Impact of phase separation of whey proteins/hydroxypropylmethylcellulose mixtures on gelation dynamics and gels properties

This work constitutes a study of the impact of phase separation behaviour on the gels properties of a low viscosity hydroxypropylmethylcellulose and whey protein concentrate (WPC) mixed system. The phase separation was characterized by drawing the limit of thermodynamic compatibility, i.e. binodal curve, at pH 6.5 and room temperature (25 °C). Gelling properties were studied under thermodynamic compatibility (WPC 12% (w/w)/E50LV 0.25% (w/w) mixed system) and incompatibility conditions (WPC 12% (w/w)/E50LV 4% (w/w) and WPC 20% (w/w)/E50LV 4% (w/w) mixed systems)Under thermodynamic compatibility the WPC/E50LV mixed system shows gelling parameters similar to WPC. Confocal scanning laser microscopy (CSLM) micrographs showed a regular pattern of microdomains of proteins imbibed into E50LV matrix.Confocal microscopy of WPC/E50LV mixture under thermodynamic incompatibility offered details about the constitution of continuous and non-continuous phase and characteristics of non-continuous phase domains. Related to gelling parameters, the solid character upon heating was reinforced in mixed systems since they reflected the concentrating effect arising from phase separation. On the other hand, the solid character of gels upon cooling correlated with the component constituting the continuous phase, and the gelation temperature was similar to polysaccharide-rich phase predicted gelation temperature.Regarding to textural properties, the presence of the polysaccharide diminished the hardness of the mixed gels inducing less resistance to small and large deformation. WPC 20% (w/w)/E50LV 4% (w/w) mixed gel presented an interesting particulated macrostructure. This result would find application in food design and technology if the E50LV concentration is chosen to finely control the rate and extent of WPC aggregation-gelation-particulation. These results could be used in microparticulation or microencapsulation application of whey proteins.     

Contribution of sarcoplasmic proteins to myofibrillar proteins gelation

Surimi, a refined protein extract, is produced by solubilizing myofibrillar proteins during the comminuting and salting stages of manufacturing. The resulting paste gels on heating to produce kamaboko or a range of analog shellfish such as crab claw, filament sticks, fish mushroom, and so on. The myosin molecule is the major myofibrillar protein in gelation. It is believed that washing steps during the traditional surimi process play an important role in enhancing the gel properties of the resultant kamaboko by removing water-soluble (sarcoplasmic, Sp-P) proteins. By contrast, some researchers claim that retaining Sp-P or adding it into the surimi gel network not only does not interfere with the action of myofibrillar proteins during the sol-gel transition step but also improves the gel characteristics of the resultant kamaboko. It seems that retention of Sp-P or their addition into raw surimi does enhance the textural properties of kamaboko gel perhaps by functioning as a proteinase inhibitor, particularly against trypsin and trypsin-like proteinases but this depends on the type of applied surimi process. Among different types of Sp-P, it has been claimed that some proteins such as endogenous transglutaminase (TGase) play a more important role than other Sp-P in bond formation, by catalyzing the cross-linking of myosin heavy chain (MHC) molecules during low-temperature setting of surimi, resulting a more elastic kamaboko gel.     

Hot topic: Sonication increases the heat stability of whey proteins

The thickening or gelling of protein-based dairy streams and ingredients upon exposure to heat has been an ongoing problem in dairy processing for many decades. This phenomenon can restrict the range of dairy product options and reduce manufacturing efficiencies by limiting the type and extent of heat treatment that can be used. In this report, we outline a novel approach to overcoming this problem. The use of preheating treatments to induce whey protein aggregate formation in whey products is well known in the field. However, we show that the application of ultrasound for a very short duration after such a heating step breaks down these aggregates and prevents their reformation on subsequent heating, thereby reducing the viscosity increase that is usually associated with this process. This novel technique has the potential to provide significant economic benefit to the dairy manufacturing industry.     

