Rapid analysis of acylglycerols in low molecular weight milk fat fractions.

A suitable analytical method was required to facilitate development of an industrial-scale short-path distillation (SPD) process. Short-path distillation produces milk fat distillates (MFD) enriched in low molecular weight milk fat components-viz. free fatty acids, monoacylglycerols, diacylglycerols, cholesterol and low molecular weight triacylglycerols. In this case, solid-phase extraction (SPE) was considered a better alternative than thin-layer chromatography for separating polar and apolar lipid components in MFD samples due to its speed and near-complete recoveries. Solid-phase extraction of MFDs yielded two fractions, both of which are sufficiently pure for subsequent analysis by gas chromatography. This procedure provided rapid and complete chemical characterization (including mass balances) of low-molecular weight milk-fat fractions.

Evidence for fibril-like structure in bovine casein micelles.

The addition of Congo red (CR) dye to diluted raw skim milk resulted in a red shift indicative of the presence of fibril-like structures. Thioflavin T (ThT) is another dye that very specifically binds to protein fibrils, and when added to undiluted raw skim milk, the classic 485 nm fluorescence peak of a ThT-fibril complex was observed. Repeating these experiments with various raw milk components showed that the CR red shift and ThT fluorescence peak were due to the presence of casein micelles, and to a lesser extent, sodium caseinate. Fluorescent peaks were also observed when ThT was added to solutions of purified alpha(S)- and kappa-casein, but not beta-casein, in 0.5 M HEPES buffer (pH = 6.8). The addition of 25 mM Ca(2+) had no effect on beta-casein fluorescence, and significantly reduced the kappacasein peak. However, adding 25 mM Ca(2+) to alpha(S)-casein produced a turbid solution and a 6-fold increase in fluorescence, indicating that the aggregates formed contain fibril-like structure. Casein micelle images obtained by transmission electron microscopy showed the presence of short (7 to 10 nm) fibers cross-linked by dense aggregate junction zones. The observed fibers closely resemble protofibrils, intermediate structures that are observed during the formation of amyloid fibrils.

Towards an optimum mixing protocol for on-farm bulk milk sampling.

This paper describes the application of a bottom-sampling technique to dynamically monitor creaming and mixing in bulk milk tanks and determine optimum mixing protocols for milk sampling. Creaming onset in field experiments occurred between 40 and 50 min. Bottom-sampling data determined after 3 h of creaming indicate that the mean mixing time required to ensure a homogenous sample for composition testing is 57 s, and there is a less than 1% probability that an individual tank would require more than 2 min of agitation. Bottom and top-sampling statistics determined after 1 h of creaming indicate mean mixing times of 20 and 34 s, respectively, and predict that individual tank mixing times will exceed 46 and 64 s, respectively, less than 1% of the time. Bacterial cell counts were directly correlated with fat content, but somatic cell counts were independent of fat content. Based on these results, it is recommended that hourly agitation of bulk tanks as currently prescribed in many jurisdictions should be maintained, but the duration of intermittent agitation should be reduced from 5 to 2 min to reduce the impact of agitation on fat globule stability. If hourly agitation is effected during milk storage, agitation time before sampling can be reduced from 5 to 2 min. This will save time for drivers and trucks and reduce the potential impact of agitation on fat globule stability.

A review of international standards and the scientific literature on farm milk bulk-tank sampling protocols.

Adequate agitation is required to ensure homogeneity before sampling from on-farm bulk tanks, but excessive agitation may cause churning with a resulting loss of milk quality. Homogeneity can be assured by thorough mixing before a sample is taken and can also be combined with intermittent agitation of the bulk tank. There is general but qualified agreement among various countries and agencies, such as the IDF, that 5 min of agitation for small, and 10 min for large, quiescent farm-milk bulk tanks is required to ensure sample homogeneity. However, no empirical studies are cited to support these standards. The few studies that examined bulk-tank mixing estimate required agitation times of 8 to 10 min or longer, depending on the size of the tank. If intermittent agitation is practiced, mixing for 1 to 2 min before sampling is considered acceptable in some jurisdictions but, once again, empirical supporting evidence is absent. Automatic samplers decrease the amount of time needed to obtain a sample from the bulk tank, but both intermittent agitation and agitation during milk transfer are still recommended to minimize fat residue accumulation in the bulk tank. Systematic studies are needed to establish mixing protocols that assure accurate sampling for all tanks in a given jurisdiction.

Solvent effects on the crystallization behavior of milk fat fractions.

