Increased Lactose Concentration Research Articles

Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum.

The use of environmentally damaging petrochemical feedstocks can be displaced by fermentation processes based on engineered microbial chassis that recycle biomass-derived carbon into chemicals and fuels. The stable retention of introduced genes, designed to extend product range and/or increase productivity, is essential. Accordingly, we have created multiply marked auxotrophic strains of Clostridium acetobutylicum that provide distinct loci (pyrE, argH, purD, pheA) at which heterologous genes can be rapidly integrated using allele-coupled exchange (ACE). For each locus, ACE-mediated insertion is conveniently selected on the basis of the restoration of prototrophy on minimal media. The Clostridioides difficile gene (tcdR) encoding an orthogonal sigma factor (TcdR) was integrated at the pyrE locus under the control of the lactose-inducible, bgaR::PbgaL promoter to allow the simultaneous control of genes/operons inserted at other disparate loci (purD and pheA) that had been placed under the control of the PtcdB promoter. In control experiments, dose-dependent expression of a catP reporter gene was observed with increasing lactose concentration. At the highest doses tested (10mM) the level of expression was over 10-fold higher than if catP was placed directly under the control of bgaR::PbgaL and over 2-fold greater than achieved using the strong Pfdx promoter of the Clostridium sporogenes ferredoxin gene. The utility of the system was demonstrated in the production of isopropanol by the C. acetobutylicum strain carrying an integrated copy of tcdR following the insertion of a synthetic acetone operon (ctfA/B, adc) at the purD locus and a gene (sadh) encoding a secondary dehydrogenase at pheA. Lactose induction (10mM) resulted in the production of 4.4g/L isopropanol and 19.8g/L Isopropanol-Butanol-Ethanol mixture.

Kinetic Model for Galacto-Oligosaccharide Synthesis

A new micro-kinetic model is proposed for galacto-oligosaccharide (GOS) synthesis with β-galactosidase from Aspergillus oryzae. Several kinetic models with similar reaction mechanisms, i.e., steps and kinetic constants, have been investigated covering a range of initial lactose (20–40 wt %) and enzyme concentrations (0.5–2 mg/mL). The kinetic parameters were estimated simultaneously for all experimental results using a hybrid genetic algorithm. A detailed analysis of the effect of process parameters on the concentrations of lactose and GOS is performed. The proposed model has a very good quantitative fit of the experimental data, with an average error of 9.34%. More importantly, qualitative trends for the change in the concentration of lactose and GOS are predicted excellently, especially if one takes into account the variation of both concentrations of enzyme and lactose in a range wider than reported in the literature to date. Also, the analysis between the two comparative reactions of transglycosylation and lactose hydrolysis confirmed the positive effect of increasing lactose concentration on the production of GOS. On the other hand, a higher enzyme concentration resulted in a faster production of GOS but with more intensive decomposition at longer reaction times.

Inline Milk Lactose Concentration as Biomarker of the Health Status and Reproductive Success in Dairy Cows

In this study, cow reticulorumen content pH and temperature together with the activity were registered using smaXtec boluses, specially designed for animal care. Body weight, rumination time, milk fat/protein ratio, milk yield, milk lactose, milk electrical conductivity, milk somatic cell count and consumption of concentrated feed were registered by Lely Astronaut® A3 milking robots. The cows in this study were assigned into two groups according to the milk lactose concentration: group 1—milk lactose < 4.70% (n = 20), group 2—milk lactose ≥ 4.70% (n = 15). The following cows were further classified according to milk fat and protein ratio: F/P < 1.2 (class 1), F/P = 1.2 (class 2) and F/P > 1.2 (class 3). According to our results, we can conclude that inline registered milk lactose concentration can be used to indicate the health status and reproductive success of fresh dairy cows. Cows with an increased lactose concentration (≥4.70%) showed more activity (54.47%) and had less risk of mastitis (determined by lower milk electrical conductivity (EC) and somatic cell counts (SCC)) and metabolic disorders, determined by milk F/P. A higher glucose concentration was also apparent in the cows with higher lactose concentration. Registered lower levels of milk lactose can be used for early identification of metabolic disorders and mastitis (set at milk SCC ≥ 100 thousand/mL). Lactose levels in cows’ milk were positively associated with their reproductive success.

Increasing lactose concentration is a strategy to improve the proliferation of Lactobacillus helveticus in milk.

