Thermal modifications of structure and codenaturation of α‐lactalbumin and β‐lactoglobulin induce changes of solubility and susceptibility to proteases

Study of heat denaturation of major whey proteins (β‐lactoglobulin or α‐lactalbumin) either in separated purified forms, or in forms present in fresh industrial whey or in recomposed mixture respecting whey proportions, indicated significant differences in their denaturation depending on pH, temperature of heating, presence or absence of other co‐denaturation partner, and of existence of a previous thermal pretreatment (industrial whey). α‐Lactalbumin, usually resistant to tryptic hydrolysis, aggregated after heating at ⪈85°C. After its denaturation, α‐lactalbumin was susceptible to tryptic hydrolysis probably because of exposure of its previously hidden tryptic cleavage sites (Lys‐X and Arg‐X bonds). Heating over 85°C of β‐lactoglobulin increased its aggregation and exposure of its peptic cleavage sites. The co‐denaturation of α‐lactalbumin with β‐lactoglobulin increased their aggregation and resulted in complete exposure of β‐lactoglobulin peptic cleavage sites and partial unveiling of α‐lactalbumin tryptic cleavage sites. The exposure of α‐lactalbumin tryptic cleavage sites was slightly enhanced when the α‐lactalbumin/β‐lactoglobulin mixture was heated at pH 7.5. Co‐denaturation of fresh whey by heating at 95°C and pH 4.5 and above produced aggregates stabilized mostly by covalent disulfide bonds easily reduced by β‐mercaptoethanol. The aggregates stabilized by covalent bonds other than disulfide arose from a same thermal treatment but performed at pH 3.5. Thermal treatment of whey at pH 7.5 considerably enhanced tryptic and peptic hydrolysis of both major proteins.     

Effect of different treatments on the stability of lysozyme,lactoferrin and β‐lactoglobulin in donkey's milk

The aim of this study was to investigate the effects of freezing and heat treatments at different temperatures on the stability of the main whey proteins of donkey milk in comparison with those obtained from colostrum and raw milk. Samples subjected to heat treatment at 85 °C showed greater loss of stability, with levels decreasing by 60% and 87% for lysozyme and β‐lactoglobulin, respectively. Lactoferrin completely disappeared at heat treatments higher than 65 °C. Colostrum contained the highest lactoferrin and β‐lactoglobulin concentrations, however, lysozyme was found to be present at similar concentrations in colostrum, raw, frozen and heat‐treated milk at temperatures lower than 85 °C.     

Thermal stability of plasminogen activators and plasminogen activation in heated milk

Thermal stabilities of bovine plasminogen (PG) activators, tissue and urokinase type (t-PA and u-PA), and their impact on activation of PG, were studied after application of various heating conditions (65, 75, 85, or 90 °C for 15, 20, or 30 s), in both milk and buffer systems. Both t-PA and u-PA were thermally stable in milk heated at ⩽75 °C. However, almost half of the t-PA activity and 30% of u-PA activity was lost after heating milk at 85 °C for 30 s. A lower heat stability of both t-PA and u-PA was observed in buffer than in milk. The decrease in level of PG was more pronounced than that of active plasmin in milk heated at ⩾85 °C for 30 s; however, the residual level of PG was considerably higher than the residual level of active PL. Overall, results indicated that u-PA plays a more significant role than t-PA in the activation of PG during storage of heated milk.     