The high- and medium-melting fractions of milk fat (HMF and MMF, respectively) were crystallized in the presence of various solvents, including the low-melting fraction of milk fat (LMF), canola oil (CO), hexane, and ethyl acetate. Choice of solvent was shown to have a strong influence on phase behavior and crystallization kinetics. Dilution and solubilization effects were observed for all the blends. More solids were formed in the HMF and MMF blends with LMF than with CO, and complexes were formed between the milk fat fractions possibly because of molecular complementarity. Solids were slightly higher for the more polar ethyl acetate than for hexane. Crystallization proceeded more rapidly in the presence of LMF and ethyl acetate than in the presence of CO and hexane, respectively. According to the Hildebrand equation, HMF and MMF were ideally soluble in LMF and CO. X-ray diffraction spectroscopy (XRD) revealed the existence of liquid-state structure in mixtures of HMF/CO, HMF/LMF, MMF/CO, and MMF/LMF. The observed liquid-state structure was reminiscent of liquid crystals. No differences were observed in the structure of the liquid phase between LMF- and CO-containing mixtures.

Influence of protein surface morphology on the ultrafiltration flux resistance of bovine serum albumin.

The effect of added ethanol and (NH(4))(2)SO(4) on the flux decline index (FDI) of bovine serum albumin (BSA) and a fatty acid-poor derivative (BSA/FAP) was examined. Ternary phase diagrams of the two protein species indicated that the concentration polarization (CP) layer on the surface of a nonadsorbing 10 000 MWCO regenerated cellulose membrane had principally a packed bed structure up to 33 wt % ethanol and 21 wt % (NH(4))(2)SO(4). Intrinsic viscosity and turbidity analysis were conducted to determine the degree of intra- and interprotein interactions within this packed bed morphology. With BSA/FAP, the effects of these two interactions tended to counterbalance each other, so the FDI of this protein was not strongly influenced by solute addition. In contrast, the adsorption of fatty acids to BSA caused the protein to expand, producing a less rigid CP layer with a higher FDI. However, the addition of ethanol led to protein compression, reducing this effect. The presence of fatty acids also produced a more associated BSA in salt solution, which increased flux resistance. The results obtained for both proteins indicate that an FDI minimum is observed when a noninteraction hard sphere structure is present in the CP layer.

Lipid modification strategies in the production of nutritionally functional fats and oils.

Rapid improvements in the understanding of the nutritional requirements of both infants and adults has led to new developments in the modification of fats and oils. Specific targets include the improvement in growth and development of infants, treatment of disease in adults, and disease prevention. Efforts have been focussed on the production of structured lipids using medium-chain acids and long-chain polyunsaturated fatty acids (PUFAs), as well as the concentration of long-chain PUFAs from new and existing sources. Short- and medium-chain fatty acids are metabolized differently than long-chain fatty acids and have been used as a source of rapid energy for preterm infants and patients with fat malabsorption-related diseases. Long-chain PUFAs, specifically docosahexaenoic acid and arachidonic acid, are important both in the growth and development of infants, while n-3 PUFAs have been associated with reduced risk of cardiovascular disease in adults. Based on the requirements for individual fat components by different segments of the population, including infants, adults, and patients, ideal fats can be formulated to meet their needs. By using specific novel fat sources and lipid modification techniques, the concentrations of medium-chain, long-chain saturated, and long-chain polyunsaturated fatty acids as well as cholesterol can be varied to meet the individual needs of each of these groups. While genetic modification of oilseeds and other novel sources of specific lipid components are still being developed, chemical and lipase-catalyzed interesterification reactions have moved to the forefront of lipid modification technology. Fractionation of fats and oils to provide fractions with different nutritional properties has potential, but little work has been performed on the nutritional applications of this method. The choice of suitable lipid modification technologies will depend on the target lipid structure, production costs, and consumer demand. A combination of some or all of the present lipid modification techniques may be required for this purpose.

Effect of ethanol, ammonium sulfate, fatty acids, and temperature on the solution behavior of bovine serum albumin.

Ternary phase diagrams (TPDs) for aqueous bovine serum albumin (BSA) solutions containing ethanol or (NH4)2SO4 were determined for temperatures ranging from 20 to 70 degrees C. At 20 degrees C, ethanol destabilized BSA, resulting in gel formation at moderate solute concentrations. In contrast, (NH4)2SO4 concentrations above 20 wt % precipitated BSA from solution. Raising the temperature led to gel formation at increasingly lower BSA and solute concentrations. The removal of adsorbed fatty acids from BSA had little effect on the ethanol TPDs but reduced protein solubility in (NH4)2SO4. Moreover, the salt TPDs of fatty-acid-poor BSA were similar to those previously observed with S-ovalbumin.

Low-temperature stress induces transient oscillations in sucrose metabolism in Solanum tuberosum.