The aim of the research was to explore the effect of carbon sources on the proliferation of Lactobacillus helveticus. In this study, lactose was added to skim milk medium and the effects of carbon sources on the growth and proliferation of Lactobacillus helveticus in low‐ and high‐sugar media were compared from the aspects of metabolism‐related enzyme activity, proteomics, and transcriptomics. The results showed that under high‐sugar conditions, the rate of substance transport to cells and the Embden–Meyerhof–Parnas (EMP) pathway were significantly accelerated. The synthesis and metabolism of cells were significantly enhanced, which was beneficial to the rapid proliferation of cells. By increasing the lactose concentration in the medium and optimizing the culture method, the cell density of Lactobacillus helveticus reached 3.98 × 109 CFU/ml; a good proliferation effect was obtained.

Effects of maternal supplementation with fully oxidised β-carotene on the reproductive performance and immune response of sows, as well as the growth performance of nursing piglets.

The present study was conducted to evaluate the impact of dietary fully oxidised β-carotene (OxBC, C40H60O15) supplementation during the perinatal period on immune status and productivity in a sow model. At day 85 of pregnancy, 150 sows were allocated to one of three dietary treatments with fifty sows per treatment. The three experimental diets were supplemented with 0, 4 or 8 mg/kg OxBC in the basal diet. The feeding trial was conducted from gestation day 85 until day 21 of lactation. Dietary OxBC supplementation greatly enhanced colostrum IgM, IgA and IgG levels, and the IgM and IgG content of 14-d milk. Dietary OxBC supplementation decreased the TNF-α and IL-8 levels in colostrum, as well as the TNF-α and IL-18 levels in 14-d milk. There was also a tendency towards an increase in the soluble CD14 level in 14-d milk. Although dietary treatments did not affect average daily feed intake nor backfat thickness loss during lactation, dietary OxBC supplementation tended to enhance litter weight and individual piglet weight at weaning. There was a trend towards increased lactose concentration in 14-d milk with increasing dietary OxBC. It is concluded that dietary supplementation with OxBC during the perinatal period enhances the lactose concentration of sow milk and the immune status of sows, which is reflected by improved cytokine status and immunoglobulin concentrations in colostrum and milk, and thus tending to increase litter weight and individual piglet weight at weaning. The results also provide a scientific nutritional reference for perinatal mothers due to the biological similarity between pigs and humans.

Processing Human Milk to Increase Nutrient Density for Preterm Infants

Breast milk is the optimal food for infants. It is tailor‐made for each species and each birth. Preterm birth can cause problems with initiation of lactation especially if it is the first pregnancy of the mother, and infants may have inadequate suckling reflexes. In this situation direct intragastric feeding can use donor breast milk from a milk bank, infant formula, or a combination. Concerns regarding the choice of feed are catch‐up growth and health effects. Formula might cause necrotizing enterocolitis and negative long term health effects but has faster catch‐up growth. Preterm infants have better tolerance for human milk, but lower caloric density of term mothers' milk or donor milk might not meet preterm infant growth needs. Preterm infants have higher needs for calories and protein and have a very limited stomach capacity.Aims of this study were to concentrate donor breast milk to have a higher caloric density and protein, but at the same time avoiding side effects of higher lactose concentration and hyperosmolarity by precipitating lactose at low temperature.Donor breast milk was obtained from WakeMed Mothers' Milk Bank. For preliminary results 10 human milk samples of 50 mL were concentrated by evaporation (Rotavap Buchi R‐300) to different concentration levels, held at 0°C overnight, followed by refrigerated centrifugation for lactose removal. Measurement of lactose was performed with enzymatic analysis via Roche Lactose/D‐Galactose kit on skim milk supernate and remixed skim milk.Preliminary data found that low temperature removal of lactose from unprocessed human milk was minimal. Therefore milk was concentrated to varying extents prior to freezing. Volume reductions were 63%, 48%, 40%, 30% and 28%. Macronutrients were concentrated in the milks during the evaporation procedure in proportion to the amount of water removed, so caloric density was higher. Volume reduction (%) vs. Lactose concentration (g/L) yielded a linear relationship (Lactose =1.25 * volume‐reduction +3.50, r2=.77, p<0.05). Lactose was not consistently precipitated at the lower levels of volume reduction.Water can be effectively removed from human milk by low pressure evaporation at temperatures that are unlikely to denature bioactive substances. Regression analysis found that every percent of concentration increased 1.2 g/L of lactose in milk, but the effectiveness of low temperature lactose precipitation was not statistically significant. Similar concentration should be true for protein and other bioactive factors. Additional research is needed to devise techniques to further reduce the osmotic activity in the donor milk concentrate generated by the increased lactose concentration.Support or Funding InformationUSDA HatchProject NC02435; Fulbright Foundation Scholarship to HZU.