A Simple and Fast Detection Method for Bovine Milk Residues in Foods: A 2‐Site Monoclonal Antibody Immunochromatography Assay

The ingredient declaration on food labels assumes paramount importance in the protection of food‐allergic consumers. China has not implemented Food allergen labeling. A gold immunochromatography assay (GICA) was developed using 2 monoclonal antibodies (mAb) against the milk allergen β‐lactoglobulin in this study. The GICA was specific for pure milk samples with a sensitivity of 0.2 ng/mL. Milk protein traces extracted from 110 food products were detected by this method. The labels of 106 were confirmed by our GICA method: 57 food samples originally labeled as containing milk were positive for β‐lactoglobulin and 49 food samples labeled as not containing milk were negative for β‐lactoglobulin. However, 3 food samples falsely labeled as containing milk were found to contain no β‐lactoglobulin whereas 1 food sample labeled as not containing milk actually contained β‐lactoglobulin. First, these negatives could be because of the addition of a casein fraction. Second, some countries demand that food manufacturers label all ingredients derived from milk as “containing milk” even though the ingredients contain no detectable milk protein by any method. Our GICA method could thus provide a fast and simple method for semiquantitatation of β‐lactoglobulin in foods. Practical Application: The present method provides a fast, simple, semiquantitative method for the determination of milk allergens in foods.     

Effect of Heating and Homogenization on the Stability of Coconut Milk Emulsions

: The effects of homogenization and heat treatment on the colloidal stability of coconut milk were studied. Fresh coconut milk (15% to 17% fat, 1.5% to 2% protein) was extracted and stored at 30 °C before homogenization at 40/4 MPa (stage I/stage II). Both homogenized and non‐homogenized samples were heated at 50 °C, 60 °C, 70 °C, 80 °C, and 90 °C for 1 h. Homogenization reduced the size of the primary emulsion droplets from 10.9 to 3.0 μm, but increased the degree of flocculation, presumably via a bridging mechanism. This flocculation was also responsible for increased viscosity of the homogenized samples. Heating increased the degree of flocculation in both non‐homogenized and homogenized samples. A slight amount of coalescence was also observed after heating above 80 °C. All samples creamed after 24 h of storage, but the heated samples formed a larger cream layer, presumably because the flocculated droplets packed together less efficiently. Optical microscopy was used to confirm the combination of flocculation and creaming responsible for changes in coconut milk quality. The information obtained from this study provides a better understanding of the emulsion science important in controlling coconut milk functionality.     

Instant infusion pasteurisation of bovine milk. II. Effects on indigenous milk enzymes activity and whey protein denaturation

Direct heat treatment of two milk types, skimmed and nonstandardised full‐fat, was performed by instant steam infusion and compared with indirect heating. Infusion conditions were temperatures of 72–120°C combined with holding times of 100–700 ms, and indirect heat conditions were 72°C/15 s and 85°C/30 s. The activity of indigenous enzymes such as alkaline phosphatase, lactoperoxidase, xanthine oxidase and γ‐glutamyl transpeptidase was evaluated. Infusion temperature was the main determinant of inactivation. Whey protein denaturation represented by β‐lactoglobulin increased significantly with infusion temperature. The nonstandardised milk had a higher denaturation rate than skimmed milk. The effect of instant infusion on pH and milk fat globule size in relation to whey protein denaturation and association is discussed.     

Effect of pH on the formation of serum heat‐induced protein aggregates in heated yak milk

Serum heat‐induced protein aggregates at various pH levels were isolated from heated yak milk by size‐exclusion fast protein liquid chromatography. Analysis by reversed‐phase high‐performance liquid chromatography showed that β‐lactoglobulin and κ‐casein were the two main proteins in these aggregates. The amount of serum heat‐induced protein aggregates increased from pH 6.6 to 7.4, whereas the content of β‐lactoglobulin in aggregates decreased from 61.9% at pH 6.6 to 50.4% at pH 7.2, and then increased to 51.3% at pH 7.4. The content of κ‐casein, αs₁‐casein and β‐casein increased from pH 6.6 to pH 7.4. These results show that serum heat‐induced protein aggregates are pH dependent.     

