Milk Β-lactoglobulin Research Articles

Binding of Cationic Lipids to Milk β-Lactoglobulin

We determined the bindings of several lipids such as cholesterol (CHOL), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethyl-ammoniumbromide (DDAB), and dioleoylphosphatidylethanolamine (DOPE) to β-lactoglobulin (β-LG) at physiological conditions. FTIR, CD, and fluorescence spectroscopic methods as well as molecular modeling were used to determine the binding of lipid-protein complexes. Structural analysis showed that lipids bind β-LG via both hydrophilic and hydrophobic interactions with overall binding constants of K(CHOL-β-LG) = 6.0 (±0.6) × 10(3) M(-1), K(DOPE-β-LG) = 6.5 (±0.7) × 10(3) M(-1), K(DDAB-β-LG) = 1.6 (±0.3) × 10(4) M(-1), and K(DOTAP-β-LG) = 2.2 (±0.67) × 10(4) M(-1). The number of lipid bound per protein molecule (n) was 0.8 (CHOL), 0.7 (DOPE), 1.0 (DDAB), and 1.3 (DOTAP). Molecular modeling showed the participation of several amino acid residues in lipid-protein complexation with the order of binding DOTAP > DDAB > DOPE > CHOL. Alterations of the protein conformation were observed in the presence of lipids with a minor decrease in β-sheet and an increase in turn structure.

The Hypocholesterolemic Activity of Transgenic Rice Seed Accumulating Lactostatin, a Bioactive Peptide Derived from Bovine Milk β-Lactoglobulin

Lactostatin is a novel pentapeptide (IIAEK) derived from bovine milk β-lactoglobulin with greater hypocholesterolemic activity than β-sitosterol, the drug commonly used to treat hypercholesterolemia. We developed transgenic rice expressing lactostatin as a fusion protein with seed storage protein (SSP) glutelins under the control of three different endosperm-specific promoters. Lactostatin accumulated in transgenic rice seed at approximately 1.6 mg/g seeds (dry seeds) without any apparent influence on seed traits such as endogenous SSP expression levels or alterations in the intracellular structures of endosperm cells. Short-term (three day) oral administration of the glutelin fraction containing lactostatin (namely three times of 300 mg/kg body weight/day) extracted from transgenic rice seeds resulted in hypocholesterolemic activity in rats; namely, the serum low-density-lipoprotein (LDL) cholesterol level was significantly reduced accompanied by a significant increase in beneficial serum high-density-lipoprotein (HDL) cholesterol.

Milk β-lactoglobulin complexes with tea polyphenols

The effect of milk on the antioxidant capacity of tea polyphenols is not fully understood. The complexation of tea polyphenols with milk proteins can alter the antioxidant activity of tea compounds and the protein secondary structure. This study was designed to examine the interaction of β-lactogolobulin (β-LG) with tea polyphenols (+)-catechin (C), (−)-epicatechin (EC), (−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG) at molecular level, using FTIR, CD and fluorescence spectroscopic methods as well as molecular modelling. The polyphenol binding mode, the binding constant and the effects of polyphenol complexation on β-LG stability and secondary structure were determined. Structural analysis showed that polyphenols bind β-LG via both hydrophilic and hydrophobic interactions with overall binding constants of KC–β-LG=2.2 (±0.8)×103M−1, KEC–β-LG=3.2 (±1)×103M−1, KECG–β-LG=1.1 (±0.6)×104M−1 and KEGCG–β-LG=1.3 (±0.8)×104M−1. The number of polyphenols bound per protein molecule (n) was 1.1 (C), 0.9 (EC), 0.9 (ECG) and 1.3 (EGCG). Molecular modelling showed the participation of several amino acid residues in polyphenol–protein complexation with extended H-bonding network. The β-LG conformation was altered in the presence of polyphenols with an increase in β-sheet and α-helix suggesting protein structural stabilisation. These data can be used to explain the mechanism by which the antioxidant activity of tea compounds is affected by the addition of milk.

