MILK PROTEIN/STAINLESS STEEL INTERACTION RELEVANT TO THE INITIAL STAGE OF FOULING IN THERMAL PROCESSING

Effect of temperature and surface preconditioning on β-lactoglobulin (β-lg) adsorption onto stainless steel have been studied using column adsorption chromatography. As temperature increased from 25 to 85C, amount of adsorbed β-lg increased gradually and eventually reached a plateau value. Above 85C, slope of the adsorption boundary curve was significantly less than that at lower temperatures suggesting that the association constant for Langmuir adsorption isotherm increased. Conformation change caused by heat treatment also resulted in higher amount of adsorption at equilibrium (1.65 mgm^?2) than native β-lg (0.7 mgm^?2) at 25C. Adsorption behavior of k-casein was different from that of β-lg as reflected in the shape of the adsorption curve as well as the amount of adsorption at equilibrium. A significant amount of k-casein was adsorbed onto a β-lg saturated surface, whereas no additional adsorption of β-lg occurred onto a k-casein saturated surface. These observations were in agreement with previous work. Favorable adsorption of β-lg onto calcium phosphate-deposited stainless steel suggests the formation of calcium phosphate/β-lg matrix in multilayer deposit during the thermal processing of milk.     

Adsorption of β-Lactoglobulin variants A and B to the air–water interface

Summary The adsorption of proteins to interfaces is a vital and complex process for the formation and stabilization of multiphase food systems (emulsions and foams). The process of protein adsorption is generally understood only at the phenomenological level, as the complexity of protein unfolding during adsorption is very difficult to predict and model. By comparing proteins with very similar structures, it is possible to attribute observed changes in adsorption behaviour. The A and B genetic variants of β-lactoglobulin (β-lg) differ by only two amino acids (Asp-64, Val-118 in A, and Gly-64, Ala-118 in B), thus making them ideal candidates for this type of comparison. In this study we monitored the surface behaviour of β-lg A and B, measuring the surface tension and surface dilational modulus of adsorbed protein, and the compression behaviour of spread protein films. At pH 7, variant B lowered the surface tension and increased the surface dilational modulus more rapidly than variant A. Raising the pH to 7.8 should increase the level of dissociation into monomers. Indeed, this was confirmed by the rate of adsorption, which increased in both cases. Also, the surface tension of both variants was much lower than at pH7. Variant B was less sensitive to the change of pH than A. Regardless of pH, after 3 h adsorption the difference between the variants in surface tension or surface dilational modulus was negligible. The differences in surface behaviour between the variants are discussed in terms of interactions between monomers at and with the interface, and the dimer : monomer equilibrium in the bulk solution.     

Adsorption characteristics of tryptic fragments of bovine beta-lactoglobulin on a stainless steel surface

As a strategy for the analysis of the mode of protein adsorption onto stainless steel surfaces, peptides obtained by tryptic digestion of bovine beta-lactoglobulin were subjected to adsorption experiments after identification of their primary structures. In the presence of 1 mM KOH, the peptides were scarcely adsorbed onto the surfaces of stainless steel particles from the peptide mixture. The adsorption experiments on isolated peptides showed that the affinities of the peptides for stainless steel surfaces in the presence of 1 mM HNO3 were significantly different from each other. Peptides without any acidic amino acid residues were scarcely adsorbed onto the surface, whereas some peptides with acidic amino acid residues were found to be irreversibly adsorbed onto the surfaces in the acidic pH region. As for the latter peptides, the amount adsorbed on the surface increased with increasing ionic strength. These results indicated that the carboxyl groups on the side chains of the peptides play an important role in the adsorption. Furthermore, the adsorption behavior of beta-lactoglobulin itself was found to be very similar to that of one of the latter peptides.     

INFLUENCE OF SOLID SURFACE ENERGY ON PROTEIN ADSORPTION

Surface energy characteristics of a solid surface influence the extent and rate of protein adsorption. However, critical surface tension determined by contact angle measurements, cannot be used to predict the extent or rate of milk whey protein fouling onto a particular surface. Radioisotopic iodination of α-la and β-lg was used to show that protein adsorption onto a surface is dependent on protein concentration and temperature. Multilayer adsorption was observed with a single protein solution and with sequential exposure to the two whey proteins.     