The rheological properties of exudates from cured porcine muscle: effects of added carrageenans and whey protein concentrate/carrageenan blends

Meat exudates were collected from massaged cured porcine M semimembranosis using a model massaging unit. Exudates were used to observe changes in gelation properties of test exudates containing carrageenans and whey protein concentrate (WPC) and carrageenan blends. Three carrageenan powders iota (ι ), kappa (κ), as well as a kappa/locust bean gum mix, were assessed at a 1% residual level, both individually and as blends. WPCs assessed included high gelling A‐35, B‐75 and C 55% protein β‐lactoglobulin powders, as well as a regular 76% protein, WPC D. All WPCs were incorporated at a 2% residual powder level in the final meat. Treatment and control meat samples and resulting exudates were prepared in duplicate with analysis performed in triplicate. The viscoelastic properties of control and test meat exudate samples (n=6) were analysed using control stress rheology in oscillatory mode. All exudates were heated from 20 to 80°C at 1°C min⁻¹ , and subsequently cooled after 30 min back down to 20°C at 1°C min⁻¹. Combinations of high gelling WPCs, especially β‐lactoglobulin together with iota and kappa‐carrageenans A and B, were found to increase storage modules G′ (Pa) values when compared with control values. Significant (p<0.05) synergies were observed on blending high gelling WPCs with carrageenans A and B. © 1999 Society of Chemical Industry     

The rheological properties of exudates from cured porcine muscle: effects of added polysaccharides and whey protein/polysaccharide blends

Meat exudates collected from massaged cured porcine M semimembranosus were used to observe changes in gelation properties of test exudates containing added polysaccharides, both on their own and in combination with selected whey protein concentrates (WPCs). Three polysaccharide powders, namely sodium alginate, low‐methoxy (LM) pectin and modified potato starch, were assessed at a residual powder level of 2% with Na alginate used at a 0.5% level. Polysaccharides were evaluated both individually and as dry blends with selected WPCs. WPCs assessed included high‐gelling A 35%, B 75% and C 55% protein β‐lactoglobulin powders, as well as a regular 76.5% protein, WPC D. All WPCs were incorporated at a 2% residual powder level in the final meat. Treatment and control meat samples and resulting exudates were prepared in duplicate with analysis performed in triplicate. Viscoelastic properties of control and test meat exudate samples (n = 6) were analysed using control stress rheology in oscillatory mode. Exudates were heated from 20 to 80 °C at 1 °C min⁻¹ with subsequent cooling after 30 min to 20 °C at 1 °C min⁻¹. Combinations of high‐gelling WPCs (especially β‐lactoglobulin) together with modified starch or pectin were found to increase storage modulus G′ (Pa) values compared with control values, with significant (P < 0.05) synergies being observed on dry blending these ingredients. Sodium alginate was found to have a negative effect on G′ (Pa) results, giving lower values compared with control treatments. © 1999 Society of Chemical Industry     

Contribution of somatic cell-associated activation of plasminogen to caseinolysis within the goat mammary gland

Functional regression of the mammary gland is partly reflected by proteolysis of milk protein and tissue protein. The involvement of the plasminogen activation system in degradation of milk protein and mammary tissue damage has been demonstrated under inflammatory conditions. In this study, mammary secretion from 23 dairy goats primarily grouped as lactation (milking twice daily) or involution (milking once daily or less) was used to determine the ratio of gravity-precipitated casein to total milk protein (casein ratio) as an index of caseinolysis, and activities of components of plasminogen activation system as well as their expressions on somatic cells. Based on the casein ratio, lactation goats were subcategorized as very active (71.8 ± 1.0%) or less active (29.9 ± 1.0%) in mammary function; involution goats were subcategorized as gradual (21.7 ± 1.0%) or acute (5.9 ± 0.2%) involution. This result suggests that caseinolysis occurred during regular lactation as well as during involution. On the other hand, activities of components of the plasminogen activation system in mammary secretion were increased along with the decreasing casein ratio, in contrast to the similar activities of their counterparts in circulation throughout various mammary statuses. Correlation analysis between casein ratio and activities of plasminogen activation system of goat milk indicated a significant negative relationship for plasmin (r = −0.64), plasminogen (r = −0.69), and urokinase-type plasminogen activator (uPA; r = −0.78) during involution but not during lactation. As for the cellular components of plasminogen activation system, there was an increase in immunoreactivity on somatic cells toward both monoclonal antibodies of human uPA and human uPA receptor under involution conditions suggesting their upregulation relative to lactation condition. Collectively, these results suggest that plasminogen activation system within the mammary gland differentially contribute to milk caseinolysis along the various stages of goat lactation. Meanwhile, a somatic cell-mediated local elevation of plasmin activity may be committed to extensive caseinolysis during involution.     