Evaluation of elisa method to detection of cow β-lactoglobulin in sheep milk and sheep milk products

The aim of work was to optimalize the ELISA method to detect the adulteration of sheep milk and sheep milk products by cow milk in the laboratory. We have focused on laboratory testing of ELISA kit (β-Lactoglobulin ELISA Set, SEDIUM R&D) for detection of cow β-Lg in sheep milk order to obtain high-quality, reliable and economically advantageous method suitable for routine use in practice. The results shown that for the quality of adulteration determination it is necessary to verify the sensitivity of applied kit by the samples dilution in accordance with the producer declared quantification range contained in the manual ELISA kit. The starting point for obtaining of relevant data was to create separate regression curves with high deter­mination coefficient, which allowed to quickly and easily detect the cow milk additions in sheep milk, cloddish sheep and Slovak sheep cheese.

Mapping a quantitative trait locus for the concentration of β-lactoglobulin in milk, and the effect of β-lactoglobulin genetic variants on the composition of milk from Holstein-Friesian x Jersey crossbred cows

AIM: To identify quantitative trait loci (QTL) affecting the concentration of β-lactoglobulin in milk, and to evaluate the effect of β-lactoglobulin genetic variants on the concentration of fat, protein and casein in bovine milk. METHODS: A herd of 850 F2 Holstein-Friesian x Jersey crossbred cows was produced through mating six Holstein-Friesian x Jersey F1 bulls of high genetic merit with F1 cows from the national herd. A total of 1,610 herd-test records from 556 second-parity crossbreds were analysed. The concentration of fat, protein and casein in milk was measured at peak, mid- and late lactation, during the production seasons of 2003–2004 and 2004–2005. Liveweight was measured daily. DNA from the F2 animals, their F1 dams and sires, and selected grandsires was genotyped across the genome, initially with 285 microsatellite markers, and subsequently with 6,634 single nucleotide polymorphisms (SNP). RESULTS: A highly significant QTL for the concentration of β-lactoglobulin in milk was identified, which coincided with the position of the β-lactoglobulin gene on bovine Chromosome 11. No other consistently significant QTL for the concentration of β-lactoglobulin in milk were detected. Cows with the BB β-lactoglobulin genotype produced milk with a 30% lower concentration of β-lactoglobulin than cows with the AA genotype. The β-lactoglobulin polymorphism also explained variation in the proportion of casein in total protein. In addition, the percentage of fat was higher for BB than AA animals, whereas the percentage of total protein, mean daily milk yield and liveweight did not differ between AA and BB animals. CONCLUSIONS: A significant QTL determining the concentration of β-lactoglobulin in milk was identified. Selection of animals for the β-lactoglobulin B-allele may enable the production of milk naturally enriched for casein, thus allowing a potential increase in the yield of cheese. There may be additional future value in production of bovine milk more like human milk, where decreasing the concentration of β-lactoglobulin is desirable.

Structure of a secondary vitamin D3binding site of milk β-lactoglobulin for vitamin D transport

Structure of a secondary vitamin D₃binding site of milk β-lactoglobulin for vitamin D transport

Effect of genetic polymorphism of milk proteins on rheology of acid-induced milk gels

Individual milk samples from 80 cows in mid-lactation of the Swedish Red and Swedish Holstein breeds with known protein genotypes of β- and κ-casein and β-lactoglobulin were analysed for acid coagulation properties. Glucono-δ-lactone (1.5%) was added to defatted, heated (90–95 °C) samples and rheological properties of the gels were measured using a Bohlin VOR Rheometer. Coagulation time (CT) and curd firmness after 4, 8 and 10 h ( G 4′, G 8′, and G 10′ were registered for each sample. Milk protein composition was analysed by reversed phase HPLC. Concentration of β-lactoglobulin in milk was found to be an important factor for the variation in CT and G′. The A allele of β-lactoglobulin was associated with higher concentrations of β-lactoglobulin in milk compared with B. When no adjustment for β-lactoglobulin concentration was made, there was a significant overall effect of β-lactoglobulin genotype on acid coagulation, where the AA and AB genotypes were associated with better curd firmness compared with BB, whereas at equal β-lactoglobulin concentrations a tendency in the opposite direction was found with a significant and positive effect of BB compared with AB. Lactose concentration of milk had a positive effect on acid coagulation and was shown to improve G′ in milk with low β-lactoglobulin concentrations.