Optimization of Thermal Pretreatment Conditions for the Separation of Native α-Lactalbumin from Whey Protein Concentrates by Means of Selective Denaturation of β-Lactoglobulin

ABSTRACT: In this study a method to obtain native α-lactalbumin with a high degree of purity of 98% (m/m) and recovery of 75% (m/m) by selective denaturation of β-lactoglobulin was developed. To achieve this goal, the thermal pretreatment of whey protein concentrate was optimized varying the composition of the liquid whey protein concentrate in terms of total protein, lactose and calcium content, and pH value. The kinetics of the thermal denaturation of α-la and β-lg were then investigated at predetermined optimal composition (protein content 5 to 20 g/L, lactose content 0.5 g/L, calcium content 0.55 g/L, and pH 7.5). Using the activation energies and reaction rate constants obtained, lines of equal effects for targeted denaturation degrees of α-la and β-lg were calculated. Depending on total protein content, an area of optimal heating temperature/time conditions was identified for each protein concentration level.     

Antimicrobial activity of nisin adsorbed to surfaces commonly used in the food industry

The adsorption isotherms of nisin to three food contact surfaces, stainless steel, polyethyleneterephthalate (PET), and rubber at 8, 25, 40, and 60 degrees C, were calculated. For all surfaces, the increase in temperature led to a decrease in the affinity between nisin and the surface. The rubber adsorbed a higher amount of nisin (0.697 microg/cm2) in comparison with PET (0.665 microg/cm2) and stainless steel (0.396 microg/cm2). Adsorption of nisin to the stainless steel surface described L-2 type curves for all temperatures assayed. However, for PET and rubber surfaces, the isotherms were L-2 type (at 40 and 60 degrees C) and L-4 type curves (at 8 and 25 degrees C). Nisin retained its antibacterial activity once adsorbed to the food contact surfaces and was able to inhibit the growth of Enterococcus hirae CECT 279 on Rothe agar medium. The attachment of three Listeria monocytogenes strains to the three surfaces was found to be dependent on the surface, the strain, and the initial bacterial suspension in contact with the surface. The adsorption of Nisaplin on surfaces reduced the attachment of all L. monocytogenes strains tested. The effect of PET-based bioactive packaging in food was very encouraging. When applied to a food system, nisin-adsorbed PET bottles reduced significantly (P < 0.05) the levels of the total aerobic plate counts in skim milk by approximately 1.4 log units after 24 days of refrigerated storage (4 degrees C), thus extending its shelf life.     

Core-Shell Biopolymer Nanoparticles Produced by Electrostatic Deposition of Beet Pectin onto Heat-Denatured β-Lactoglobulin Aggregates

ABSTRACT:  The purpose of this study was to produce and characterize core-shell biopolymer particles based on electrostatic deposition of an anionic polysaccharide (beet pectin) onto amphoteric protein aggregates (heat-denatured β-lactoglobulin [β-lg]). Initially, the optimum conditions for forming stable protein particles were established by thermal treatment (80 °C for 15 min) of 0.5 wt%β-lg solutions at different pH values (3 to 7). After heating, stable submicron-sized ( d = 100 to 300 nm) protein aggregates could be formed in the pH range from 5.6 to 6. Core-shell biopolymer particles were formed by mixing a suspension of protein aggregates (formed by heating at pH 5.8) with a beet pectin solution at pH 7 and then adjusting the pH to values where the beet pectin is adsorbed (< pH 6). The impact of pH (3 to 7) and salt concentration (0 to 250 mM NaCl) on the properties of the core-shell biopolymer particles formed was then established. The biopolymer particles were stable to aggregation from pH 4 to 6, but aggregated at lower pH values because they had a relatively small ζ-potential. The biopolymer particles remained intact and stable to aggregation up to 250 mM NaCl at pH 4, indicating that they had good salt stability. The core-shell biopolymer particles prepared in this study may be useful for encapsulation and delivery of bioactive food components or as substitutes for lipid droplets.     

Heat-induced denaturation and aggregation of β-Lactoglobulin: kinetics of formation of hydrophobic and disulphide-linked aggregates

Summary Solutions of a whey protein mixture were subjected to various time/temperature treatments, at pH 6.7. Kinetic and thermodynamic activation parameters for the rates of irreversible denaturation/aggregation of the principal whey protein component—β-lactoglobulin (β-lg) were followed by gel permeation. Fast Protein Liquid Chromatography (non-dissociating, non-reducing conditions) and by SDS-PAGE (dissociating, non-reducing conditions). The rate of loss of native β-lg owing to the formation of disulphide linked protein aggregates (k_sds-page) and the rate of formation of aggregates via both covalent and non-covalent bonds (k_gp-fplc) showed similar biphasic Arrhenius plots. However, the break of the plot occurred at different points. The k_gp-fplc values were higher than values of k_sds-page for all the temperatures examined. There was a similar trend for the thermodynamic activation parameters implying that not all of the β-lg aggregates through thiol–disulphide interactions. Hydrophobically driven associations occur within the aggregates.     