Microbiological and enzymatic activity of bovine whole milk treated by pulsed electric fields

Pulsed electric field (PEF)‐treated milk (25.7 kV/cm for 34 μs after preheating to 55 °C and holding for 24 s) was microbiologically stable for 21 days at 4 °C, and similar to thermally treated milk (63 °C for 30 min or 73 °C for 15 s). Alkaline phosphatase inactivation was comparable after PEF (preheating followed by PEF) and both thermal treatments. PEF treatment initially reduced xanthine oxidase (30%) and plasmin (7%) activities, but after 21 days of refrigerated storage these activities were similar to the initial untreated milk. During refrigerated storage of PEF (preheating followed by PEF) and thermally treated milk, lipolytic activity increased and pH levels decreased.     

Effect of thermal treatment on the activation of bovine plasminogen

The effect of heat on plasminogen (PG) was studied in both buffer and milk. The denaturation temperature for bovine PG was determined. Kinetic analysis of heated PG was conducted using a coupled assay and Spectrozyme^®PL. The effect of heat on PG activation was studied in milk samples with and without addition of PG activator. The temperature range for denaturation of PG was between 50.1 and 61.6 °C. The overall catalytic efficiency increased for PG heated to 60 °C and above, as indicated by the significant increase in k_cat/K_M. In a milk system, a significant increase in activation of PG was measured at 57 °C. A drop in activation of PG was observed at higher temperatures, suggesting counteracting effects of other milk components that were affected by heat. Results indicated that the increased activation of heated bovine PG observed in buffer and milk systems was probably due to the unfolding of PG as a result of thermal denaturation.     

Milk protein polymorphisms in Holstein cattle†

Milk samples from 203 Holstein cows were phenotyped for genetic variants α_s1‐casein, β‐casein, κ‐casein and β‐lactoglobulin using starch‐gel electrophoresis. All of the four milk protein loci exhibited polymorphism with allele frequencies of 0.862 ± 0.017 for α_s1‐casein B, 0.966 ± 0.0009 for β‐casein A, 0.712 ± 0.0224 for κ‐casein A and 0.567 ± 0.0245 for β‐lactoglobulin B. The mean heterozygosity estimated over all the four milk protein loci was 0.3015. Genetic equilibrium was observed among all of the loci investigated, except κ‐casein. Chi‐squared tests revealed that there was no significant linkage among studied milk protein phenotypes.     

Studies of plasmin activity in whey

The presence of the indigenous milk alkaline proteinase, plasmin, in whey products may adversely affect the quality of food products that utilise such ingredients; factors promoting the release of plasmin into whey were therefore investigated. Acidification of pasteurised skim milk (PSM) with glucono-δ-lactone (GDL) or HCl resulted in acid whey with significantly higher (P<0.05) plasmin activity than PSM. Plasmin activity in such whey was inversely correlated with rate of acidification; thus, activity in the whey made with GDL was significantly higher (P<0.05) than in whey made with HCl. Negligible levels of plasminogen in acid whey suggested that activation of the zymogen, plasminogen, contributed to the elevated plasmin activity observed. Sweet whey, manufactured by addition of rennet to PSM, had plasmin and plasminogen-derived activities significantly lower (P<0.001) than those in PSM; the release of plasmin from renneted casein micelles into whey increased in a linear manner with cooking temperature in the range 45–65°C. However, dissociation of plasmin from casein micelles into acid whey reached a maximum at a cooking temperature of 50°C. Analysis of plasmin and plasminogen levels in ultracentrifugal supernatants of pH-adjusted milk indicated that plasmin was released from the micelles at pH>5.0, while plasminogen was released more gradually over the pH range 6.6–4.6. Overall, the technology of manufacture has significant implications for plasmin activity in whey and hence, inversely, for casein products and cheese.     

