Freeze-Dried Curdlan/Whey Protein Isolate-Based Biomaterial as Promising Scaffold for Matrix-Associated Autologous Chondrocyte Transplantation-A Pilot In-Vitro Study.

The purpose of this pilot study was to establish whether a novel freeze-dried curdlan/whey protein isolate-based biomaterial may be taken into consideration as a potential scaffold for matrix-associated autologous chondrocyte transplantation. For this reason, this biomaterial was initially characterized by the visualization of its micro- and macrostructures as well as evaluation of its mechanical stability, and its ability to undergo enzymatic degradation in vitro. Subsequently, the cytocompatibility of the biomaterial towards human chondrocytes (isolated from an orthopaedic patient) was assessed. It was demonstrated that the novel freeze-dried curdlan/whey protein isolate-based biomaterial possessed a porous structure and a Young's modulus close to those of the superficial and middle zones of cartilage. It also exhibited controllable degradability in collagenase II solution over nine weeks. Most importantly, this biomaterial supported the viability and proliferation of human chondrocytes, which maintained their characteristic phenotype. Moreover, quantitative reverse transcription PCR analysis and confocal microscope observations revealed that the biomaterial may protect chondrocytes from dedifferentiation towards fibroblast-like cells during 12-day culture. Thus, in conclusion, this pilot study demonstrated that novel freeze-dried curdlan/whey protein isolate-based biomaterial may be considered as a potential scaffold for matrix-associated autologous chondrocyte transplantation.

Proteomics Analysis Reveals Altered Nutrients in the Whey Proteins of Dairy Cow Milk with Different Thermal Treatments.

Thermal treatments of milk induce changes in the properties of milk whey proteins. The aim of this study was to investigate the specific changes related to nutrients in the whey proteins of dairy cow milk after pasteurization at 85 °C for 15 s or ultra-high temperature (UHT) at 135 °C for 15 s. A total of 223 whey proteins were confidently identified and quantified by TMT-based global discovery proteomics in this study. We found that UHT thermal treatment resulted in an increased abundance of 17 proteins, which appeared to show heat insensitivity. In contrast, 15 heat-sensitive proteins were decreased in abundance after UHT thermal treatment. Some of the heat-sensitive proteins were connected with the biological immune functionality, suggesting that UHT thermal treatment results in a partial loss of immune function in the whey proteins of dairy cow milk. The information reported here will considerably expand our knowledge about the degree of heat sensitivity in the whey proteins of dairy cow milk in response to different thermal treatments and offer a knowledge-based reference to aid in choosing dairy products. It is worth noting that the whey proteins (lactoperoxidase and lactoperoxidase) in milk that were significantly decreased by high heat treatment in a previous study (142 °C) showed no significant difference in the present study (135 °C). These results may imply that an appropriately reduced heating intensity of UHT retains the immunoactive proteins to the maximum extent possible.

Limited hydrolysis of glycosylated whey protein isolate ameliorates the oxidative and physical stabilities of conjugated linoleic acid oil-in-water emulsions.

Conjugated linoleic acid contains unsaturated fatty acids with multiple bioactivities, but it has poor oxidative and physical stabilities. Its emulsion was fabricated with glycosylated whey protein isolate and hydrolysates of glycosylated whey protein isolate to enhance its stability. An obvious decrease in peroxide value, thiobarbituric acid-reactive substances, particle size and creaming index of emulsion loaded by hydrolysates of glycosylated protein isolate with the increase of hydrolysis time. However, the absolute value of zeta-potential and interfacial adsorption rate of emulsion stabilized by hydrolysates of glycosylated whey protein isolate, were increased by 10.99 and 16.94% at hydrolysis time of 120 min, compared with emulsion loaded by glycosylated whey protein isolate. Thus, limited hydrolysis of glycosylated whey protein isolate as an effective method, remarkably improved the oxidative and physical stability of emulsion.