Original Papers and Proceedings

The relationship between the denaturation of β-lactoglobulin (β-lg) and the deposition of milk constituents in a heat exchanger was investigated. Experiments were carried out on a plate heat exchanger with skim milk by applying different temperature-time combinations in the temperature range 70 to 122°C. The aggregation of β-lg was measured by high performance gel permeation chromatography, and the amount of deposits on the plates of the heat exchanger was determined by chemical oxygen demand measurements. Based on the results of the experiments, a fouling model at temperatures up to 100°C is proposed and some measures to reduce the amount of deposits by more than 50% are given.     

Stability of β-lactoglobulin/micellar casein mixtures on heating in simulated milk ultrafiltrate at pH 6.0

The stability of β-lactoglobulin (β-lg)/micellar casein (MC) mixtures was examined on heating at pH 6.0 in increasing levels of lactose-free simulated milk ultrafiltrate (SMUF). Heated β-lg associated with MC to form stable particles (up to 771 nm in size in SMUF × 0.5). Higher levels of SMUF induced reductions in the charge on particles, resulting in greater aggregation and precipitation. Results indicated that the nonthiol-containing α_s1- and β-casein fractions showed greater interaction with β-lg on heating than the thiol-containing fractions (α_s2-casein and κ-casein). Casein proteins and their fractions have potential application in the development of heat-stable dairy-based beverages.     

Short communication: Evaluation of a sol-gel–based stainless steel surface modification to reduce fouling and biofilm formation during pasteurization of milk

Milk fouling and biofilms are common problems in the dairy industry across many types of processing equipment. One way to reduce milk fouling and biofilms is to modify the characteristics of milk contact surfaces. This study examines the viability of using Thermolon (Porcelain Industries Inc., Dickson, TN), a sol-gel-based surface modification of stainless steel, during thermal processing of milk. We used stainless steel 316L (control) and sol-gel-modified coupons in this study to evaluate fouling behavior and bacterial adhesion. The surface roughness as measured by an optical profiler indicated that the control coupons had a slightly smoother finish. Contact angle measurements showed that the modified surface led to a higher water contact angle, suggesting a more hydrophobic surface. The modified surface also had a lower surface energy (32.4 ± 1.4 mN/m) than the control surface (41.36 ± 2.7 mN/m). We evaluated the susceptibility of control and modified stainless steel coupons to fouling in a benchtop plate heat exchanger. We observed a significant reduction in the amount of fouled layer on modified surfaces. We found an average fouling weight of 19.21 mg/cm² and 0.37 mg/cm² on the control and modified stainless steel coupons, respectively. We also examined the adhesion of Bacillus and biofilm formation, and observed that the modified stainless steel surface offered greater resistance to biofilm formation. Overall, the Thermolon-modified surface showed potential in the thermal processing of milk, offering significantly lower fouling and bacterial attachment than the control surface.     

Factors Affecting Rheological Characteristics of Fibril Gels: The Case of β-Lactoglobulin and α-Lactalbumin

ABSTRACT:  Some of the factors that affect the rheological characteristics of fibril gels are discussed. Fibrils with nanoscale diameters from β-lactoglobulin (β-lg) and α-lactalbumin (α-la) have been used to create gels with different rheological characteristics. Values of the gelation time, t_c , the critical gel concentration, c ₀, and the equilibrium value of the storage modulus, G , such as     at long gelation times, derived from experimental rheological data, are discussed. Fibrils created from β-lg using solvent incubation and heating result in gels with different rheological properties, probably because of different microstructures and fibril densities. Partial hydrolysis of α-la with a serine proteinase from Bacillus licheniformis results in fibrils that are tubes about 20 nm in diameter. Such a fibril gel from a 10% (w/v) α-la solution has a higher modulus than a heat-set gel from a 10% (w/w) β-lg, pH 2.5 solution; it is suggested that one reason for the higher modulus might be the greater stiffness of α-la fibrils. However, the gelation times of α-la fibrils are longer than those of β-lg fibrils.     

OPTIMIZING STABILITY OF ORANGE JUICE FORTIFIED WITH WHEY PROTEIN AT LOW pH VALUES

Abstract The influence of temperature, heating time and pH on the stability of whey protein-fortified Valencia orange juice was determined by uronic acid content, degree of esterification (DE), % transmission measurements (%T) and capillary electrophoretic analysis of the juice-protein supernatants. Uronic acid content and charge of pectins showed no significant change in heat-treated samples with added proteins. The %T decreased with decreasing pH and increasing temperature and heating time for α-lactalbumin (α-lac), β-lactoglobulin (β-lg) and whey protein isolate (WPI). The lowest transmission values were shown at pH 3.0 and 85C. Capillary electropherograms confirmed more extensive juice-protein interactions in WPI and β-lg added juices than in those containing α-lac, especially at low pH, resulting in more stable juice-protein mixtures.     