Exposure to low but nonfreezing temperatures induces the net breakdown of starch and the accumulation of sucrose, glucose and fructose in potato tuber tissue, a complex phenomenon known as low-temperature sweetening (LTS). When transferred to 4 degrees C storage, tissue sucrose levels in LTS-sensitive potato tubers (Solanum tuberosum cv. Norchip) did not change monotonically to a new steady state, but rather transiently oscillated about the trajectory to the new steady state. The dynamic patterns observed in sensitive tubers grown in 1993 and 1994 were qualitatively similar. Quantitatively, however, the transient oscillation had a period of 11.5 days in 1993, whereas a period of 80 days was observed in 1994. In contrast, the sucrose levels of the LTS-tolerant potato tubers (Solanum tuberosum seedling ND860-2) increased monotonically to a higher level upon exposure to low temperatures.

Effect of shear on the inactivation kinetics of the enzyme dextransucrase.

An inactivation model previously developed to characterize the rate of enzyme activity loss in unstirred solutions was extended to take into account orthokinetic interactions resulting from convective mixing. A synergistic relationship between shear rate and temperature was observed; the rate of inactivation of the enzyme dextransucrase was unaffected by the action of shear below 25 degrees C, but was increased by the shear rate at 30 degrees C. Shear rate does not appear to influence the equilibrium between native and denatured dextransucrase either directly in solution or indirectly by augmenting the turnover of the gas-liquid interface. However, a second-order plot of the inverse of relative activity (A(O)/A) versus Gt (shear rate x time) of dextransucrase at a constant temperature was linear because of the influence of shear on the coagulation of the denatured enzyme. The addition of 0.01 g L(-1) of polyethylene glycol (MW 20,000) blocked this coagulation reaction, thereby completely inhibiting the shear-induced inactivation of dextransucrase at 30 degrees C.

Effect of subunit dissociation, denaturation, aggregation, coagulation, and decomposition on enzyme inactivation kinetics: I. First-order behaviour.

Enzyme inactivation kinetics typically follows what would appear to be simple first-order behavior. However, the inactivation process is known to involve a number of reversible (decomposition, denaturation) as well as irreversible (decomposition, aggregation, and coagulation) reactions. These reactions can combine to form a wide variety of reaction pathways which can potentially demonstrate complex inactivation kinetics. However, it was shown that with appropriate assumptions with regard to the relative magnitudes of the various reaction rates, many complex inactivation pathways can demonstrate apparent first-order behavior. Thus, with this analysis, a more accurate interpretation of the slope of an activity versus time semi-log plot can be obtained.

Effect of subunit dissociation, denaturation, aggregation, coagulation, and decomposition on enzyme inactivation kinetics: II. Biphasic and grace period behavior.

A model previously developed to characterize enzymatic in activation behavior was used to explain the non-first-order biphasic and grace period phenomena that are often observed with oligomeric enzymes. Luciferase and urease were used as model enzyme such as luciferase, the oligomer initially dissociates reversibly into two native monomer species. These native monomers can then reversibly denature and irreversibly aggregate and coagulate. With the hexamer, urease, two trimers are formed that can subsequently aggregate to form an inactive hexamer. The dissociated monomer species of luciferase do not possess catalytic activity, so the inactivation mechanism, is biphasic; the first slope of a first-order kinetic plot is influenced by the reversible oligomer/monomer/denatured-monomer transition. Whereas the second slope is associated with either irreversible aggregation or coagulation. In contrast, the trimer of urease has the same activity as the hexamer; therefore, during the intitial hexamer-trimer transition, little activity loss occurs. However, as the trimer concentration increases, activity decreases as a result of trimer aggregation. As a result, grace period inactivation behavior is observed.

Absorption of CO2 in algal mass culture systems: a different characterization approach.

For the characterization of CO(2) absorption in aerated microalgal culture systems, a different approach based on K(L)a(O(2)) determination and transformation was studied. To confirm the validity of this method, the influence of reactions between CO(2) and compounds (OH(-), H(2)O, and NH(3)) present in the culture medium upon the absorption mechanism was evaluated under different physical and chemical culture conditions. Under these conditions, knowledge of the relative magnitudes of the diffusion and reaction kinetics permitted the evaluation of their relative importance. For the determination of the parameters required for the calculation of the CO(2) absorption constant, empirical correlations for K(L) (0) and a were used that had been previously verified with experimental data for O(2) absorption. Since, for the conditions studied, the absorption rate was shown to be independent of the chemical reactions taking place in the liquid phase, the K(L)a for CO(2) could be directly related to the K(L)a for O(2) by a simple factor that took into account the difference in aqueous diffusivity of the two gases. Thus, using methods developed for determining O(2) absorption in gas-liquid contactors, it is possible to adequately characterize CO(2) absorption for laboratory and pilot scale algal production systems.