Inhibition of Collagenolytic Cathepsins by β-Lactoglobulin in Milk and Its Suppressive Effect on Bone Resorption

It is well known that the collagenolytic cathepsins play an important role in the degradation of bone matrix. Therefore, the purpose of this study was to clarify the prevention effect of bone resorption by milk components. Using double-layer reverse zymography, we found a 20 kDa protein in milk which inhibited cysteine proteases. This inhibitory protein was identified as beta-lactoglobulin B. The inhibitory activity of beta-lactoglobulin B against cathepsin K was stronger than that of beta-lactoglobulin A. Beta-lactoglobulin B specifically inhibited papain type cysteine proteases such as cathepsins K and L, but not serine proteases, aspartic proteases or metallo proteases. Beta-lactoglobulin B inhibited cathepsin K non-competitively and the Ki value was 10(-5) M. The formation of osteoclastic pits in the culture system was effectively inhibited by 10(-4)-10(-5) M beta-lactoglobulin B in vitro. Furthermore, we demonstrated that beta-lactoglobulin B inhibited degradation of type I-collagen by collagenolytic cathepsins. Using the everted-sac method in rat small intestines, it was found that beta-lactoglobulin was more easily absorbed from the intestines of young rats (5 wk-old) than from those of older rats (more than 20 wk-old). The digested products of beta-lactoglobulin B with lysyl-endopeptidase showed a similar inhibitory activity against cathepsin K to the intact beta-lactoglobulin B did. Therefore, peroral intake of beta-lactoglobulin in milk and its digested peptides are expected to help protect osteoclastic bone resorption via inhibition of collagenolytic cathepsins K and L.

Donkey milk production: state of the art

Milk is one of the most common causes of food allergies among children under one year of age. No specific therapy exists for this allergy, and thus the only feasible response is to avoid assumption of milk and derived products. Studies conducted on the serum of children with hypersensitivity to milk have shown that caseins are the proteins with the greater allergenic potential. However, in some cases, children have also shown hypersensitivity to the β-lactoglobulines and to the α-lactal-bumins. When food intolerance is diagnosed in an infant, it is often necessary to impose a period of total parenteral feeding, followed by breast feeding, considered the most correct method of re-feeding. When human milk can not be given, alternative food sources must be sought. Clinical studies have demonstrated that donkey milk could substitute breast feeding in infants affected by severe Ig-E mediated milk allergies. In these subjects, donkey milk is not only useful, but also safer than other types of milk. In fact donkey milk composition in lipids (high levels of linoleic and linolenic acid) and proteins (low caseins content) is very close to human milk. Lysozyme content in donkey milk resulted to be very high (mean value 1.0 mg/ml) if compared to bovine (traces), caprine (traces) and human milk. The high lysozyme content of donkey milk may be responsible of the low bacterial count reported in literature and also makes this milk suitable to prevent intestine infections to infants. Among seroproteins, β-lactoglobulin and α-lactalbumin content in donkey milk was respectively 3.75 and 1.80 mg/ml and remained substancially the same during the different stages of lactation.