Pressure-Induced Denaturation of β-Lactoglobulin in Skim Milk: Effect of Milk Concentration

The effect of milk concentration (10-40% TS) on the kinetics of the pressure-induced denaturation of β-lactoglobulin (β-LG) was studied. The denaturation was found to be a second-order process at all milk concentrations and pressures. There was a change in pressure dependence of the rate constants for denaturation at about 300 MPa, and this effect became more pronounced as the milk concentration increased. At pressures ≥300 MPa, a small effect of milk concentration was observed, with small decreases in the rate of denaturation as the milk concentration was increased above 20% TS. This was attributed to the lower pH as the milk concentration was increased. In contrast, at 200 MPa, β-LG denaturation was markedly retarded as the milk solids concentration was increased. This was attributed to the increased lactose concentration at higher milk concentrations. This would promote β-LG dimerization at this pressure and this would stabilize the β-LG to denaturation.

Persistency of the effect of long-term administration of a whey protein gel composite of soybean and linseed oils on performance and milk fatty acid composition of dairy cows

To study the persistency of the effect of supplementing a whey protein emulsion gel (WPEG) of a mixture of linseed and soybean oil on milk fatty acids, a 10-wk experiment was carried out with 32 lactating Holstein Friesian dairy cows. During the first 5 wk, all cows were managed on a restricted grazing regime with pasturing during the daytime and receiving a mixed ration of maize silage, grass hay, soybean expeller and a mineral mix during night-time. During the last 5 wk, all cows were indoors throughout the day and received a mixed ration containing grass silage, maize silage, grass hay, and concentrates. Cows were randomly allotted to one of two treatments: a control treatment with no supplementation and a “Gel” treatment supplementing WPEG at a rate of approximately 1.5 kg/d per cow. Feed intake and milk production were monitored and samples of a.m. milking were analyzed for fat, protein, lactose and fatty acids. Supplementing WPEG had no significant effect on monitored dry matter intake (14 kg DM/d during restricted grazing — grass intake not determined; 21 kg DM/d during the last 5-wk period). Supplementing WPEG had no effect on milk yield (average 35 kg/d) and on concentrations and yields of milk fat and milk protein. Supplementing WPEG increased lactose concentration. Supplementing WPEG decreased the proportions in milk of medium-chain fatty acids and increased the proportions of most C18 fatty acids, except cis9 C18:1 and trans10, cis12 C18:2. Supplementing WPEG led to doubling of cis9, cis12 C18:2 (from 1.8 to 4.0 g/100 g of fatty acid) and a fourfold increase above control in cis9, cis12, cis15 C18:3 (from 0.5 to 2.2 g/100 g of fatty acids). From these results it is concluded that feeding vegetable oils as WPEG to dairy cattle has a persistent effect on the proportions of poly-unsaturated fatty acids in milk.

The processing of nanoparticles containing protein for suspension in hydrofluoroalkane propellants

Nanoparticles delivered from pressurized metered dose inhalers (pMDIs) potentially offer a means of efficiently delivering proteins to the lung. Nanoparticles containing the model protein lysozyme have been produced using microemulsion and nanoprecipitation methods. Freeze-drying water in oil emulsions, with chloroform as the organic solvent, followed by washing of excess surfactant (lecithin) led to the production of lactose nanoparticles having approximately 300 nm mean size. Substitution of lactose with lysozyme led to a significant increase in the mean size of nanoparticles (645–750 nm). This may have been due to the surface activity of lysozyme which altered the emulsification properties. The retained biological activity of lysozyme increased with increased lactose concentration in the formulation, and approximately 99% biological activity was retained when 20% (w/w) lactose was used. Ethanol used in the formulation in place of chloroform changed the production process from emulsification to nanoprecipitation. A monodisperse system (mean size approximately 275 nm, polydispersity index approximately 0.1) of spherical nanoparticles containing 80% (w/w) bioactive lysozyme (retained activity 99%) was generated. The nanoparticles washed with ethanol containing DPPC, oleic acid or Span 85 (2%, w/v) could readily be dispersed in HFA 134a without further processing, and a stable suspension was formed. Lysozyme remained stable (retained biological activity 98%) even after the nanoparticles were suspended in HFA 134a. This indicates the potential of nanoparticles for delivery of proteins from HFA-based pressurized metered dose inhaler formulations.