Rheology and microstructure of cross-linked waxy maize starch/whey protein suspensions

The objective of the present work was to investigate the effect of the heating process on the structural and rheological properties of whey protein isolate/cross-linked waxy maize starch (WPI/CWMS) blends depending upon the concentration and the starch/whey protein ratio. Starch concentration ranged from 3 to 4% (w/w) and the protein content was of 0.5, 1 and 1.5% (w/w). The blend (pH 7, 100 mM ionic strength) was heated using a jacketed vessel at two pasting temperatures: 90 and 110 °C. The particle size distribution of the WPI suspension (1.5%) displayed three distinct classes of aggregates (0.3, 65 and 220 μm), whereas the size of swollen starch granules varied from 48 to 56 μm according to the pasting temperature. When the two components were mixed together, the peak attributed to swollen starch granules was attenuated and broadened towards higher values (up to 88 μm) due to protein aggregates (260–410 μm). This effect was more pronounced as the protein concentration increased. When compared to starch alone, the rheology of the mixed system was dramatically modified for the flow behaviour as well as for the viscoelastic properties which changed from a solid-like (3–4% starch) to a liquid-like behaviour (3–4% starch/1.5% protein). Microscopic observations showed aggregated proteins located in the continuous phase and swollen starch granules as the dispersed phase. Protein aggregates were of different sizes, part of them appeared adsorbed onto swollen starch granules while another part was unevenly distributed in the continuous phase, yielding discontinuous network which could explain the peculiar viscoelastic behaviour of such suspensions.     

Heat and/or high-pressure treatment of skim milk: changes to the casein micelle size, whey proteins and the acid gelation properties of the milk

Skim milk was subjected to heat, pressure or combined processes. In general, higher levels of whey protein denaturation were observed for milk subjected to combined processes than those heat- or pressure-treated only. Heat treatment caused small changes to the casein micelle size. Pressure treatment decreased the casein micelle size; however, the effect was less marked when heat and pressure treatments were combined. Acidification of the skim milks produced gels with a range of firmness, yield stresses and yield strains depending on the treatments applied. These changes in acid gel properties were not related only to whey protein denaturation levels in the milks.     

Interactions between whey proteins and salivary proteins as related to astringency of whey protein beverages at low pH

Whey protein beverages have been shown to be astringent at low pH. In the present study, the interactions between model whey proteins (β-lactoglobulin and lactoferrin) and human saliva in the pH range from 7 to 2 were investigated using particle size, turbidity, and ζ-potential measurements and sodium dodecyl sulfate-PAGE. The correlation between the sensory results of astringency and the physicochemical data was discussed. Strong interactions between β-lactoglobulin and salivary proteins led to an increase in the particle size and turbidity of mixtures of both unheated and heated β-lactoglobulin and human saliva at pH ∼3.4. However, the large particle size and high turbidity that occurred at pH 2.0 were the result of aggregation of human salivary proteins. The intense astringency in whey protein beverages may result from these increases in particle size and turbidity at these pH values and from the aggregation and precipitation of human salivary proteins alone at pH <3.0. The involvement of salivary proteins in the interaction is a key factor in the perception of astringency in whey protein beverages. At any pH, the increases in particle size and turbidity were much smaller in mixtures of lactoferrin and saliva, which suggests that aggregation and precipitation may not be the only mechanism linked to the perception of astringency in whey protein.     

Evaluation of proteolytic and ACE-inhibitory activity of Lactobacillus acidophilus in soy whey growth medium via response surface methodology

Soy whey is a rich by-product of tofu-manufacturing industries. We have previously optimized the growth of Lactobacillus acidophilus FTCC 0291 in soy whey upon supplementation of meat extract, vegetable extract and peptone using response surface methodology (RSM). The present study evaluated the proteolytic and angiotensin-I converting enzyme (ACE)-inhibitory activities of L. acidophilus FTCC 0291 in the optimized soy whey medium. The probiotic-fermented soy whey exhibited growth-associated proteolysis and ACE-inhibitory activity. Proteolysis was highly correlated with ACE-inhibitory activity, indicating that peptides liberated via fermentation may have exerted in vitro antihypertensive properties. Of the three nitrogen sources studied, peptone was found to have the highest influence on growth performance and ACE-inhibitory activity. Our results strongly indicated that probiotic-fermented soy whey produced in vitro antihypertensive bioactivity, and hence could be further developed into a carrier for probiotics with enhanced functional properties.     