Effect of high-pressure treatment at various temperatures on indigenous proteolytic enzymes and whey protein denaturation in bovine milk

The objective of the present study was to determine the effect of high pressure (HP) processing (200, 450 and 650 MPa) at various temperatures (20, 40 and 55 degrees C) on the total plasmin plus plasminogen-derived activity (PL), plasminogen activator(s) (PA) and cathepsin D activities and on denaturation of major whey proteins in bovine milk. Data indicated that transfer of both PL and PA from the casein micelles to milk serum occurred at all pressures utilized at room temperature (20 degrees C). In addition to the transfer of PL and PA from micelles, there were reductions in activities of PL (16-18%) and PA (38-62%) for the pressures 450 and 650 MPa, at room temperature. There were synergistic negative effects between pressure and temperature on residual PL activity at 450 and 650 MPa and on residual PA activity only at 450 MPa. Cathepsin D activity in the acid whey from HP-treated milk was in general baroresistant at room temperature. The residual activity of cathepsin D decreased significantly at 650 MPa and 40 degrees C and at the pressures 450 and 650 MPa at 55 degrees C. Synergistic negative effects on the amount of native beta-lactoglobulin were observed at 450 and 650 MPa and on the amount of native alpha-lactalbumin at 650 MPa. There were significant correlations between enzymatic activities (PL, PA and cathepsin D) and the residual native beta-lactoglobulin and alpha-lactalbumin in bovine milk. In conclusion, HP significantly affected the activity of indigenous proteolytic enzymes and whey protein denaturation in bovine milk. Reduction in activity of indigenous enzymes (PL, PA and cathepsin D) and transfer of PL and PA from the casein to milk serum induced by HP is expected to have a profound effect on cheese yield, proteolysis during cheese ripening and quality of UHT milk during storage.

Impact of γ-Radiation on Antigenic Properties of Cow's Milk β-Lactoglobulin

This study evaluated the effects of γ-radiation on the antigenic properties of β-lactoglobulin in cow's milk. Liquid and lyophilized samples of cow's milk and whey were irradiated with γ-cells (60Co) at dose levels of 3, 5, and 10 kGy, at room temperature in the presence of air. Effects of treatment on proteins were monitored by Lowry's method, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and enzyme-linked immunosorbent assay. Radiation did not affect the molecular-weight distributions of proteins, but it did reduce their solubility. Furthermore, results showed that irradiation at 10 kGy increased the recognition of milk and whey powders by anti–β-lactoglobulin (β-Lg) rabbit immunoglobulin G, with the other samples remaining antigenically stable. These results indicate that γ-rays do not reduce cow's milk β-lactoglobulin antigenicity.

Selective Separation of the Major Whey Proteins Using Ion Exchange Membranes

Synthetic microporous membranes with functional groups covalently attached were used to selectively separate β-lactoglobulin, BSA, and α-lactalbumin from rennet whey. The selectivity and membrane performance of strong (quaternary ammonium) and weak (diethylamine) ion-exchange membranes were studied using breakthrough curves, measurement of binding capacity, and protein composition of the elution fraction to determine the binding behavior of each membrane. When the weak and strong anion exchange membranes were saturated with whey, they were both selective primarily for β-lactoglobulin with less than 1% of the eluate consisting of α-lactalbumin or BSA. The binding capacity of a pure β-lactoglobulin solution was in excess of 1.5 mg/cm2 of membrane. This binding capacity was reduced to approximately 1.2 mg/cm2 when using a rennet whey solution (pH 6.4). This reduction in protein binding capacity can be explained by both the competitive effects of other whey proteins and the effect of ions present in whey. Using binary solution breakthrough curves and rennet whey breakthrough curves, it was shown that α-lactalbumin and BSA were displaced from the strong and weak anion exchange membranes by β-lactoglobulin. Finally, the effect of ionic strength on the binding capacity of individual proteins for each membrane was determined by comparing model protein solutions in milk permeate (pH 6.4) and a 10mM sodium phosphate buffer (pH 6.4). Binding capacities of β-lactoglobulin, α-lactalbumin, and BSA in milk permeate were reduced by as much as 50%. This reduction in capacity coupled with the low binding capacity of current ion exchange membranes are 2 serious considerations for selectively separating complex and concentrated protein solutions.