Combination of rehydrated whey protein isolate aqueous solution with blackcurrant concentrate and the formation of encapsulates via spray-drying and freeze-drying: Alterations to the functional properties of protein and their anticancer properties.

Novel protein ingredients were produced by encapsulating blackcurrant concentrate (BC) with whey protein through spray-, or freeze-, drying strategies. The effects of encapsulation strategies and the addition of BC on the physical and functional characteristics, and anticancer activity of the ingredients were evaluated. The mechanistic interactions between the blackcurrant anthocyanins (BAs) with the whey protein components were predicted via in silico studies. HPLC results revealed that spray-dried and freeze-dried whey protein-BC encapsulates have effectively delivered the BAs. The physical and functional properties of the proteins were altered by drying strategies and the addition of BC. Anticancer effects were linked to reactive oxygen species production and cell apoptosis towards HepG2. Molecular docking results showed that hydrogen bonds were the main binding forces between BAs and various whey protein molecules, resulting in the formation of complexes. These findings are relevant to the formulation of powdered products to be used as ingredients in practical food matrix.

Fabrication and characterization of whey protein isolates- lotus seedpod proanthocyanin conjugate: Its potential application in oxidizable emulsions.

Emulsified ω-3 polyunsaturated fatty acid have expanding application in different food matrix with improved water solubility while still prone to oxidation. Lotus seedpod proanthocyanidin (LSPC) was grafted to whey protein isolate (WPI) to create nature-derived antioxidant emulsifiers. 1HNMR, SDS-PAGE and multiple spectrometry showed that the structure of protein was changed after grafting. DPPH and FRAP measurements showed that WPI-LSPC conjugate (90.53 ± 1.48% of DPPH scavenging, 691.85 ± 4.54 µg/mL for FRAP assay) possessed a much better antioxidant ability than WPI (17.06 ± 3.34% of DPPH scavenging, 10.43 ± 0.26 µg/mL for FRAP assay). Ultrasonic emulsification and DSC experiments showed that WPI-LSPC conjugate were more effective at forming and stabilizing the flaxseed oil emulsions than pure WPI, with higher thermostability. Likewise, low levels of primary and secondary oxidation products were formed for the conjugate than the pure protein in O/W systems after storage, again suggesting WPI-LSPC could be used as fine antioxidant emulsifiers in oxidizing delivery systems.

High-intensity ultrasound pretreatment influence on whey protein isolate and its use on complex coacervation with kappa carrageenan: Evaluation of selected functional properties.

The aim of this work was to evaluate the influence of high-intensity ultrasound (HIUS) treatment on whey protein isolate (WPI) molecular structure as a previous step for complex coacervation (CC) with kappa-carrageenan (KC) and its influence on CC functional properties. Protein suspension of WPI (1% w/w) was treated with an ultrasound probe (24 kHz, 2 and 4 min, at 50 and 100% amplitude), non HIUS pretreated WPI was used as a control. Coacervation was achieved by mixing WPI and KC dispersions (10 min). Time and amplitude of the sonication treatment had a direct effect on the molecular structure of the protein, FTIR-ATR analysis detected changes on pretreated WPI secondary structure (1600-1700 cm-1) after sonication. CC electrostatic interactions were detected between WPI positive regions, KC sulfate group (1200-1260 cm-1), and the anhydrous oxygen of the 3,6 anhydro-D-galactose (940-1066 cm-1) with a partial negative charge. After ultrasound treatment, a progressive decrease in WPI particle size (nm) was detected. Rheology results showed pseudoplastic behavior for both, KC and CC, with a significant change on the viscosity level. Further, volume increment, stability, and expansion percentages of CC foams were improved using WPI sonicated. Besides, HIUS treatment had a positive effect on the emulsifying properties of the CC, increasing the time emulsion stability percentage. HIUS proved to be an efficient tool to improve functional properties in WPI-KC CC.