Galactooligosaccharide production by a thermostable β-galactosidase from Sulfolobus solfataricus

A recombinant β-galactosidase from Sulfolobus solfataricus produced galactooligosaccharides (GOS) from lactose by transgalactosylation. The enzyme activity for GOS production was maximal at pH 6.0 and 85°C. The half-lives of the recombinant β-galactosidase at 70, 75, 80, 85, and 90°C were 700, 111, 72, 43, and 2.4 h, respectively, and its deactivation energy was 213 kJ mol−1. The optimal amount of enzyme for effective GOS production was 3.6 U of enzyme ml−1. GOS production increased with increasing lactose concentration, whereas the yield of GOS from lactose was almost constant. The rates of hydrolysis and transgalactosylation reactions increased with increasing temperature but the final concentration of GOS was maximal at 80°C. Under the conditions of pH 6.0, 80°C, 600 g lactose l−1, and 3.6 U enzyme ml−1, 315 g GOS l−1 were obtained for 56 h with a yield of 52.5% (w/w). The β-galactosidase from S. solfataricus produced GOS with the highest concentration and yield among thermostable β-galactosidases reported to date.

Effect of lactose and glycerol on the motility, normal apical ridge, chromatin condensation and chromatin stability of frozen boar spermatozoa

The effect of lactose and glycerol concentration, as well as the equilibration time with glycerol was studied on motility, normal apical ridge (NAR), and chromatin state of boar spermatozoa after the freezing and thawing process. In the first experiment, samples were frozen in first and second extenders containing different concentrations of lactose (11, 12 and 14%). In the second experiment samples were frozen using second extenders with different concentrations of glycerol (4, 6, 8 and 10%) and were incubated at 5 °C for 0 and 30 min. Motility, motility after caffeine treatment, NAR, chromatin condensation and stability (susceptibility to decondense after heparin treatment) were evaluated. The results indicated that freezing spermatozoa in extenders with increasing concentrations of lactose adversely affected motility but provided a protective effect on acrosomes. Increased lactose concentration induced higher chromatin condensation but maintained the same stability. Increasing the glycerol concentration in the second extender from 4–6 to 8% led to higher motility and NAR as well as lower chromatin condensation and stability. When 30 min equilibration time was allowed after dilution with the same extenders, spermatozoa showed higher NAR and lower chromatin condensation and stability. The longer equilibration time was detrimental for motility when freezing in the 8% glycerol extender but favourable when using the 4% glycerol extender. Compared to the 8% glycerol, spermatozoa frozen in the 10% glycerol extender showed similar motility and increased chromatin condensation and stability, as well as low values of NAR that did not improve by longer incubation time.

ANALYSIS OF HUMAN BREAST MILK AT MICROWAVE FREQUENCIES

In this communication a comprehensive study of the dielectric properties of human breast milk at microwave frequencies is reported. The samples are collected at weekly intervals following child birth. Measurements are made at the S-band of microwave frequency employing the rectangular cavity perturbation technique. The dielectric constants of the breast milk samples are found to increase as weeks elapse, which is attributed to the reduced fat content and increased lactose concentration. The conductivity of the breast milk samples is similarly found to increase due to the increased dilution.

Dielectric Properties of Human Colostrum at Microwave Frequencies

This article communicates the study of both the dielectric properties of human Colostrums and breast milk at microwave frequencies. The colostrum samples were taken immediately after child birth and breast milk samples were collected at weekly intervals following the delivery. Rectangular cavity perturbation technique is used for the measurements of dielectric properties at the S-band of microwave frequency. The dielectric constants of the colostrums samples and breast milk samples are found to increase as weeks elapse, which is attributed to the reduced fat content and increased lactose concentration. The conductivity of these samples is similarly found to increase due to the increased dilution.