Silver‐ and Coomassie‐staining protocols: Detection limits and compatibility with ESI MS

Staining protocols for PAGE have to be sensitive and should not impair further MS analysis of selected samples. In this study, the MS compatibility of different silver- and Coomassie-staining protocols with a nano-LC-MS/MS system was systematically elucidated. Altogether, 13 different silver-staining, 1 imidazole-staining and 2 Coomassie-staining protocols were used and compared to each other for their achieved sequence coverage and their detection sensitivity. Three proteins were used as model proteins (bovine serum albumin, rabbit L-lactate dehydrogenase, bovine milk beta-lactoglobulin) in decreasing concentration (12 pmol down to 30 fmol) and different staining protocols were applied. The conclusion of this study is that two silver-staining protocols (Blum, H. et al.,. Electrophoresis 1987, 8, 93-99 and Shevchenko, A. et al.,. Anal. Chem. 1996, 68, 850-858) combine good sequence coverage and good sensitivity and are recommended for nano-LC-MS/MS analysis.

A novel regulatory pathway for cholesterol degradation via lactostatin

Our group previously discovered a novel hypocholesterolemic pentapeptide (IIAEK: Ile-Ile-Ala-Glu-Lys, or what we describe as “lactostatin”) derived from bovine milk β-lactoglobulin. To clarify the mechanism of the hypocholesterolemic action of lactostatin, we screened the target gene and signal transducing pathway induced by lactostatin in HepG2, a human liver cell line. Unexpectedly, we found that water-soluble lactostatin can activate cholesterol 7α-hydroxylase (CYP7A1) gene expression. Treatment with mitogen-activated protein kinase (MAPK) inhibitor or calcium (Ca 2+) channel blocker blocked this activation. We also found that lactostatin regulates the phosphorylation of extracellular signal-regulated kinase (ERK) and intracellular Ca 2+ concentration. Here, we show the involvement of a new regulatory pathway in the calcium-channel-related MAPK signaling pathway of lactostatin-mediated cholesterol degradation. Oligopeptide shows promise as a new molecule for the development of medicines and functional foods to prevent and improve hypercholesterolemia and atherosclerosis.

Effect of β-lactotensin on acute stress and fear memory

β-Lactotensin (β-LT) is a bioactive peptide derived from bovine milk β-lactoglobulin and is a natural ligand for neurotensin receptors. We examined the effect of β-LT on restraint stress and fear memory in mice. Mice subjected to acute restraint stress exhibited a decreased number of head-dips and increased head-dip latency compared to non-stressed controls in the hole-board test, reflecting increased stress-induced behaviors. However, prior administration of β-LT improved the behaviors caused by stress. The anti-stress effect of β-LT was blocked by levocabastine, a neurotensin receptor subtype 2 (NTR2) antagonist. In the fear-conditioning test, the duration of freezing responses by cued fear conditioning was significantly reduced in mice administered β-LT compared with control mice. These results suggest that β-LT has an anti-stress effect and promotes the extinction of fear memory, which may be mediated by NTR2.

Screening for lactic acid bacteria with potential to reduce antigenic response of β-lactoglobulin in bovine skim milk and sweet whey

