Isolation of lactoperoxidase using different cation exchange resins by batch and column procedures

Lactoperoxidase (LP) was isolated from whey protein by cation-exchange using Carboxymethyl resin (CM-25C) and Sulphopropyl Toyopearl resin (SP-650C). Both batch and column procedures were employed and the adsorption capacities and extraction efficiencies were compared. The resin bed volume to whey volume ratios were 0.96:1.0 for CM-25C and ≤ 0.64:1.0 for SP-650 indicating higher adsorption capacity of SP-650 compared to CM-25C. The effluent LP activity depended on both the enzyme activity in the whey and the amount of whey loaded on the column within the saturation limits of the resin. The percentage recovery was high below the saturation point and fell off rapidly with over-saturation. While effective recovery was achieved with column extraction procedures, the recovery was poor in batch procedures. The whey-resin contact time had little impact on the enzyme adsorption. SDS PAGE and HPLC analyses were also carried out, the purity was examined and the proteins characterised in terms of molecular weights. Reversed phase HPLC provided clear distinction of the LP and lactoferrin (LF) peaks. The enzyme purity was higher in column effluents compared to batch effluents, judged on the basis of the clarity of the gel bands and the resolved peaks in HPLC chromatograms.

A further study of the recovery and purification of surfactin from fermentation broth by membrane filtration

Surfactin is a bacterial lipopeptide produced by Bacillus subtilis and is a powerful surfactant, having also antiviral, antibacterial and antitumor properties. The recovery and purification of surfactin from complex fermentation broths is a major obstacle to its commercialization; therefore, a two-step membrane filtration process was developed using a lab scale tangential flow filtration (TFF) unit with 10 kDa MWCO regenerated cellulose (RC) and polyethersulfone (PES)membranes at three different transmembrane pressure (TMP) of 1.5 bar, 2.0 bar and 2.5 bar. Two modes of filtrations were studied, with and without cleaning of membranes prior to UF-2. In a first step of ultrafiltration (UF-1), surfactin was retained effectively by membranes at above its critical micelle concentration (CMC); subsequently in UF-2, the retentate micelles were disrupted by addition of 50% (v/v) methanol solution to allow recovery of surfactin in the permeate. Main protein contaminants were effectively retained by the membrane in UF-2. Flux of permeates, rejection coefficient (R) of surfactin and proteinwere measured during the filtrations. Overall the three different TMPs applied have no significant effect in the filtrations and PES is the more suitable membrane to selectively separate surfactin from fermentation broth, achieving high recovery and level of purity. In addition this two-step UF process is scalable for larger volume of samples without affecting the original functionality of surfactin, although membranes permeability can be affected due to exposure to methanolic solution used in UF-2.

Effects of fat on the properties of halloumi cheese

Halloumi cheese was produced from 11 bovine milks with fat contents of 1.61-4.04%, giving a range of 32-53% fat in dry matter (FDM) in the cheeses. Starter culture and/or microparticulated whey protein (Simplesse((R)) 100(E)) was also added to selected batches of milk. Hardness decreased with increasing FDM, with increase in moisture and with lower pH. On sensory evaluation, there was an increase in preference score with FDM (R-2 = 0.8). Inclusion of microparticulated whey protein may have had a fat mimetic effect, as preference scores otherwise decreased with increasing protein levels (R-2 = 0.75).

Textural and microstructural properties of urfa cheese (a white-brined Turkish cheese)

The texture and microstructure of white-brined cheeses similar to urfa (a traditional Turkish cheese) were studied. One batch of cheeses was made in the traditional manner and one batch was made from ultrafiltered (UF) milk. Samples from each batch were either ripened in brine after production or scalded in whey for 3 min at 90degreesC prior to ripening. The results showed only marginal differences in the ripening profiles of both batches of unscalded cheeses, but scalding slowed down the extent of proteolysis in both batches. The scalded cheeses had a firmer texture than the unscalded ones, and the unscalded UF cheese had a more 'springy' body than the unscalded traditional cheese. Overall, scalding resulted in a more homogeneous structure, but the unscalded UF cheese had a close texture that resembled the scalded samples. It was concluded that, with respect to texture and structure, cheeses made with UF milk do not need to be scalded after production.