Effects of native and denatured whey proteins on plasminogen activator activity

The plasmin system native to bovine milk consists of the caseinolytic serine proteinase plasmin; its inactive zymogen, plasminogen; plasminogen activators; and inhibitors. Evidence in the literature indicates that whey proteins may inhibit plasmin activity, but there is very little mention of their effect on plasminogen activators. The objective of this research was to determine the effect of both unheated and heat-denatured beta-lactoglobulin (beta-LG), alpha-lactalbumin (alpha-LA), and BSA on plasminogen activators. Plasminogen activator activity was significantly stimulated by non-heat treated and denatured alpha-LA as well as by denatured beta-LG. The stimulation effect by these whey proteins was kinetically characterized, which showed that all 3 significantly increased the rate of plasminogen activation. The stimulation effect was shown to be independent of any effect of the whey proteins on plasmin activity by testing 2 different substrates, d-Val-Leu-Lys p-nitroanilide (S-2251) and Spectrozyme PL (Spec PL), in a plasmin assay. Results using S-2251 confirmed the inhibitory effect of whey proteins on plasmin observed by several researchers. However, use of SpecPL did not suggest inhibition. Ligand binding studies showed this discrepancy to be due to significant interaction between S-2251 and the whey proteins. Overall, this study indicates that whey protein incorporation into cheese may not hinder plasmin activity and may stimulate plasminogen activation. Furthermore, the results indicate the need for careful consideration of the type of synthetic substrate chosen for model work involving whey proteins and the plasmin system.     

Low‐field NMR: A tool for studying protein aggregation

Low‐field nuclear magnetic resonance (NMR) spin–spin relaxation (T₂) measurements were used to study the denaturation and aggregation of β‐lactoglobulin (β‐LG) solutions of varying concentrations (1–80 g L^?1) as they were heated at temperatures ranging from ambient up to 90 °C. For concentrations of 1–10 g L^?1, the T₂ of β‐LG solutions did not change, even after heating to 90 °C. A decrease in T₂ was only observed when solutions having higher concentrations (20–80 g L^?1) were heated. Circular dichroism (CD) spectroscopy and fluorescence tests using the dye 1‐anilino‐8‐naphthalene sulfonate (ANS) on 0.2 and 1 g L^?1 solutions, respectively, indicated there were changes in the protein's secondary and tertiary conformations when the β‐LG solutions reached 70 °C and above. In addition, dynamic light scattering (DLS) showed that protein aggregation occurred only at concentrations above 10 g L^?1 and for heating at 70 °C and above. The hydrodynamic radius increased as T₂ decreased. When excess 2‐mercaptoethanol was added, the changes in both T₂ and the hydrodynamic radius followed the same trend for all β‐LG protein concentrations between 1 and 40 g L^?1. These observations led to the conclusion that the changes in T₂ were due to protein aggregation, not protein unfolding. Copyright © 2007 Society of Chemical Industry     

Survivability and β‐galactosidase activity of bifidobacteria stored at low temperatures

