Comparison of isolation methods using commercially available kits for obtaining extracellular vesicles from cow milk

Extracellular vesicles (EV) are important for delivering biologically active substances to facilitate cell-to-cell communication. Milk-derived EV are widely known because of their potential for immune enhancement. However, procedures for isolating milk-derived EV have not been fully established. To obtain pure milk-derived EV and accurately reveal their function, such procedures must be established. The aim of the present study was to compare methods using commercially available kits for isolating milk-derived EV. Initially, we investigated procedures to remove casein, which is the major obstacle in determining milk-derived EV purity. We separated whey using centrifugation only, acetic acid precipitation, and EDTA precipitation. Then, we isolated milk-derived EV by ultracentrifugation, membrane affinity column, size exclusion chromatography (SEC), polymer-based isolation, or phosphatidylserine-affinity isolation. Using EV count per milligram of protein, which is a good indicator of purity, we determined that acetic acid precipitation was the best method for removing casein. Using nanoparticle tracking analysis, protein quantity analysis, and RNA quantity analysis, we comprehensively compared each isolation method for its purity and yield. We found that SEC-based qEV column (Izon Science) could collect purer milk-derived EV at higher quantities. Thus, a combination of acetic acid precipitation and qEV can effectively isolate high amounts of pure extracellular vesicles from bovine milk.     

The effect of fertilizer nitrogen input to grass-clover swards and calving date on the productivity of pasture-based dairy production

The objective of this systems-scale study was to investigate grazing season timeframes on pasture and milk production and on milk processability of dairy systems with compact spring-calving dairy cows grazing white clover (Trifolium repens L.) based grassland. Fifty-four primiparous and multiparous Holstein-Friesian dairy cows were used in a one-factor study with 3 systems (n = 18) and repeated over 2 yr (2008/09 and 2009/10). The 3 systems were: early spring calving with annual fertilizer N input of 100 kg·ha^?1 applied in spring (ES100N; 2.1 cows·ha^?1; grazing February to November), early spring calving without fertilizer N (ES0N; 1.6 cows·ha^?1; grazing February to November) and late spring calving without fertilizer N (LS0N; 1.53 cows·ha^?1; grazing April to January). Annual pasture production was affected by an interaction between grazing system and year: Mean annual pasture yields for 2008 and 2009 were ES100N; 10.35 and 9.88, ES0N; 8.88 and 8.63, LS0N; 9.18 and 10.31 t of dry matter (DM)·ha^?1 (SEM 0.39). LS0N had higher pasture DM yield in 2009 due to higher clover DM production and biological N fixation compared with the other systems. Clover stolon and root mass in the following February was correlated with stolon and root mass in the previous November with 64% of stolon mass present on LS0N in February (R² = 0.84). There were no detectable differences in per-lactation milk yield (6,335 kg·cow^?1), fat, protein and lactose yields (271, 226, 297 kg·cow^?1, respectively), cow liveweight (585 kg) or body condition score (3.02). Although winter grazing favored subsequent clover DM production, biological N fixation and pasture DM production, delaying calving date in spring and extending lactation into the following winter led to inefficient use of this pasture by the grazing herd and lowered the quality of late-lactation milk for processing purposes. Hence, a mean calving date in mid- to late-February is recommended for zero-fertilizer N input clover-based grassland.     

Influence of konjac flour on foaming properties of milk protein concentrate and quality characteristics of gluten‐free cookie

The effects of konjac flour (KF) for modifying the foaming properties of milk protein concentrate (MPC) and characteristics of gluten‐free rice cookies were investigated. The three variables of konjac concentration (0.1–0.4%), pH (pH 5–9) and NaCl concentration (0.2–0.6 м) were studied. Both KF and pH had considerable effects on the foaming capacity (FC) and foaming stability (FS) of MPC. The NaCl had a negligible effect. Lowered FC and FS observed at pH 9 and pH 5 were increased with the increase in KF addition. Batter characteristics (flowability and consistency), cookie quality (moisture, yield, spread ratio, specific volume, hardness and colour) and sensory acceptance were evaluated in gluten‐free cookies with 0%, 0.25% and 0.4% KF. Konjac supplementation increased batter characteristics, yield, specific volume and hardness, but decreased lightness. Panellist acceptance, especially regarding texture and overall acceptability, increased in gluten‐free cookies with 0.4% KF. A higher purchasing decision was found for cookies with added KF.     