The whey protein β-lactoglobulin (β-lg) is in about 80% of all cases the main elicitor of milk allergies for children and infants. β-lg is the major whey protein in milk and milk products and it is of particular interest since it is the only whey protein of cow's milk not present in human milk. Lactic acid bacteria were screened for their ability to reduce the β-lg antigenity in skim milk and sweet whey. The antigenic response, measured as β-lg concentration equivalent was determined by means of an indirect competitive ELISA, using polyclonal antibodies from chicken egg yolk. Lactic acid fermentation was found to be suitable for decreasing milk immunoreactivity. In addition, synergism regarding the reduction of the antigenity were observed when using 1:1 mixtures of lactic acid bacteria with Streptococcus thermophilus subsp. salivarius strains. A 90% reduction of the initial β-lg antigenity of skim milk was observed for some of the lactic acid bacteria. In sweet whey more than 70% reduction was achieved. In general, lactic acid fermentation can attenuate β-lg antigenity in skim milk and sweet whey, but did not eliminate antigenity. Industrial relevance The research demonstrated that some lactic acid bacteria feature potential to reduce the antigenic response of β-lg in sweet whey and skim milk. The antigenity was quantified by an ELISA specific to some of the epitopes responsible for allergenic reaction according to the literature. Reduction of more than 70% in sweet whey and more than 90% in skim milk compared to the initial value was detected. Some of the strains studied reduced the β-lg antigenity significantly better in sweet whey, some in skim milk. The enzymes are more or less specific and some enzymes released from microorganisms are even better for reducing antigenity in milk and/or whey, as well as the different composition of milk and whey is of note. These results are promising for new fermented milk products with reduced antigenic properties. In addition, synergism regarding the reduction of antigenity was observed when using 1:1 mixtures of lactic acid bacteria with S. thermophilus subsp. salivarius strains. However, only the antigenity of β-lg was tested by means of an ELISA and not the allergenicity. The laboratory experiments demonstrate that fermented milk products or milk based formula may be prepared with reduced antigenity. Further research will be addressed to produce such products in pilot scale for clinical tests in order to test allergenicity or the allergy-reduced characteristic of the product.

Comparative Study on Shelf Life of Whole Milk Processed by High-Intensity Pulsed Electric Field or Heat Treatment

The effect of high-intensity pulsed electric fields (HI-PEF) processing (35.5 kV/cm for 1,000 or 300μ with bipolar 7-μs pulses at 111Hz; the temperature outside the chamber was always<40° C) on microbial shelf life and quality-related parameters of whole milk were investigated and compared with traditional heat pasteurization (75° C for 15s), and to raw milk during storage at 4° C. A HIPEF treatment of 1,000μ ensured the microbiological stability of whole milk stored for 5 d under refrigeration. Initial acidity values, pH, and free fatty acid content were not affected by the treatments; and no proteolysis and lipolysis were observed during 1 wk of storage in milk treated by HIPEF for 1,000μ. The whey proteins (serum albumin, β-lactoglobulin, and α-lactalbumin) in HIPEF-treated milk were retained at 75.5, 79.9, and 60%, respectively, similar to values for milk treated by traditional heat pasteurization.

Changes in Chemical Composition of Alxa Bactrian Camel Milk During Lactation

Changes in chemical composition of Alxa bactrian camels reared in Inner Mongolia (China) during lactation were investigated. Colostrum and milk samples from 10 nomadic female camels in their first season of lactation were collected periodically from parturition until 90 d postpartum (PP). The average contents of gross composition were 14.23% protein, 4.44% lactose, 0.27% fat, 0.77% ash, and 20.16% total solids in colostrum at 2h PP, and the respective mean values were 3.55, 4.24, 5.65, 0.87, and 14.31% for regular milk on d 90. A 15-fold increase was shown in fat content during the first 24h, whereas a sharp decrease was shown during the first 12h of lactation in protein, ash, and total solids contents. Variation in lactose content was small (4.24 to 4.71%) throughout the study period. Total N, nonprotein N, casein N, and whey protein N were found to be 2.23, 0.06, 0.86, and 1.31 g/100mL for the colostrum at 2h PP; and 0.56, 0.04, 0.45, and 0.07 g/100mL for the milk at 90 d PP. Percentages of caseins increased steadily, whereas whey proteins declined gradually until 3 mo of lactation. Gas liquid chromatography analysis of milk fat showed that the content of even-numbered saturated fatty acids (C12:0-C18:0) in camel colostrum (2h to 7 d PP) was lower than that of regular milk (15 to 90 d PP). The predominant saturated fatty acids were C14:0, C16:0, and C18:0, regardless of the stage of lactation. There was a considerable level of polyunsaturated fatty acids (mainly C18:1) in Alxa camel's milk fat. The levels of Ca, P, Na, K, and Cl were 222.58, 153.74, 65.0, 136.5, and 141.1 mg/100g, respectively, at 2h PP; the values of the minerals were 154.57, 116.82, 72.0, 191.0, and 152.0 mg/100g, respectively, for the regular milk on d 90. The levels of vitamins A, C, E, B1, B2, B6, and D were 0.97, 29.60, 1.45, 0.12, 1.24, 0.54 mg/L, and 640 IU/L, respectively, in Alxa camel milk at 90 d PP. Vitamin A and C contents were higher and vitamins E and B1 were lower than those in colostrum. Sodium dodecyl sulfate-PAGE and densitometry results demonstrated that Alxa camel colostrum is rich in immunoglobulins, serum albumin, and 2 unknown fractions, which are reduced in amount (%) within 2 d of lactation. It seems that there is lack of β-lactoglobulin in Alxa camel milk, whereas casein and α-lactalbumin start at a low level and increase gradually until they reach their regular levels in the milk.