Influence of protein and mineral composition on the formation of whey protein heat-induced microgels

The aim of the present study was to investigate the effect of the initial protein and mineral composition on the formation of whey protein microgels at a protein concentration of 4 wt%. As a model system, both demineralized α-lactalbumin (α-la) and β-lactoglobulin (β-lg) were mixed at 20/80, 50/50 and 80/20 α-la/β-lg ratios and heated (85 °C, 15 min) in the pH range 5.7–6.2. Pure dispersions of α-la were not forming microgels but rather precipitates (12%) and a majority of soluble aggregates (83%). Upon mixing with β-lg, microgels were only obtained for the 20/80 ratio at pH 5.7 or for pure β-lg at pH 5.8. Interestingly, the latter ratio was close to the natural composition occurring in most of the commercial whey protein isolates (WPIs). For higher ratios, particles were more or less aggregated and not spherical. As for α-la alone, most of the denatured proteins formed soluble aggregates. These results might be explained by the fact that α-la reduced the hydrophobic attractive forces involved in microgel formation, shifting the equilibrium towards the soluble aggregates pathway. This assumption was confirmed by the very low surface hydrophobicity of the soluble aggregates determined by ANS binding experiments. The conversion yield from native protein into microgels was about 51% for the 20/80 α-la/β-lg model mixture, whereas it was higher (70–85%) for commercial WPIs under similar experimental conditions. To test the possible effect of the initial mineral composition of the commercial WPIs, these products were demineralized and their ability to form microgels was tested again. After demineralisation, WPIs behaved very similarly to the 20/80 α-la/β-lg model system, the yield of conversion into microgels dropping to about 65% at pH 5.7. It was therefore concluded that in addition to the α-la/β-lg mixing ratio that was controlling hydrophobic interactions between denatured proteins that was accounting for about 50% of the microgel conversion yield, the initial mineral composition of WPIs contributed to another 20–35% yield as it was modulating the repulsive forces between the denatured proteins, promoting aggregation.     

SURFACE PROPERTIES OF PROTEIN LAYERS ADSORBED FROM MIXTURES OF GELATIN WITH VARIOUS CASEINS

We report surface tensions and surface shear viscosities of protein layers adsorbed from mixtures of gelatin +α_s11-casein, β-casein, k-casein or sodium caseinate. Under conditions where the two protein components carry opposite net charges, the combined experimental data at 25C and neutral pH are consistent with a two-layer model of the mixed protein film. The surface properties of sodium caseinate lie intermediate between those for the two major individual caseins (α_s1 and β).     

In-needle Microextraction Coupled with Gas Chromatography/Mass Spectrometry for the Analysis of Phthalates Generating from Food Containers

In this study, we fabricated a new device for headspace in-needle microextraction using a stainless steel wire coated with multiwalled carbon nanotube/polyaniline composite by coulometric technique. Then, the coated wire was placed into a stainless steel needle inside. This headspace in-needle microextraction device was applied for the determination of phthalates dissolved in aqueous solution by gas chromatography-mass spectrometry. Multiwalled carbon nanotube/polyaniline composite film of 10 mm length on the surface of stainless steel wire was optimized to polymerize electrochemically when constant potential of 2.0 V was applied during 500 s. Atomic force microscopy and Auger electron spectroscopy showed that polymerization on the surface of wire had smooth, homogeneous, and constant coating thickness of 1.5?±?0.004 μm. The optimum molar ratio of multiwalled carbon nanotube and aniline (C:N) for polymerization was determined at 1: 2 M ratio. The optimum conditions of the proposed method were extraction temperature 50 °C, saturation time 60 min, extraction time 30 min, desorption temperature 230 °C, and desorption time 3 min, respectively. Validation of headspace in-needle microextraction-multiwalled carbon nanotube/polyaniline coupled with GC/MS was also performed including limit of detection, limit of quantitation, dynamic range, recovery, and reproducibility. The proposed device is found to be inexpensive, easy, and rapid to fabricate and could be utilized effectively as the solventless microextraction method for the analysis of phthalates generating from food containers.     