OPTIMIZATION OF GALACTOOLIGO-SACCHARIDE PRODUCTION FROM LACTOSE USING β-GLYCOSIDASES FROM HYPERTHERMOPHILES

The maximal production of galactooligosaccharides (GOS) from lactose by (β-glycosidases from the hyperthermophilic archaea, Sulfolobus solfataricus (LacS) (derived from lacS gene) and Pyrococcus furiosus (CelB) (derived from celB gene) was optimized. The performance of these enzymes under extreme reaction conditions, temperatures up to 95°C and lactose concentrations up to 90% (w/v), were studied. The highly thermostable enzymes were shown to be very well suited for oligosaccharide synthesis. For both LacS and CelB the maximum yield of GOS increased with increasing lactose concentration, up to 70% (w/v). The maximum yield of GOS also increased with increasing temperature, and the optimal pH for synthesis was different at different temperatures. The sum of tri- and tetrasaccharides yielded 37% (w/w) in an optimally designed reaction for LacS, and a maximum yield of 40% (w/w) was attained for CelB. Compared to aqueous solution, an increase of the tetrasaccharide/trisaccharide ratio was obtained in two-phase systems with heptane and nonane. These two enzymes from hyperthermophilic organisms were shown to give higher GOS yields at high substrate concentrations than a β-galactosidase from a thermophilic/mesophilic organism (Aspergillus oryzae).

Non-equilibrium thermodynamics analysis of the transport properties of formed-in-place Zr(IV) hydrous oxide-polyacrylate membranes: III. Ternary lactose-NaCl-water solutions

A non-equilibrium thermodynamics approach using practical transport coefficients has been used to evaluate frictional coefficients for the dominant solute near infinite dilution of the minor solute in the high pressure nanofiltration of ternary lactose-NaCl-water solutions with Zr(IV) hydrous oxide-polyacrylate membranes at pH 8.0. In terms of the frictional coefficients, the observed increase in the passage of NaCl with increasing lactose concentration at low concentrations of lactose is attributed to larger decreases in the NaCl-membrane and NaCl-solvent couplings than the decrease in the solvent-membrane coupling. The observed small decrease in the passage of lactose with increasing NaCl concentration at low concentrations of NaCl is attributed to a larger increases in the lactose-membrane and lactose-solvent couplings than the increase in the solvent-membrane coupling. The use of practical transport coefficients to evaluate the frictional coefficients by this method is limited to solutions with large differences in the concentrations of the two solutes.

Optimization of alkyl β‐D‐galactopyranoside synthesis from lactose using commercially available β‐galactosidases

Commercially available lactase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) enzymes produced from Kluyveromyces fragilis and Kluyveromyces lactis were accessed as catalysts for use in the production of beta-galactopyranosides of various alcohols using lactose as galactosyl donor. The yield of galactoside was enhanced by using the highest practical concentrations of both lactose and alcohol acceptor. The concentrations and thus yield, were limited by the solubility of the substrates. The increase in galactoside yield with increasing lactose concentration appeared to be specific to the lactose substrate and not due to water activity alterations, because addition of maltose to a fixed concentration of lactose had no effect. During the course of the reaction, the yield of galactoside peaked after around 70% to 80% of the lactose was consumed, due to hydrolysis of the product by the enzyme. A wide variety of compounds with primary or secondary hydroxyl groups could act as acceptors, the essential requirement being at least some water solubility. Addition of organic cosolvents had little effect on galactoside yield except when it increased the water solubility of sparingly soluble alcohols. Some galactosides were synthesized on a gram scale to determine practical product recoveries and improve purification methods for large-scale synthesis. Initial purification by hydrophobic chromatography (for galactosides of hydrophobic alcohols) or strong anion-exchange chromatography (for galactosides of hydrophilic alcohols) separated galactosides, galactobiosides, and higher oligomers from reducing sugars. A facile separation of the galactoside and galactobioside could then be effected by flash chromatography on silica gel.

Regulation of nisin biosynthesis by continuous cultures and by resting cell of Lactococcus lactis subsp. lactis