Denaturation of β-Lactoglobulin in Pressure-Treated Skim Milk

The kinetics of beta-lactoglobulin (beta-LG) denaturation in pressure-treated reconstituted skim milk samples over a wide pressurization range (100-600 MPa) and at various temperatures (10-40 degrees C) was studied. Denaturation was extremely dependent on the pressure and duration of treatment. At 100 MPa, no denaturation was observed regardless of the temperature or the holding time. At higher pressures, the level of denaturation increased with an increasing holding time at a constant pressure or with increasing pressure at a constant holding time. At 200 MPa, there was only a small effect of changing the temperature at pressurization. However, at higher pressures, increasing the temperature from 10 to 40 degrees C markedly increased the rate of denaturation. The two major genetic variants of beta-LG (A and B) behaved similarly to pressure treatment, although the B variant appeared to denature slightly faster than the A variant at low pressures (< or =400 MPa). The denaturation could be described as a second-order process for both beta-LG variants. There was a marked change in pressure dependence at about 300 MPa, which resulted in markedly different activation volumes in the two pressure ranges. Evaluation of the kinetic and thermodynamic parameters suggested that there may have been a transition from an aggregation-limited reaction to an unfolding-limited reaction as the pressure was increased.

Evaluation of IP mouse models for assessing the allergenic potential of proteins

RATIONALE: The objective of this exercise was to evaluate the utility of mouse models for differentiating between allergens and putative non-allergens in the context of assessing novel proteins from biotechnology. METHODS: Known human allergenic proteins (peanut Ara h 1 and Ara h 2, cow's milk β-lactoglobulin [B-LG]) and putative non-allergenic proteins (spinach RUBISCO and soy lipoxygenase [SLG]) were purified to > 90% and provided to each laboratory. Each of four participating laboratories employed the intraperitoneal (ip) route of exposure using mouse strains including BALB/c, BDF-1, C3H/HeJ, A/J, and C57/Bl6 to evaluate the test proteins. Other experimental variables, which differed for each laboratory, included the presence or absence of adjuvant (i.e., alum), the number and length of time between ip injections, protein doses, and the use of homologous or heterologous PCA to assess IgE. One laboratory also systematically evaluated clinical signs. RESULTS: All the laboratories demonstrated significant PCA responses to RUBISCO in the absence of adjuvant. In two laboratories, the relative response via PCA to RUBISCO was significantly greater than the response to Ara h 2. In another laboratory, a much higher relative clinical score was seen with SLG than was seen with B-LG. In addition, the nature and magnitude of the responses varied considerably between the different mouse strains. CONCLUSIONS: In general, it was not possible to accurately differentiate between the known allergens and the putative non-allergens in any laboratory using the strains and endpoints evaluated. Further work appears necessary to evaluate the allergenic potential of proteins using mouse models.