Influence of soy and whey protein, gelatin and sodium caseinate on carotenoid bioaccessibility.

Proteins could alter carotenoid bioaccessibility through altering their fate during digestion, due to emulsifying properties of resulting peptides, or influencing access of digestion enzymes to lipid droplets. In this investigation, we studied whether whey protein isolate (WPI), soy protein isolate (SPI), sodium caseinate (SC) and gelatin (GEL), added at various concentrations (expressed as percentage of recommended dietary allowance (RDA): 0, 10, 25 and 50%) would influence the bioaccessibility of lycopene, ß-carotene or lutein, added as pure carotenoids solubilized in oil, during simulated gastro-intestinal (GI) digestion. Protein and lipid digestion as well as selected physico-chemical parameters including surface tension, ζ-potential and micelle size were evaluated. Adding proteins influenced positively the bioaccessibility of ß-carotene, by up to 189% (p < 0.001), but it resulted in generally decreased bioaccessibility of lutein, by up to 50% (p < 0.001), while for lycopene, the presence of proteins did not influence its bioaccessibility, except for a slight increase with WPI, by up to 135% (p < 0.001). However, the effect depended significantly on the type of protein (p < 0.001) and its concentration (p < 0.001). While ß-carotene bioaccessibility was greatly enhanced in the presence of SC, compared to WPI and GEL, the presence of SPI strongly decreased carotenoid bioaccessibility. Neglecting individual carotenoids, higher protein concentration correlated positively with carotenoid bioaccessibility (R = 0.57, p < 0.01), smaller micelle size (R = -0.83, p < 0.01), decreased repulsive forces (ζ-potential, R = -0.72, p < 0.01), and higher surface tension (R = 0.44, p < 0.01). In conclusion, proteins differentially affected carotenoid bioaccessibility during digestion depending on carotenoid and protein species, with both positive and negative interactions occurring.

pH-Induced structural transitions in whey protein isolate and ultrasonically solubilized Persian gum mixture.

The present work evidently reports that ultrasonic depolymerization strongly enhanced complex coacervation between Persian gum (PG) and whey protein isolate (WPI). PG was sonicated at 60 °C, operating frequency of 20 kHz and nominal power output of 800 W for various times followed by mixing with WPI. Acid-induced interaction between the two biopolymers was studied by turbidity, light scattering, zeta potential and viscosity measurements over a wide pH range. Sonication of intact PG (IPG) for 10 min considerably reduced the molecular weight from 4.12 × 106 to 0.76 × 106 g/mol. Besides, ultrasonic fragmentation of water insoluble fraction of PG drove protein containing chains into the soluble phase. Sonicated PG (SPG) was shown to be more flexible with higher number of carboxyl groups available for electrostatic interaction with WPI, such that the complete neutralization did not occur even at protein to polysaccharide ratio of 50: 1. Additionally, scattered light intensity and viscosity measurements revealed two maxima in the pH ranges of 4.4-4.85 and 3.27-4.0, being highly intense for the gum sonicated for 10 min and longer. Considering the pH-behavior of WPI components, the former peak was related to interpolymer complex formation between ß-lactoglobulin and long chain fraction of SPG, while the latter was attributed to intrapolymer association of α-lactalbumin with the short chain oligosaccharides arising from ultrasonic degradation of PG.

Chemical composition as an indicator for evaluating heated whey protein isolate (WPI) and sugar beet pectin (SBP) systems to stabilize O/W emulsions.

We investigated changes in the chemical composition of WPI as a result of heating (60 °C, 72 h) with SBP in solution (pH 6.75). The concentration of WPI was kept at a constant (3%), whereas the level of SBP was varied at 1, 1.5, and 3%. The reaction products were examined using the Ellman's reagent, ninhydrin assay, and gel electrophoresis. The results demonstrated that the losses of the free sulfhydryl (-SH) and primary amine (-NH2) contents in WPI were less severe compared to those occurring in the dry-state at similar conditions (mass ratio, temperature, and reaction duration). The mixtures were used as emulsifiers in an O/W emulsion system at pH 3.20 and 6.75 and showed an improved ability to stabilize the average size of the droplets than WPI alone at acidic pH. The mixtures at higher levels of SBP, ≥ 1.5%, however, adversely affected the emulsion stability at neutral pH.