Abstract

This work evaluated the ability of strains representing six species of Bifidobacterium with probiotic potential to survive and maintain β‐galactosidase activity through a two‐step, low‐temperature storage period. Cultures were also evaluated for their ability to ferment skim milk and retain viability during storage at 4°C. Bifidobacterium longum ATCC 15707, B. breve 15700, and B. bifidum 29521 maintained the greatest viabilities at > 1 x 10⁷ CFU/mL, and B. infantis 15702 maintained the highest β‐galactosidase activity at > 1 U/ml (with < 1 × 10⁵ CFU/mL) after ‐60 to 4°C storage. In fermented skim milk, B. breve 15700, B. bifidum 29521, and B. animalis 25527 tolerated a final product pH of 4.75 with > 1 × 10⁸ CFU/mL remaining after 14 days of storage at 4°C. Overall, it was found that highest levels of β‐galactosidase activities did not necessarily correlate to the highest plate‐count populations.     

Effects of fermentation by lactic acid bacteria on the antigenicity of bovine whey proteins

BACKGROUND: The main whey proteins α‐lactalbumin (α‐LA) and β‐lactoglobulin (β‐LG) are considered as the major allergens in cow's milk. Microbial fermentation can produce some proteolytic enzymes, which can induce the degradation of milk protein allergens. In this study, the effects of fermentation by lactic acid bacteria on the antigenicity of α‐LA and β‐LG were investigated using indirect competitive ELISA. Meanwhile, the proteolysis of milk proteins was detected by TNBS assay and SDS‐PAGE electrophoresis. RESULTS: Fermentation by lactic acid bacteria could significantly reduce the antigenicity of α‐LA and β‐LG in skim milk. Combined strains of Lactobacillus helveticus and Streptococcus thermophilus were the most effective in reducing the antigenicity of both whey proteins. In addition, α‐LA and β‐LG antigenicity decreased to a lower value at 6 h of fermentation and at 0.5 d of cold storage by fermentation with the combined strains. The results of TNBS assay and SDS‐PAGE electrophoresis showed that lactic acid bacteria strains used in this study hydrolysed whey proteins only to a limited extent. CONCLUSION: The fermentation with lactic acid bacteria is an effective way to reduce whey proteins antigenicity. Copyright © 2010 Society of Chemical Industry     

Protein stability function relations: native β‐lactoglobulin sulphhydryl–disulphide exchange with PDS

Intermolecular sulphhydryl–disulphide exchange with β‐lactoglobulin dimer occurs when this dissociates to form monomers exposing two SH groups. This notion is re‐evaluated in the light of recent structural data suggesting that the degree of SH group exposure in β‐lactoglobulin is unaffected by dissociation. β‐Lactoglobulin was treated with 2,2′‐dipyridyl disulphide (PDS). The rate of sulphhydryl–disulphide exchange was measured at sub‐denaturation temperatures of 25–60 ° C. Parallel studies were conducted by reacting PDS with reduced glutathione (GSH). The SH group of GSH was up to 31 000 times more reactive than β‐lactoglobulin. At pH 7 the reaction activation enthalpy (ΔH^#) and entropy (ΔS^#) was 26 kJ mol⁻¹ and −100 J mol⁻¹ K⁻¹ respectively for GSH. For β‐lactoglobulin, ΔH^# was 157.2 kJ mol⁻¹ and ΔS^# was 254 J mol⁻¹ K⁻¹. At pH 2.6, ΔH^# was 14.4 kJ mol⁻¹ and ΔS^# was −213 J mol⁻¹ K⁻¹ for GSH. The corresponding results for β‐lactoglobulin were 20.3 kJ mol⁻¹ and −147 J mol⁻¹ K⁻¹. These and other thermodynamic results are discussed in terms of the effects of β‐lactoglobulin conformational structure and stability on SH group reactivity. For native β‐lactoglobulin at neutral pH, intermolecular sulphhydryl–disulphide exchange appears to involve the dissociated monomer. SH group activation probably arises from the lower structural stability of the monomer relative to the dimer. At pH 2.6 the mechanism of SH–disulphide exchange does not require protein dissociation and probably involves breathing motions or localised changes in protein structure. © 2000 Society of Chemical Industry