experiment 1, DM intake was greater for the germ (24.3 kg/d) and DDGS treatments (23.8 kg/d), but DDGS was not different from the control (22.9 kg/d) and HPDDG treatments (22.4 kg/d). Milk production paralleled DM intake and tended to be greater for the germ (32.1 kg/d) and DDGS treatments (30.9 kg/d), but the DDGS treatment was not different from the control (30.6 kg/d) and HPDDG treatments (30.3 kg/d). However, yields of milk fat, milk protein, and 3.5% FCM were similar and averaged (±SEM) 1.1 ± 0.1, 0.9 ± 0.03, and 31.7 ± 1.3 kg/d. Milk urea nitrogen was greater for the HPDDG (15.9 mg/dL) and germ treatments (15.5 mg/dL) than for the control (15.0 mg/dL) and DDGS treatments (14.9 mg/dL). In experiment 2, DM intake and milk production were not different across treatments and averaged 26.1 ± 2.3 and 28.3 ± 3.9 kg/d. Ruminal pH (6.26 ± 0.08) and total concentration of volatile fatty acids (125.3 ± 4.2 mM) were similar. Acetate concentration was higher for the control treatment than the DDGS, germ, and HPDDG treatments (81.7 vs. 75.8, 75.0, and 78.4 mM). Concentrations of propionate and butyrate were not different and averaged 27.8 ± 1.2 and 14.3 ± 0.9 mM across treatments. The acetate:propionate ratios for the control, germ, and HPDDG treatments were greater than for the DDGS treatment (3.02, 2.88, and 2.91 vs. 2.62). Dry matter, organic matter, and neutral detergent fiber digestibilities were similar across treatments and averaged 63.5 ± 2.7, 67.3 ± 2.2, and 43.5 ± 4.2%. Milk production followed DM intake in experiment 1, and yield of major milk components was not affected. Results of these experiments indicate that dairy rations can be successfully formulated to include 15% of diet DM as corn-milling coproducts while maintaining or increasing DM intakes and yields of milk and milk components.     

The effect of dietary iodine supplementation in dairy goats on milk production traits and milk iodine content

Dairy products offer an important source of iodine for humans, particularly infants and children. An adequate iodine content in the diet of lactating animals must guarantee a suitable milk iodine concentration. In this experiment, the effects of iodine supplementation of dairy goat diets on the iodine concentration, milk yield, and milk composition of goat milk were studied. Thirty crossbred dairy goats of the Sarda population were divided into 3 groups supplemented with 0 (control group), 0.45 (group 1), or 0.90 (group 2) mg of KI/d per goat. The dose of KI (76.5% of iodine) was dissolved in water and orally administered with a syringe every day for 10 wk. Mean milk iodine concentrations were 60.1 ± 50.5, 78.8 ± 55.4, and 130.2 ± 62.0 μg/L (mean ± SD) in the control group, group 1, and group 2, respectively. The extent of iodine enrichment in milk was approximately 31% in group 1 and 117% in group 2 compared with the control group. Milk yield was not influenced by KI supplementation and averaged 1,229, 1,227, and 1,179 g/d in groups 0, 1, and 2, respectively. Milk urea nitrogen concentration was significantly lower in the KI-supplemented groups (32 and 33 mg/dL in groups 1 and 2, respectively) than in the control group (37 mg/dL). Iodine supplementation of dairy goat diets can increase milk iodine content without adverse effects on milk production traits.     

Prediction of coagulation properties, titratable acidity, and pH of bovine milk using mid-infrared spectroscopy