Influence of heated, unheated whey protein isolate and its combination with modified starch on improvement of encapsulated pomegranate seed oil oxidative stability.

This study aimed at encapsulating pomegranate seed oil (PSO) by emulsification followed by spray drying using whey protein isolate (WPI) in its natural form, heated (Pickering), and combined with modified starch (WPI:Capsul®) as emulsifiers/wall materials. Emulsions were stable under different stress conditions. Pickering emulsions presented bigger droplet size (6.49-9.98 µm) when compared to WPI (1.88-4.62 µm) and WPI:Capsul® emulsions (1.68-5.62 µm). Sixteen fatty acids were identified in PSO. WPI treatment was considered the best formulation since it presented the highest fatty acid retention (68.51, 65.47, 47.27, 53.68, 52.95, and 52.28% for linoleic, oleic, punicic, α-eleostearic, catalpic, and ß-eleostearic acids after 30 days-storage, respectively) and protected the oil against volatile compound formation (heptanal, (E,E)-2,4-heptadienal, (Z)-2-heptenal, octanal, pentanal, (E)-2-hexenal, (E)-2-octenal, nonanal, (E)-2-decenal, and (E,E)-2,4-octadienal), which did not occur with free PSO. Overall, encapsulation protected PSO against oxidation over time, which may allow the development of new functional foods.

Impact of Preheating Temperature on the Separation of Whey Proteins When Combined with Chemical or Bipolar Membrane Electrochemical Acidification.

Separation of α-lactalbumin and ß-lactoglobulin improves their respective nutritional and functional properties. One strategy to improve their fractionation is to modify their pH and ionic strength to induce the selective aggregation and precipitation of one of the proteins of interest. Electrodialysis with bipolar membrane (EDBM) is a green process that simultaneously provides acidification and demineralization of a solution without adding any chemical compounds. This research presents the impact on whey proteins separation of different preheating temperatures (20, 50, 55 and 60 °C) combined with EDBM or chemical acidification of 10% whey protein isolate solutions. A ß-lactoglobulin fraction at 81.8% purity was obtained in the precipitate after EDBM acidification and preheated at 60 °C, representing a recovery yield of 35.8%. In comparison, chemical acidification combined with a 60 °C preheating treatment provides a ß-lactoglobulin fraction at 70.9% purity with a 11.6% recovery yield. The combination of EDBM acidification with a preheating treatment at 60 °C led to a better separation of the main whey proteins than chemical acidification.

Interactions of Whey Proteins with Metal Ions.

Whey proteins tend to interact with metal ions, which have implications in different fields related to human life quality. There are two impacts of such interactions: they can provide opportunities for applications in food and nutraceuticals, but may lead to analytical challenges related to their study and outcomes for food processing, storage, and food interactions. Moreover, interactions of whey proteins with metal ions are complicated, requiring deep understanding, leading to consequences, such as metalloproteins, metallocomplexes, nanoparticles, or aggregates, creating a biologically active system. To understand the phenomena of metal-protein interactions, it is important to develop analytical approaches combined with studies of changes in the biological activity and to analyze the impact of such interactions on different fields. The aim of this review was to discuss chemistry of ß-lactoglobulin, α-lactalbumin, and lactotransferrin, their interactions with different metal ions, analytical techniques used to study them and the implications for food and nutraceuticals.

Mechanisms of whey protein isolate interaction with basil seed gum: Influence of pH and protein-polysaccharide ratio.