Effect of pH at heat treatment on the hydrolysis of κ-casein and the gelation of skim milk by chymosin

Skim milk was adjusted to pH values between 6.5 and 7.1 and heated at 90 °C for times from 0 to 30 min. After heat treatment, the samples were re-adjusted to the natural pH (pH 6.67) and allowed to re-equilibrate. High levels of denatured whey proteins associated with the casein micelles during heating at pH 6.5 (about 70-80% of the total after 30 min of heating). This level decreased as the pH at heating was increased, so that about 30%, 20% and 10% of the denatured whey protein was associated with the casein micelles after 30 min of heating at pH 6.7, 6.9 and 7.1, respectively. Increasing levels of κ-casein were transferred to the serum as the pH at heating was increased. The loss of κ-casein and the formation of para-κ-casein with time as a consequence of the chymosin treatment of the milk samples were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The loss of κ-casein and the formation of para-κ-casein were similar for the unheated and heated samples, regardless of the pH at heating or the heat treatment applied. Monitoring the gelation properties with time for the chymosin-treated milk samples indicated that the heat treatment of the milk markedly increased the gelation time and decreased the firmness (G^′) of the gels formed, regardless of whether the denatured whey proteins were associated with the casein micelles or in the milk serum. There was no effect of pH at heat treatment. These results suggest that the heat treatment of milk has only a small effect on the primary stage of the chymosin reaction (enzymatic phase). However, heat treatment has a marked effect on the secondary stage of this reaction (aggregation phase), and the effect is similar regardless of whether the denatured whey proteins are associated with the casein micelles or in the milk serum as nonsedimentable aggregates.     

Development and validation of a methodology to qualitatively screening veterinary drugs in porcine muscle via an innovative extraction/clean-up procedure and LC-MS/MS analysis

A qualitative multiresidue method that facilitates rapid monitoring of veterinary drugs in porcine muscle is described. The method comprises the application of an innovative extraction/clean-up procedure, namely liquid–liquid extraction with partition at very low temperature (LLE-FPVLT), and analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Besides the high selectivity, sensitivity and specificity, this high-throughput method proved to be quite general as 34 veterinary drugs (from six distinct classes: tetracyclines, sulfonamides, penicillins, quinolones, macrolides and benzimidazoles) could be successfully detected. The whole screening procedure was validated according to the directives from European Commission Decision 2002/657/EC and guidelines for the validation of screening methods. Acceptable values for the evaluation parameters were achieved for all analytes (except for ampicillin, clindamycin and erythromycin). Finally, these very promising results have strengthened the possibility of inclusion of such a methodology as an integral part of the National Residue Control Plan scope of the Ministry of Agriculture, Livestock and Food Supply of Brazil.     

Impacts of glucosamine/oligochitosan glycation and cross‐linking by transglutaminase on the structure and in vitro antigenicity of whey proteins

With the fixed and selected conditions, whey protein concentrate (WPC) was glucosamine‐/oligochitosan‐glycated and cross‐linked by transglutaminase, resulting in glucosamine conjugation of 4.18–5.88 g/kg. Electrophoretic analysis showed cross‐linking and glycation of whey proteins, while circular dichroism analysis indicated that the two reactions contributed less ordered secondary structure to the two products. Enzyme‐linked immunosorbent assay results showed that the two products lost 64–95% antigenic responses of WPC, and oligochitosan was more powerful than glucosamine to reduce the responses. Rocket immuno‐electrophoretic analysis also evidenced antigenicity loss. This applied treatment is efficient to modify the structure and to decrease the allergenicity of whey proteins.     

Formation of sunflower oil emulsions stabilized by whey proteins with high-pressure homogenization (up to 350 MPa): effect of pressure on emulsion characteristics

A new ultra-high-pressure homogenizer was used to make very fine oil in water emulsions. The effect of pressures up to 350 MPa on sunflower oil (20%) in water emulsions was studied. The emulsifier used was whey protein concentrate (1.5%). The properties of the emulsions were characterized by laser light scattering (droplet size distribution) and coaxial cylinders rheometry (rheological behaviour). The protein adsorption fraction was obtained by a spectrophotometric method using bicinchoninic acid reagent.
Significant modifications in the structure and the texture of the emulsions were observed as the pressure increased. No change was revealed by polyacrylamide gel electrophoresis of the whey protein within the pressure range studied. Microdifferential scanning calorimetry scans indicated that the changes of the structural and textural properties may be because of changes in the protein conformation.     

Flow cytometric assessment of Bacillus spore response to high pressure and heat

The physiological response of Bacillus licheniformis spores to high pressure and thermal inactivation in sodium citrate buffer and nutrient broth was investigated using multiparameter flow cytometry. Spores were treated by heat-only at 121 °C, by high pressure at 150 MPa (37 °C), or by a combined high pressure and heat treatment at 600 MPa and 77 °C, and then dual stained with the fluorescent dyes SYTO 16 and propidium iodide (PI). For pressure treated spores, but not heat-only treated spores, four distinct sub-populations were detected by flow cytometry, and for these we suggest a three step model of inactivation involving a germination step following hydrolysis of the spore cortex, an unknown step, and finally an inactivation step with physical compromise of the spore's inner membrane.