This study investigated the potential application of mid-infrared spectroscopy (MIR 4,000-900 cm⁻¹) for the determination of milk coagulation properties (MCP), titratable acidity (TA), and pH in Brown Swiss milk samples (n = 1,064). Because MCP directly influence the efficiency of the cheese-making process, there is strong industrial interest in developing a rapid method for their assessment. Currently, the determination of MCP involves time-consuming laboratory-based measurements, and it is not feasible to carry out these measurements on the large numbers of milk samples associated with milk recording programs. Mid-infrared spectroscopy is an objective and nondestructive technique providing rapid real-time analysis of food compositional and quality parameters. Analysis of milk rennet coagulation time (RCT, min), curd firmness (a₃₀, mm), TA (SH°/50 mL; SH° = Soxhlet-Henkel degree), and pH was carried out, and MIR data were recorded over the spectral range of 4,000 to 900 cm⁻¹. Models were developed by partial least squares regression using untreated and pretreated spectra. The MCP, TA, and pH prediction models were improved by using the combined spectral ranges of 1,600 to 900 cm⁻¹, 3,040 to 1,700 cm⁻¹, and 4,000 to 3,470 cm⁻¹. The root mean square errors of cross-validation for the developed models were 2.36 min (RCT, range 24.9 min), 6.86 mm (a₃₀, range 58 mm), 0.25 SH°/50 mL (TA, range 3.58 SH°/50 mL), and 0.07 (pH, range 1.15). The most successfully predicted attributes were TA, RCT, and pH. The model for the prediction of TA provided approximate prediction (R² = 0.66), whereas the predictive models developed for RCT and pH could discriminate between high and low values (R² = 0.59 to 0.62). It was concluded that, although the models require further development to improve their accuracy before their application in industry, MIR spectroscopy has potential application for the assessment of RCT, TA, and pH during routine milk analysis in the dairy industry. The implementation of such models could be a means of improving MCP through phenotypic-based selection programs and to amend milk payment systems to incorporate MCP into their payment criteria.     

Fate of lysostaphin in milk from individual cows through pasteurization and cheesemaking

Transgenic cows secreting over 3 μg of lysostaphin/ mL of milk are protected against mastitis caused by Staphylococcus aureus, but it is unknown if active lysostaphin persists through dairy processing procedures or affects the production of fermented dairy foods. The objective of this study was to determine the fate of lysostaphin as milk was pasteurized and then processed into cheese. Raw milk from transgenic cows was heat treated at 63°C for 30 min, 72°C for 15 s (high temperature, short time), or 140°C for 2 s (UHT). Portions of the high temperature, short-time milk were manufactured into semi-hard cheeses. Aliquots taken at each processing step were assayed to determine the quantity (ELISA) and activity (ability to inhibit S. aureus growth) of lysostaphin. Results indicated that most of the lysostaphin was present in the aqueous portion of the milk and was not affected by pasteurization, although UHT treatment reduced enzyme concentration by 60%. The quantity and activity of the lysostaphin decreased during cheesemaking. Based on the amount of lysostaphin present in the starting cheesemilk, 10 to 15% of the lysostaphin was recovered in the whey, 21 to 55% in the cheese curd at d 1, and 21 to 36% in cheese stored at 4°C for 90 d. Enough of the lysostaphin secreted into milk by transgenic cows survived typical dairy processing conditions to impart potential value as a bioprotective agent against staphylococci in dairy foods.     

The influence of temperature and pressure factors in supercritical fluid extraction for optimizing nonpolar lipid extraction from buttermilk powder

The milk fat globule membrane, readily available in buttermilk, contains complex lipids claimed to be beneficial to humans. Phospholipids, including sphingolipids, exhibit antioxidative, anticarcinogenic, and antiatherogenic properties and have essential roles in numerous cell functions. Microfiltration coupled with supercritical fluid extraction (SFE) may provide a method for removing triacylglycerols while concentrating these nutritionally valuable lipids into a novel ingredient. Therefore, SFE as a method for phospholipid concentration needs to be optimized for triacylglycerol removal in buttermilk. The SFE conditions were assessed using a general full factorial design; the experimental factors were pressure (15, 25, and 35 MPa) and temperature (40, 50, and 60°C). Particularly interesting is that only triacylglycerols were removed from buttermilk powder. Little to no protein loss or aggregation was observed compared with the untreated buttermilk powder. Calculated theoretical values showed a linear increase for lipid solubility as pressure, temperature, or both were increased; however, experimental values showed nonlinearity, as an effect of temperature. In addition, the particular SFE parameters of 35 MPa and 50°C displayed enhanced extraction efficiency (70% total lipid reduction).     