Viability of Salmonella enterica subsp. enterica during the preparation and cold storage of Egyptian soft cheeses and ice-cream

Changes occurring in the viability of Salmonella enterica subsp. enterica during the preparation and cold storage of Domiati cheese, Kariesh cheese and ice-cream were examined. A significant decrease in numbers was observed after whey drainage during the manufacture of Domiati cheese, but Salmonella remained viable for 13 weeks in cheeses prepared from milks with between 60 and 100 g/L NaCl; the viability declined in Domiati cheese made from highly salted milk during the later stages of storage. The method of coagulation used in the preparation of Kariesh cheese affected the survival time of the pathogen, and it varied from 2 to 3 weeks in cheeses made with a slow-acid coagulation method to 4-5 weeks for an acid-rennet coagulation method. This difference was attributed to the higher salt-in-moisture levels and lower pH values of Kariesh cheese prepared by the slow-acid coagulation method. A slight decrease in the numbers of Salmonella resulted from ageing ice-cream mix for 24 h at 0degreesC, but a greater reduction was evident after one day of frozen storage at -20degreesC. The pathogen survived further frozen storage for four months without any substantial change in numbers.

Calcium removal from milk by ion exchange

Calcium removal, using Duolite C433 ion exchange resin, was faster from permeate than from milk. Almost all calcium could be removed, suggesting a fairly rapid conversion from both soluble calcium phosphate and from micellar calcium to ionic calcium. Calcium reduction from milk is accompanied by an increase in pH, a reduction in ionic calcium, an increase in ethanol stability and an increase in the rennet coagulation time. There is a gradual increase in the average casein micelle size with calcium removal, up to a point where the micelle size increases dramatically. Zeta potential becomes more negative with calcium removal. At higher levels of calcium removal, the changes are not reversible, on reducing pH to its original value. For goat's milk, over the range 0-20% total calcium removal, relatively small reductions in total calcium gave rise to proportionally larger reductions in ionic calcium in a ratio of about 1:3.2.

Recovery and purification of surfactin from fermentation broth by a two-step ultrafiltration process

Surfactin is a bacterial lipopeptide produced by Bacillus subtilis and it is a powerful surfactant, having also antiviral, antibacterial and antitumor properties. The recovery and purification of surfactin from complex fermentation broths is a major obstacle to its commercialization; therefore, two-step membrane filtration processes were evaluated using centrifugal and stirred cell devices while the mechanisms of separation were investigated by particle size and surface charge measurements. In a first step of ultrafiltration (UF-1), surfactin was retained effectively by membranes at above its critical micelle concentration (CMC); subsequently in UF-2, the retentate micelles were disrupted by addition of 50% (v/v) methanol solution to allow recovery of surfactin in the permeate. Main protein contaminants were effective]), retained by the membrane in UF-2. Ultrafiltration was carried out either using centrifugal devices with 30 and 10 kDa MWCO regenerated cellulose membranes, or a stirred cell device with 10 kDa MWCO polyethersulfone (PES) and regenerated cellulose (RC) membranes. Total rejection of surfactin was consistently observed in UF-1, while in UF-2 PES membranes had the lowest rejection coefficient of 0.08 +/- 0.04. It was found that disruption of surfactin micelles, aggregation of protein contaminants and electrostatic interactions in UF-2 can further improve the selectivity of the membrane based purification technique. (C) 2007 Elsevier B.V. All rights reserved.

Synthesis of isomaltooligosaccharides and oligodextrans in a recycle membrane bioreactor by the combined use of dextransucrase and dextranase

A recycle ultrafiltration membrane reactor was used to develop a continuous synthesis process for the production of isomaltooligosaccharides (IMO) from sucrose, using the enzymes dextransucrase and dextranase. A variety of membranes were tested and the parameters affecting reactor stability, productivity, and product molecular weight distribution were investigated. Enzyme inactivation in the reactor was reduced with the use of a non-ionic surfactant but its use had severe adverse effects on the membrane pore size and porosity. During continuous isomaltooligosaccharide synthesis, dextransucrase inactivation was shown to occur as a result of the dextranase activity and it was dependent mainly on the substrate availability in the reactor and the hydrolytic activity of dextranase. Substrate and dextranase concentrations (50-200 mg/mL(-1) and 10-30 U/mL(-1), respectively) affected permeate fluxes, reactor productivity, and product average molecular weight. The oligodextrans and isomaltooligosaccharides formed had molecular weights lower than in batch synthesis reactions but they largely consisted of oligosaccharides with a degree of polymerization (DP) greater than 5, depending on the synthesis conditions. No significant rejection of the sugars formed was shown by the membranes and permeate flux was dependent on tangential flow velocity. (C) 2004 Wiley Periodicals, Inc.