Functional characterization of protein stabilized emulsions: Emulsifying behaviour of proteins in a valve homogenizer

Protein stabilised emulsions have been prepared in a valve homogeniser incorporated into a recirculating emulsification system, where the power input and number of passes have been varied. The food proteins studied were a soy-bean protein isolate, a whey protein concentrate (WPC) and a sodium caseinate. The emulsions obtained were characterized in terms of particle size distribution and amount of protein adsorbed on to the fat surface (protein load). Generally, the final fat surface area of the emulsions obtained increases more as a function of power input than as a function of number of passes. Distribution width, c_s, decreases mostly with increasing power supply and number of passes, but at the highest power input c_s increases. The protein load on the fat globules is largely determined by the fat surface area and by the type of protein adsorbed. The soy proteins give a high protein load and the caseinates give a low protein adsorption at small fat surface areas created. This relation is reversed at large surface areas of the fat globules. The relation between percentage protein adsorbed from bulk as a function of surface area suggests that the caseinates mainly cover the newly created interface by adsorption from the bulk, whereas the soy proteins fulfil this task mostly by spreading at the interface. Salt addition to 0.2M-NaCl enhances protein adsorption at the fat globule interface in the case of soy protein and caseinate, but for the whey proteins protein load is higher in distilled water.     

Protease susceptibility of beta-lactoglobulin adsorbed on stainless steel surface as evidence of contribution of its specific segment to adsorption

beta-Lactoglobulin (beta-Lg) is a major constituent of fouling deposits in the dairy industry. To determine the interaction between beta-Lg and stainless steel surfaces, beta-Lg irreversibly adsorbed on stainless steel particles was subjected to lysyl endopeptidase treatment and the course of fragmentation was compared with that observed for beta-Lg in solution. The results showed a distinct difference between the courses of fragmentation: a fragment (residues 102-135) was liberated readily from beta-Lg in solution but scarcely from beta-Lg irreversibly adsorbed on stainless steel particles. This result strongly suggests that residues 102-135 include a segment primarily responsible for the interaction of beta-Lg with stainless steel surfaces. This supports our previous results [Sakiyama et al., J. Biosci. Bioeng., 88, 536-541 (1999)] that showed that residues 125-135 of beta-Lg have a strong affinity toward stainless steel surfaces and probably a major contribution to the adsorption of beta-Lg.     

Interfacial dynamic properties of whey protein concentrate/polysaccharide mixtures at neutral pH

The effect of two non-surface active polysaccharides (sodium alginate, SA, and λ-carrageenan, λ-C) in the aqueous phase on the surface dynamic properties (dynamic surface pressure and surface dilatational properties) of a commercial milk whey protein concentrate (WPC) adsorbed film at the air–water interface has been studied. A whey protein isolate (WPI) was used as reference. The WPC and WPI concentration (at 1.0% wt), temperature (at 20 °C), pH (7), and ionic strength (at 0.05 M) were maintained constant, while the effect of polysaccharide (PS) was evaluated within the concentration range 0.0–1.0% wt. The surface dynamic properties of the adsorbed films were measured in an automatic pendant drop tensiometer. At short adsorption time and in the presence of PS, the rate of diffusion of WPC to the interface was affected by the interactions with PS in the aqueous phase, which could limit protein availability for the adsorption. On the other hand, at long-term adsorption, the magnitudes of the molecular penetration and configurational rearrangement rates of WPC in mixed systems (WPC/PS) reflected the viscoelastic characteristics of the adsorbed films. The attractive interactions between WPC and PS and/or the WPC aggregation in the presence of PS, which depend on the proper polysaccharide and its concentration in the aqueous phase, have an effect on the adsorption kinetic parameters, the amount of WPC adsorbed at the air–water interface, and the dilatational viscoelastic characteristics of WPC/PS mixed systems.     

转谷氨酰胺酶的固定化及其在处理米糠废水中的应用

制备醋酸纤维素-聚丙烯复合膜,并将转谷氨酰胺酶（transglutaminase,MTG）固定在膜上,制得了固定化MTG酶膜,测得其酶活为17.6 U/g。然后将酶膜固定在不锈钢网框上,并将其悬挂在烧杯中,用来处理米糠废水中的蛋白质。利用单因素试验,分别考察物料温度、物料pH值、转子转速以及酶膜面积对蛋白质聚合率的影响,并在单因素试验基础上,利用响应面试验优化,得到最佳聚合条件为物料温度43 ℃、pH 6.6、转子转速124 r/min、酶膜面积与底物含量比例为80∶1（cm2/g）、反应时间1.8 h,蛋白质聚合率为70%。固定化酶膜在使用5 次后,相对酶活力仍保持在74.3%以上,为连续聚合米糠废水中的蛋白质以及保留营养物质提供理论基础。