Effect of the inclusion of whole citrus in the ration of lactating ewes on the properties of milk and cheese characteristics during ripening

The effect of including citrus fruits (CF) in the rations of dairy ewes on the milk characteristics and biochemical changes of cheeses during ripening was evaluated. For this purpose, 48 lactating ewes (Guirra breed) were divided into 4 homogeneous groups and fed with isoenergetic and isoprotein rations containing CF at 0, 10, 20, and 30% on a dry matter basis in substitution of dry barley and pelleted beet pulp. During the experimental period, 3 batches of bulk milk were collected from each group and semi-hard cheeses were manufactured. Cheeses were sampled at 15, 30, and 60 d of ripening. Milk coagulation parameters and cheese yield were not negatively affected by the inclusion of CF in the ration. Physicochemical composition of cheeses at 60 d showed statistical differences for lower total solids and fat content of 30% CF cheeses. Proteolysis of cheeses measured by water-soluble nitrogen and total free amino acids content was not influenced by the ration. Differences between rations with respect to free fatty acids were significant for medium- and long-chain free fatty acids, and therefore for total content, but differences did not show a trend related to the increase of CF in the diet. The inclusion of CF in the ration of lactating ewes up to levels of 30% did not negatively affect the properties of milk and the biochemical and sensory characteristics of cheeses.     

Technical note: Flow cytometric identification of bovine milk neutrophils and simultaneous quantification of their viability

The aim of the present study was to develop a flow cytometric procedure for the quantification of the proportion of viable, apoptotic, and necrotic polymorphonuclear neutrophilic leukocytes (PMNL) isolated from both low- and high-somatic-cell-count quarter milk samples. Milk PMNL were differentiated from other cells by indirect fluorescent labeling using a primary anti-bovine granulocyte monoclonal antibody (CH138A) and an Alexa 647-labeled secondary antibody. Polymorphonuclear neutrophilic leukocytes were identified flow cytometrically based on their cytoplasmic granularity and CH138A-positivity. Additional labeling with annexin-V-fluorescein isothiocyanate and propidium iodide was used to determine milk PMNL viability. Thirty milk samples were run in parallel to assess the repeatability of the immunoassay and 6 repeated measurements per sample were performed to assess the instrument stability. Fluorescence microscopic verification of the CH138A staining pattern showed both a high sensitivity (90.9%) and specificity (92.3%). The combination of the side-scatter properties of granulated PMNL and CH138A-Alexa 647 positivity allows the distinction of labeled PMNL from other milk cells and particles that may bind nonspecifically, and from autofluorescent particles present in milk. Quantification of the proportion of PMNL and viable, apoptotic, and necrotic subpopulations in parallel samples gave repeatable results with concordance correlation coefficients varying between 0.93 and 0.99. The average coefficient of variation for repeated measurements in identical samples ranged between 4.2 and 9.7%. In conclusion, this is the first flow cytometric method suited for the simultaneous quantification of viable, apoptotic, and necrotic bovine milk PMNL in a straightforward manner.     

Fermentation characteristics and transit tolerance of probiotic Lactobacillus casei Zhang in soymilk and bovine milk during storage

Lactobacillus casei Zhang is a novel strain that was screened out of koumiss collected in Inner Mongolia, and our previous research showed that L. casei Zhang has health benefits such as cholesterol-reducing and immunomodulating effects. The fermentation characteristics of L. casei Zhang in soymilk and bovine milk and the transit tolerance of L. casei Zhang in fermented milk products during refrigerated storage for 28 d were assessed. A faster decrease in pH and faster growth of L. casei Zhang during fermentation were observed in soymilk compared with bovine milk at various inoculation rates, probably because of the low pH buffering capacity of soymilk. The fermented bovine milk samples had much higher final titratable acidity (TA) values (between 0.80 and 0.93%) than the soymilk samples (between 0.40 and 0.46%). Dramatic increases in TA values in the fermented soymilk samples during storage were observed, and the TA values of the fermented soymilk samples changed from <0.56% to values between 0.86 and 0.98%. On the other hand, only slight increases in TA were observed in the bovine milk samples during the 28 d of storage. The survival rates of freshly prepared cultures of L. casei Zhang in simulated gastric juice at pH 2.0 and 2.5 were 31 and 69%, respectively, and the delivery of L. casei Zhang through fermented soymilk and bovine milk significantly improved the viability of L. casei Zhang in simulated gastric transit. Lactobacillus casei Zhang showed good tolerance to simulated gastric juice and intestinal juice in the fermented soymilk and bovine milk samples, and maintained high viability (>10⁸ cfu/g) during storage at 4°C for 28 d. Our results indicated that both soymilk and bovine milk could serve as vehicles for delivery of probiotic L. casei Zhang, and further research is needed to elucidate the mechanism of the change in pH and TA of L. casei Zhang in fermented milk samples during fermentation and storage and to understand the difference between soy- and milk-based systems.