In the present work, we determined the structure-function relationships of basil seed gum (BSG) and whey protein isolate (WPI) mixtures at the start of soluble complex formation, maximum soluble complex formation and predominant thermodynamic incompatibility to understand BSG:WPI blends interaction behavior. Accordingly, turbidity and zeta potential were analyzed in the pH range of 2.0-7.0 and BSG:WPI ratios of 1:4, 1:6.6 and 1:9. Dynamic rheometry was used to evaluate samples at three different pHs. Additionally, dilute solution properties of BSG, WPI and their blends were studied at pH = 7.0. Independent of mixture ratio, all dispersions showed maximum interaction at pH = 5.0, the start of soluble complex formation around pH = 6.0 and thermodynamic incompatibility interaction behavior at pH = 7.0. Cole-Cole plots based on dynamic rheometry supported the Gibbs free energy change of mixtures based on intrinsic viscosity data. These results are important to create new structures from mixtures of proteins and polysaccharides.

Effect of isoelectric point on cheese whey wastewater treatment using a microbial electrochemical system.

In this study, a microbial electrochemical system (MES) was employed to investigate the effect of isoelectric point (IEP) on cheese whey wastewater treatment. The experiments were carried out in a bioreactor equipped with a semicircular carbon cloth and stainless steel electrodes as anode and cathode, respectively. The effects of IEP, whey protein concentration, electrical current, and time were studied. The IEP of the whey protein was determined at pH 5.9. The optimum electrical current was obtained at 6 mA for synthetic cheese whey wastewater. The results of rotary exponential doping showed that the third structure of proteins chenges to the second structure at the IEP. The highest protein removal (98%) was obtained at pH 6. The results showed that 76%, 83%, and 98% protein removal were achieved at 2, 4, and 8 h, respectively.

Impact of gum Arabic on the partition and stability of resveratrol in sunflower oil emulsions stabilized by whey protein isolate.

In protein-stabilized oil-in-water emulsions, a co-emulsifier may also be an antioxidant, increasing the oxidative stability of the oil and adding nutritional value to the formulation. We investigated the impact of gum Arabic on the partition and stability of resveratrol in sunflower oil emulsions produced using whey protein isolate in the absence and presence of calcium. Gum Arabic increased the protein and resveratrol contents at the oil-water interface and the stability of resveratrol, which was enhanced by calcium. Resveratrol increased the oxidative stability of the oil. These results indicate that resveratrol is stable in the interfacial membrane of emulsions made with whey protein isolate, calcium and gum Arabic and suggest that oil-in-water emulsions could be used as potential carriers of co-encapsulated functional oils and polyphenolic antioxidants.

Enhancing the recovery of whey proteins based on application of ultrasound in ultrafiltration and spray drying.

Improvements in the membrane separation of whey and subsequent spray drying for the recovery of Whey Protein Concentrate (WPC) have been investigated in the present work. Initially, ultrasonic pretreatment of whey was performed to reduce the microbial contamination and possibly improve the membrane separation based on inducing changes in the whey proteins. Studies involving the effect of important parameters in the membrane operation such as transmembrane pressure (TMP), type of membrane and molecular weight cutoff (MWCO) on permeate flux established that 10 kDa hydrosart membrane gives maximum permeate flux with minimum fouling at an optimum TMP of 1.2 bar. Studies with ultrasound assisted ultrafiltration established that the fouling was reduced with ultrasound also giving higher permeate flux. Effect of spray drying parameters such as air pressure, aspirator rate (rpm), feed flow rate and dilution on the WPC yield, analysed using SDS-PAGE and Kjeldahl method, has also been investigated. Additionally, the use of ultrasonic nozzle in the spray drying was investigated and the results were compared with pneumatic nozzle. Overall, it was clearly established that pretreatment, membrane characteristics, ultrafiltration operating parameters and spray drying parameters play an important role in deciding the WPC recovery from whey. Significant process intensification benefits were demonstrated to be obtained with the use of ultrasound both in membrane separation and spray drying.