Nisin production by Lactococcus lactis subsp. lactis has been investigated using lactose as carbon source. Whether or not continuous cultures were lactose-limited, maximum nisin titre was observed at an intermediate μ value with a sharp peak of activity between 0.2 and 0.3/h. The maximum specific growth rate obtained in the medium used was 0.6/h and the maximum titre of nisin at μ = 0.25/h (160 AU/ml) was about nine-fold higher as compared with activity obtained at a dilution rate of 0.05/h or 0.4/h. With a constant dilution rate of 0.25/h and varying initial lactose concentrations from 3 to 40 g/l, there is an increase in nisin biosynthesis with increasing lactose concentration correlated with higher rates of sugar consumption. A Ymax value of 0.2 g bacterial dry weight and a maintenance coefficient of 124 mg lactose/g bacterial dry weight/h were determined. Lactose consumption increased from 1 to 3.28 g of lactose /g (dry wt) of cell mass/h and the nisin titre from 12.5 to 164.2 AU/ml. At higher values, nisin production declined. This implies that biosynthesis of nisin is regulated by a system of repression and derepression. Addition of lanthionine and β-methyllanthionine precursors to the medium decreased the nisin titre when either threonine, threonine-cysteine, or cysteine-serine-threonine was added at the optimal dilution rate of 0.25/h; however, simultaneous addition of serine and cysteine elicited a slight increase in nisin activity. Studies with resting cells confirm that the biosynthesis of nisin is tightly regulated, since the production rate can be 5.6-fold higher than in cells grown in continuous culture. In addition, cell-adhered nisin appears to play a role in the production of the enzyme: low levels of cell-adhered nisin elicited high production rates, whereas high levels were not associated with nisin biosynthesis. In addition to pH, magnesium sulphate and lactose concentrations, nitrogen sources were also able to interfere in cell-adherence nisin.

Lactose--a potential dietary fiber. The regulation of its microecologic effect in the intestinal tract. 4. Dietary fiber action of lactose: evaluation with multivariate statistical analysis

The conditions and the intestinal processes responsible for the action of lactose as a potential dietary fibre are described. The beta-galactosidase activity in the rat caecum and colon is influenced by dietary factors: It declines with increasing lactose concentration and it rises with increasing protein and phosphate concentration in the diet. The enzyme activity correlates negatively with the content of lactose, and positively with the content of protein and phosphate in the chymus. The products of lactose hydrolysis are degraded by microbial glycolysis in caecum and colon. The glycolytic products are mainly absorbed and energetically utilized by the macroorganism. Phosphate stimulates the microbial metabolism and, therefore, accelerates the consumption of the energy substrate lactose. Mathematical optimization gives the necessary composition of the diet which causes an intended microecological effect. To minimize the chymus pH (5.1 in the colon ascendens; 4.6 in the colon descendens; 4.3 in the faeces) the lactose content of the diet has to be greater than or equal to 160 mumol/g, the protein content less than or equal to 10 mg/g, and the phosphate content less than or equal to 5.5 mumol/g. The minimal pH value depends to a greater extent on variations in the supply of protein and phosphorus with the diet whereas the response to changes in lactose concentration is less noticeable.

Water Mobility and Crystallization Action of Lactose‐water Systems by Oxygen‐17 and Carbon‐13 NMR

ABSTRACTSystems ranging in concentration from 10‐60g lactose/100g water were investigated by fully proton decoupled 17O and fully proton decoupled 13C Nuclear Magnetic Resonance (NMR). The relation between 17O NMR transverse relaxation rate measurements and lactose concentration showed water mobility decreased linearly with increasing lactose concentration up to 25g lactose/100g water and decreased rapidly and nonlinearly above that level. Water mobility was used to monitor lactose crystallization in supersaturated solutions with time. The 13C NMR showed no significant differences in chemical shifts of carbon atom peaks in lactose molecules during crystallization. However, depending on the method of sample preparation, mutarotation could be observed.

Factors affecting the thermostability of β-galactosidase (Streptococcus salivariussubsp.thermophilus) in milk: a quantitative study

SummaryEnhanced stability of β-galactosidase (Streptococcus salivariussubsp.thermophilus) in milk is due to several milk components acting in concert. Milk salts stabilized the enzyme 3-fold compared to phosphate buffer. K+was a better stabilizer than Na+. Omission of divalent cations, especially Mg2+, caused a marked drop in stability. Lactose stabilized enzyme stored in the frozen state but destabilized enzyme stored unfrozen. Caseinate stabilized the enzyme 8-fold in phosphate buffer but it stabilized 144-fold in the presence of lactose. In the presence of milk proteins, lactose was 25 times as effective as galactose and 100 times as effective as glucose at promoting stability; sucrose slightly destabilized the enzyme. Stability rose with increasing lactose concentration but declined with increasing enzyme concentration. In milk, soluble casein was the primary stabilizer; whey proteins and peptides had much less effect. Micellar casein had no effect on stability.