An insight into the mechanism of protein separation by colloidal gas aphrons (CGA) generated from ionic surfactants

Colloidal gas aphrons (CGA), which are surfactant stabilised microbubbles, have been previously applied for the recovery of proteins from model mixtures and a few studies have demonstrated the potential of these dispersions for the selective recovery of proteins from complex mixtures. However there is a lack of understanding of the mechanism of separation and forces governing the selectivity of the separation. In this paper a mechanistic study is carried out to determine the main factors and forces influencing the selectivity of separation of whey proteins with CGA generated from ionic surfactants. Two different separation strategies were followed: (i) separation of lactoferrin and lactoperoxidase by anionic CGA generated from a solution of sodium bis-(2-ethyl hexyl) sulfosuccinate (AOT); (ii) separation of beta-lactoglobulin by cationic CGA generated from a solution of cetyltrimethylammonium bromide (CTAB). Separation results indicate that electrostatic interactions are the main forces determining the selectivity however these could not completely explain the selectivities obtained following both strategies. Protein-surfactant interactions were studied by measuring the zeta potential changes on individual proteins upon addition of surfactant and at varying pH. Interestingly strongest electrostatic interactions were measured at those pH and surfactant to protein mass ratios which were optimum for protein separation. Effect of surfactant on protein conformation was determined by measuring the change in fluorescence intensity upon addition of surfactant at varying pH. Differences in the fluorescence patterns were detected among proteins which were correlated to differences in their conformational features which could in turn explain their different separation behaviour. The effect of conformation on selectivity was further proven by experiments in which conformational changes were induced by pre-treatment of whey (heating) and by storage at 4 degrees C. Overall it can be concluded that separation of proteins by ionic CGA is driven mainly by electrostatic interactions however conformational features will finally determine the selectivity of the separation with competitive adsorption having also an effect. (c) 2006 Elsevier B.V. All rights reserved.

Factors affecting lactoperoxidase activity

The extent to which lactoperoxidase (LP) activity was affected while varying the concentration of various compounds normally present in the reaction medium was investigated. LP activity increased with increasing concentrations of 2,2'-azino-bis-3-ethylbenz-thiazoline-6-sulphonic acid (ABTS) but decreased with increasing thiocyanate concentrations. Maximum activity was at 0.1 mm for peroxide. Activity increased in the presence of lactose, whey protein concentrate, sodium, magnesium and calcium chlorides, but decreased in the presence of casein. Activity was similar in either acetate or phosphate buffers but higher in either citrate or succinate buffers. These compounds influence LP activity and should be considered when optimum activity conditions are being established.

A novel technique for differentiation of proteins in the development of acid gel structure from control and heat treated milk using confocal scanning laser microscopy

The incorporation of caseins and whey proteins into acid gels produced from unheated and heat treated skimmed milk was studied by confocal scanning laser microscopy (CSLM) using fluorescent labelled proteins. Bovine casein micelles were labelled using Alexa Fluor 594, while whey proteins were labelled using Alexa Fluor 488. Samples of the labelled protein solutions were introduced into aliquots of pasteurised skim milk, and skim milk heated to 90 degrees C for 2 min and 95 degrees C for 8 min. The milk was acidified at 40 degrees C to a final pH of 4.4 using 20 g gluconodelta-lactone/l (GDL). The formation of gels was observed with CSLM at two wavelengths (488 nm and 594 nm), and also by visual and rheological methods. In the control milk, as pH decreased distinct casein aggregates appeared, and as further pH reduction occurred, the whey proteins could be seen to coat the casein aggregates. With the heated milks, the gel structure was formed of continuous strands consisting of both casein and whey protein. The formation of the gel network was correlated with an increase in the elastic modulus for all three treatments, in relation to the severity of heat treatment. This model system allows the separate observation of the caseins and whey proteins, and the study of the interactions between the two protein fractions during the formation of the acid gel structure, on a real-time basis. The system could therefore be a valuable tool in the study of structure formation in yoghurt and other dairy protein systems.