Industrial relevance

This preliminary study offers a simple and fast flow cytometric method for the rapid assessment of the physiological state of bacterial spores following high pressure and thermal processing. An improved understanding of the mechanisms of spore inactivation will aid in the food safety assessment of pressure assisted thermal sterilisation in particular, and also assist in the commercialisation of these processes facilitating adoption by industry.     

Effects of nisin and reutericyclin on resistance of endospores of Clostridium spp. to heat and high pressure

The effects of high pressure, temperature, and antimicrobial compounds on endospores of Clostridium spp. were examined. Minimal inhibitory concentrations (MIC) of nisin and reutericyclin were determined for vegetative cells and endospores of Clostridium sporogenes ATCC 7955, Clostridium beijerinckii ATCC 8260, and Clostridium difficile 3195. Endospores of C. sporogenes ATCC 7955 and C. beijerinckii ATCC 8260 were exposed to 90 °C and 90 °C/600 MPa in the presence of 16 mg L⁻¹ nisin or 6.4 mg L⁻¹ reutericyclin for 0–60 min in a 0.9% saline solution. Dipicolinic acid (DPA) release was measured using a terbium-DPA fluorescence assay, and endospore permeability was assessed using 4′,6-diamidino-2-phenylindole (DAPI) fluorescence. Vegetative cells of C. sporogenes ATCC 7955 exhibited higher sensitivity to nisin relative to endospores, with MIC values 0.23 ± 0.084 mg L⁻¹ and 1.11 ± 0.48 mg L⁻¹, respectively. Nisin increased DPA release when endospores were treated at 90 °C; however, only C. sporogenes ATCC 7955 exhibited higher inactivation, suggesting strain or species specific effects. Reutericyclin did not enhance spore inactivation or DPA release. Use of nisin in combination with high pressure, thermal treatments enhanced inactivation of endospores of Clostridium spp. and may have application in foods.     

Resistance of purified seed storage proteins from sesame (Sesamum indicum L.) to proteolytic digestive enzymes

Sesame has been increasingly associated with food allergy. The main seed storage proteins of sesame (the 2S albumin and the 7S and 11S globulins) were purified and subjected to proteolysis with pepsin, trypsin and chymotrypsin. The degree of proteolysis obtained was monitored by SDS–PAGE, followed by densitometry of the main bands. The 2S albumin was found to be stable to proteolysis, being extremely resistant to pepsin, and relatively resistant to trypsin and chymotrypsin. The 7S and 11S proteins were relatively labile to pepsin. Acidic polypeptides from the 11S protein were more susceptible to proteolysis than the basic polypeptides. Both 7S and 11S proteins generated what appeared to be stable polypeptides after proteolysis with trypsin and chymotrypsin. The results are discussed in relation to similar studies on related seed storage proteins, available structural information, and the potential allergenicity of the sesame proteins.     

Prediction of time to growth of Listeria monocytogenes using Monte Carlo simulation or regression analysis,influenced by sublethal heat and recovery conditions

Stochastic models, including the variability in extent and probability of microbial growth, are useful for estimating the risk of foodborne illness (i.e. Nauta, 2000). Risk assessment typically has to embrace all sources of variability. In this paper, a stochastic approach to evaluate growth of heat damaged Listeria monocytogenes cells influenced by different stresses (pH and presence of eugenol) was performed, using an individual-based approach of growth through OD measurements. Both the lag phase duration and the “work to be done” (h₀ parameter) were derived from the growth curves obtained. From results obtained histograms of the lag phase were generated and distributions were fitted. Histograms showed a shift to longer lag phases and an increase in variability with high stress levels. Using the distributions fitted, predictions of time to unacceptable growth (10² cfu/g) of L. monocytogenes were established by Monte Carlo simulation and they were compared with results from statistical methods. It was evidenced that both methods (Monte Carlo and regression analysis) gave a good indication of the probability of a certain level of growth other than the average. Tornado plots were obtained to establish a sensitivity analysis of the influence of the conditions tested (heat, pH, eugenol) applied to the microorganism and their combinations.