Epilactose production by 2 cellobiose 2-epimerases in natural milk

It was reported recently that cellobiose 2-epimerases (CE) from various aerobic microorganisms convert lactose to epilactose in defined buffer systems. In this study, we showed that CE from 2 mesophilic microorganisms, Flavobacterium johnsoniae and Pedobacter heparinus, were capable of converting lactose to prebiotic epilactose not only in buffer but also in a complex milk system. First, the 2 enzymes were separately cloned, recombinantly expressed in Escherichia coli, and purified by column chromatography. The production of F. johnsoniae CE was carried out in a stirred-tank reactor, indicating that future upscaling is possible. The bioconversions of milk lactose were carried out at an industrially relevant low temperature of 8°C to avoid undesired microbial contaminations or chemical side reactions. Both enzymes were reasonably active at this low temperature, because of their origin from mesophilic organisms. The enzymes showed different operational stabilities over a 24-h time-course. A conversion yield of about 30 to 33% epilactose was achieved with both enzymes. No side products were detected other than epilactose. Therefore, CE may introduce an added value for particular dairy products by in situ production of the prebiotic sugar epilactose.     

Invited review: Genetics and modeling of milk coagulation properties

Milk coagulation properties (MCP) are conventionally measured using computerized renneting meters, mechanical or optical devices that record curd firmness over time (CF_t). The traditional MCP are rennet coagulation time (RCT, min), curd firmness (a₃₀, mm), and curd-firming time (k₂₀, min). The milk of different ruminant species varies in terms of CF_t pattern. Milk from Holstein-Friesian and some Scandinavian cattle breeds yields higher proportions of noncoagulating samples, samples with longer RCT and lower a₃₀, and samples for which k₂₀ is not estimable, than does milk from Brown Swiss, Simmental, and other local Alpine breeds. The amount, proportion, and genetic variants (especially κ-casein) of milk protein fractions strongly influence MCP and explain variable proportions of the observed differences among breeds and among individuals of the same breed. In addition, other major genes have been shown to affect MCP. Individual repeatability of MCP is high, whereas any herd effect is low; thus, the improvement of MCP should be based principally on selection. Exploitable additive genetic variation in MCP exists and has been assessed using different breeds in various countries. Several models have been formulated that either handle noncoagulating samples or not. The heritability of MCP is similar to that of other milk quality traits and is higher than the heritability of milk yield. Rennet coagulation time and a₃₀ are highly correlated, both phenotypically and genetically. This means that the use of a₃₀ data does not add valuable information to that obtainable from RCT; both traits are genetically correlated mainly with milk acidity. Moreover, a₃₀ is correlated with casein content. The major limitations of traditional MCP can be overcome by prolonging the observation period and by using a novel CF_t modeling, which uses all available information provided by computerized renneting meters and allows the estimation of RCT, the potential asymptotic curd firmness, the curd-firming rate, and the syneresis rate. Direct measurements of RCT obtained from both mechanical and optical devices show similar heritabilities and exhibit high phenotypic and genetic correlations. Moreover, mid-infrared reflectance spectroscopy can predict MCP. The heritabilities of predicted MCP are higher than those of measured MCP, and the 2 sets of values are strongly correlated. Therefore, mid-infrared reflectance spectroscopy is a reliable and cheap method whereby MCP can be improved at the population level; this is because such spectra are already routinely acquired from the milk of cows enrolled in milk recording schemes.     

A 100-Year Review: The production of fluid (market) milk

During the first 100 years of the Journal of Dairy Science, dairy foods and dairy production dairy scientists have partnered to publish new data and research results that have fostered the development of new knowledge. This knowledge has been the underpinning of both the commercial development of the fluid milk processing industry and regulations and marketing policies for the benefit of dairy farmers, processors, and consumers. During the first 50 years, most of the focus was on producing and delivering high-quality raw milk to factories and improving the shelf life of pasteurized fluid milk. During the second 50 years, raw milk quality was further improved through the use of milk quality payment incentives. Due to changing demographics and lifestyle, whole fluid milk consumption declined and processing technologies were developed to increase the range of fluid milk products (skim and low-fat milks, flavored milks, lactose-reduced milk, long-shelf-life milks, and milks with higher protein and calcium contents) offered to the consumer. In addition, technology to produce specialty high-protein sports beverages was developed, which expanded the milk-based beverage offerings to the consumer.     