Whey protein membrane processing methods and membrane fouling mechanism analysis.

Whey is a byproduct with nutritional value and high organic and saline content. It is an important source of organic contamination in dairy industry. In this paper, we gave an overview of the current use of membrane materials and membrane processing in cheese whey protein recovery and discussed recent developments in membrane technology. Different types of membranes, such as polymers, ceramic membranes and modification membranes, are used for various purposes, such an increasing permeation flux, reducing membrane fouling, and increasing the protein rejection rate, concentration, fractionation and purification of whey protein. New membrane processing methods and integrated membrane methods to recover whey protein were reviewed. Membrane fouling factors during whey protein ultrafiltration process, which included whey protein conformation, membrane filtration conditions and the interaction between proteins and the membrane surface or pores, were also discussed and analyzed to reveal membrane fouling mechanism.

Rheological and microstructural properties of cold-set emulsion gels fabricated from mixed proteins: Whey protein and lactoferrin.

Cold-set emulsion gels were fabricated from oil droplets coated by mixed proteins: whey protein and lactoferrin. The impact of protein composition, droplet concentration, pH, and ionic strength on the microstructure, texture, and stability of the cold-set emulsion gels was determined. Protein composition had a major influence on gel strength, with the strongest emulsion gels being formed at an optimized protein composition (0.5 wt% whey protein and 1.5 wt% lactoferrin). The storage modulus of the emulsion gels increased from 149 to 1590 Pa as the droplet concentration increased from 10 to 40 wt%. The gel strength could also be modulated by adjusting pH, with the strongest gels being formed at pH = 6.5, where the net charge on the droplets was neutral. Increasing the ionic strength weakened the electrostatic interactions, which inhibited droplet aggregation and led to a decrease in gel strength. These results may be useful for designing cold-set emulsion gels with rheological properties that can be tailored for specific commercial products.

Optimization of whey protein isolate-quince seed mucilage complex coacervation.

In this study, the complex coacervation of whey protein isolate (WPI) and quince seed mucilage (QSM) was studied as a function of pH (7.0-2.0), biopolymers concentration (0.05, 0.1 and 0.5%) and WPI:QSM ratio (10:90 to 90:10), according to protolytic titration, electrical conductivity (EC) and turbidity analyses. The solution containing 0.5% biopolymers with WPI:QSM ratio of 70:30 resulted in maximum complex coacervation at the pHopt 4.0. With increasing WPI:QSM ratio, the peaks of pH-turbidity curves shifted to higher pH values, and with increasing biopolymers concentration, the optimum WPI:QSM ratio and pH shifted to higher values. The EC of biopolymers solutions (concentration 0.5%) increased by decreasing pH and WPI:QSM ratio. The aforementioned optimum condition resulted coacervates with maximum particles size (16.22 µm) and minimum ζ-potential (-5.1 mV), which were observed as densely agglomerated macro-complexes with highest coacervation yield (80.67%). The X-ray analysis showed that coacervates retain the amorphous structure of individual biopolymers. These coacervates may be useful for encapsulation and delivery of (bio-) active compounds.

Preparation and characterization of microencapsulated phytosterols for the formulation of functional foods: Scale up from laboratory to semi-technical production.

Phytosterols were microencapsulated by spray drying in a shell represented by WPI, inulin and chitosan at four different combinations through the formulation of aqueous suspensions. Moreover, two concentrations of Tween 80 (1.25% and 2.50% w/w) and two inlet temperatures (125 °C and 155 °C) were tested. The effect of the different experimental conditions on the process yield and on the microcapsules properties was evaluated. A significant effect of all variables on the microcapsule properties was found. Accordingly, the best performance, with the maximum loading capacity of 25%, was obtained by using only WPI as shell material, Tween 80 at 1.25% and inlet temperature of 155 °C. The process was successfully scaled-up from laboratory equipment to a semi-technical scale keeping the optimal shell formulation and process conditions.