Some functional properties of fractionated soy protein isolates obtained by microfiltration

Five soy proteins isolate (SPI) fractions were produced using two microfiltration membranes with different pore sizes. Fractionation was carried out on SPI produced by isoelectric precipitation of a crude protein extract. The five fractions were two retentates and two permeates from the two membranes, the fifth fraction was obtained as the retentate on the smaller-po re- sized membrane fed with the permeate from the larger-pore-sized membrane. Solubility, foaming and emulsifying properties of the collected fractionates were investigated. It was observed that in the pH range 3-8 the retentates featured superior solubility compared with permeates. There was no significant difference (p > 0.0 1) in solubility between the retentates and SPI at pH >= 6. Foaming characteristics of the fractions followed the same trend as solubility with regard to foam expansion. There was, however, no particular trend observed with regards to foam stability. Emulsions stabilised by the retentates exhibited higher values (p<0.01) of emulsion stability index (ESI) and emulsifying activity index (EAI) than those stabilised with permeates. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) profiles indicated that the fractions exhibiting high functionality in terms of solubility, foaming and emulsifying properties were also richer in 7S globulin soy protein subunits. Isoelectric focussing (IEF) profiles showed that retentates were richer in species with isoelectric points (pl) between 5.2 and 5.6 while permeates featured more prominently at pis between 4.5 and 4.8. (C) 2006 Elsevier Ltd. All rights reserved.

Effect of chymosin reduction and salt substitution on the properties of white salted cheese

A whey salts mixture was used as a partial substitute for sodium chloride to provide a modified Na:K ratio (1:3.4) in the manufacture of white salted cheese using ultrafiltration. Reduction of chymosin addition from 20 to 8 mu L kg(-1) of cheese was also investigated. Variation of salt and chymosin levels did not result in any significant differences in composition and physicochemical properties. The rates of proteolysis in terms of water-soluble nitrogen (WSN) and nitrogen soluble in 12% trichloroacetic acid (TCA-SN) were affected by chymosin levels but not by salt treatment. Urea-PAGE electrophoretic analysis of caseins from the cheeses manufactured using three levels of chymosin and two salt types showed that the hydrolysis of alpha(s1)-casein was higher than for beta-caseins but the differences between the cheeses were not significant (P > 0.05). The chymosin level did not have a significant effect (P > 0.05) on hardness and fracturability, suggesting that any variation in hardness due to the initial hydrolysis was being confounded by other variables. Cheeses including the whey salts product were harder and more fracturable (P < 0.01) than the cheese treated with NaCl only. Both hardness and fracturability values decreased (P < 0.05) over the maturation period. The scores for bitterness were low; neither the effects of salt nor chymosin levels were significant (P > 0.05). (c) 2005 Elsevier Ltd. All rights reserved.

Associations between dairy consumption and body weight: a review of the evidence and underlying mechanisms

As the incidence of obesity is reaching 'epidemic' proportions, there is currently widespread interest in the impact of dietary components on body-weight and food intake regulation. The majority of data available from both epidemiological and intervention studies provide evidence of a negative but modest association between milk and dairy product consumption and BMI and other measures of adiposity, with indications that higher intakes result in increased weight loss and lean tissue maintenance during energy restriction. The purported physiological and molecular mechanisms underlying the impact of dairy constituents on adiposity are incompletely understood but may include effects on lipolysis, lipogeneis and fatty acid absorption. Furthermore, accumulating evidence indicates an impact of dairy constituents, in particular whey protein derivatives, on appetite regulation and food intake. The present review summarises available data and provides an insight into the likely contribution of dairy foods to strategies aimed at appetite regulation, weight loss or the prevention of weight gain.

Improved process topology for membrane distillation

In membrane distillation in a conventional membrane module, the enthalpies of vaporisation and condensation are supplied and removed by changes in the temperatures of the feed and permeate streams, respectively. Less than 5% of the feed can be distilled in a single pass, because the potential changes in the enthalpies of the liquid streams are much smaller than the enthalpy of vaporisation. Furthermore, the driving force for mass transfer reduces as the feed stream temperature and vapour pressure fall during distillation. These restrictions can be avoided if the enthalpy of vaporisation is uncoupled from the heat capacities of the feed and permeate streams. A specified distillation can then be effected continuously in a single module. Calculations are presented which estimate the performance of a flat plate unit in which the enthalpy of distillation is supplied and removed by the condensing and boiling of thermal fluids in separate circuits, and the imposed temperature difference is independent of position. Because the mass flux through the membrane is dependent on vapour pressure, membrane distillation is suited to applications with a high membrane temperature. The maximum mass flux in the proposed module geometry is predicted to be 30 kg/m2 per h at atmospheric pressure when the membrane temperature is 65°C. Operation at higher membrane temperatures is predicted to raise the mass flux, for example to 85 kg/m2 per h at a membrane temperature of 100°C. This would require pressurisation to 20 bar to prevent boiling at the heating plate of the feed channel. Pre-pressurisation of the membrane pores and control of the dissolved gas concentrations in the feed and the recyled permeate should be investigated as a means to achieve high temperature membrane distillation without pore penetration and wetting.