Performance evaluation of small-batch container delivery systems used in lean manufacturing – Part 2: number of Kanban and workstation starvation

In lean manufacturing, milk run (MR) systems represent route-based, cyclic material handling systems that are used widely to enable frequent and consistent deliveries of containerised parts on an as-needed basis from a central storage area (the ‘supermarket’) to multiple line-side deposit points on the factory floor. In the first part of this two-part paper, a basic, single-tugger MR system is described, and stability conditions as well as the probability of exceeding either the physical capacity of the tugger or the prescribed cycle time are derived. Given the stability conditions, and the distribution of the number of containers requested per MR, in the second part of the paper, the number of Kanban required in the MR system is examined, and analytical approximations are derived both for the number of Kanban required and for predicting workstation starvation. The latter is a key concern when designing a MR system that will support workstations in a manufacturing plant. The performance of the analytic approximation is evaluated by simulating various MR systems. Our results suggest that, in a stable MR system, the number of Kanban and the physical capacity of the tugger have a bigger impact on workstation starvation than the prescribed cycle time.     

Characterization and Technological Properties of Lactic Acid Bacteria Isolated from Traditional Argentinean Goat’s Milk Products

A total of 286 lactic acid bacteria isolated from goat’s dairy products in northwest of Argentina were characterized. Lactobacilli (38%) and cocci (62%) were identified according to morphological, physiological, and relevant technological properties. L. plantarum (14%) and Enterococcus (34%) were the predominant species. S. thermophilus, Pediococcus and L. plantarum were the highest acid producers. Eight strains of L. fermentum produced bacteriocins or metabolites similar to bacteriocins. The API-ZYM test was applied to 39 isolates. Eight strains were selected from their both technological properties and enzymatic activities for use as starter or adjunct culture in the manufacture of artisanal goat cheeses.     

Impact of amorphous and crystalline lactose on milk chocolate properties

Chocolate mass of low viscosity is preferred for most applications. Milk powder influences processing behaviour, flow properties and taste of milk chocolate. The project aimed to investigate influences of skim milk powders containing amorphous or crystalline lactose on flow properties after producing samples by roller milling and conching or alternatively by ball milling. For the first case, it was found that mass consistency before roller milling is strongly influenced by lactose type; producers must specify it and adapt initial mass fat content. Little impact on final products was found after processing milk powders at equilibrium moisture. If predried powders are used for reducing conching time, crystalline lactose leads to chocolate with slightly lower viscosity. At ball mill processing, crystalline lactose resulted in significantly lower viscosity, for example 15% at 40 s^?1; thus, for this process, it can be recommended to use special milk powders high in crystalline lactose content.     

Organic whey as a source of Lactobacillus strains with selected technological and antimicrobial properties

Twenty‐five strains, isolated from raw, non‐pasteurised, organic whey samples, were identified phenotypically and genotypically. Biochemical tests were performed, and enzyme profiles, antibiotic resistance and antimicrobial properties were investigated. Sixteen strains were identified as genus Lactobacillus. Based on 16S rDNA gene sequence, the strains were identified as Lb. plantarum and Lb. fermentum. All of the strains had β‐galactosidase activity, and some of them reduced nitrate content. All strains utilised carbohydrates. The tested strains were characterised by low or average lipolytic and esterolytic activity. Moreover, the strains showed low proteolytic activity which is advantageous for their use as starter cultures for foods with low protein content. Strains Lb. fermentum S20, SM1, SM3, S2R and Lb. plantarum SM5 produced harmful N‐acetyl‐β‐glucosaminidase; moreover, the strain S20 produced also β‐glucuronidase. None of the strains produced α‐chymotrypsin. In phenotypic studies, most of the test strains were susceptible to gentamicin, ampicillin, tetracycline, chloramphenicol, penicillin and erythromycin. Strains Lb. plantarum S1 and Lb. fermentum S4, S7, S8, S10, SM1 and SM3 did not possess any transfer resistance genes. Antagonistic activity of the culture LAB strains was assessed as high or moderate in relation to the indicator strains, with the greatest zones of inhibition for E.coli and the smallest for L. monocytogenes ATCC 15313. This study reveals that the LAB strains isolated from organic whey have high potential for food application. Some strains of species Lb. fermentum (S4, S7, S8, S10) have been identified as the best candidates.