Low-Frequency Ultrasound Effects on Cellulose Nanocrystals for Potential Application in Stabilizing Pickering Emulsions.

Cellulose, in the form of cellulose nanocrystals (CNCs), is a promising biomaterial for stabilizing Pickering emulsions (PEs). PEs are commonly formed using low-frequency ultrasound (LFU) treatment and impact CNC properties. The present study investigated the specific effects of LFU treatment on CNCs' chemical and physical properties. CNCs were characterized using dynamic light scattering, ζ;-potential determination, Fourier transform infrared spectroscopy, X-ray diffraction, and contact angle measurement. CNC suspensions were studied using rheological analysis and static multiple light scattering. LFU treatment broke CNC aggregates and modified the rheological behavior of CNC suspensions but did not affect the CNCs' chemical or crystallographic structures, surface charge, or hydrophilic properties. During the storage of CNC suspensions and PEs, liquid crystal formation was observed with cross-polarized light. Hypotheses related to the impact of liquid crystal CNCs on PE stability were proposed.

Influence of Alginate Properties and Calcium Chloride Concentration on Alginate Bead Reticulation and Size: A Phenomenological Approach.

Two types of alginates, AlgLF and AlgP, were used in this study to produce alginate beads by electro-vibratory extrusion. AlgLF and AlgP exhibited different Mannuronate/Guluronate (M/G) ratios and molecular weights as measured by NMR and SEC-MALS. The calcium chloride concentration was found to have the greatest effect on bead size. Higher concentrations resulted in smaller beads. AlgLF with a higher molecular weight and a lower proportion of G blocks showed smaller beads. For both alginates, the bead size was also influenced by the flow rate and vibration frequency. Alginate solution aging showed a minimal effect. Alginate reticulation was modeled using a mathematical equation. The study provides insights for the optimization of alginate-based materials in different applications by shedding light on the main factors influencing bead size. The importance of the molecular weight, M/G ratio and calcium ion concentration in the gelling process is highlighted, providing opportunities for the tailoring of alginate materials through a phenomenological model.

Comparison of Electrostatic Spray Drying, Spray Drying, and Freeze Drying for Lacticaseibacillus rhamnosus GG Dehydration.

Spray drying (SD) is extensively used to encapsulate lactic acid bacteria in large-scale industrial applications; however, bacteria combat several harms that reduce their viability. In this study, a novel technique called electrostatic spray drying (ESD) was used to explore the benefits and disadvantages of using electrostatic charge and lower temperatures in the system. Freeze drying (FD) was used as a reference. The effect of different encapsulation agents, like maltodextrin, arabic gum, and skim milk, on the viability of Lacticaseibacillus rhamnosus GG (LGG) was investigated. The initial cell concentration, particle size distribution, aspect ratio, sphericity, scanning-electron-microscopy images, moisture content, water activity, glass transition, rehydration abilities, and survival during storage were compared. Skim milk was proven to be the best protectant for LGG, regardless of the drying process or storage time. A huge reduction in cell numbers (4.49 ± 0.06 log CFU/g) was observed with maltodextrin using SD; meanwhile, it was protected with minimum loss (8.64 ± 0.62 log CFU/g) with ESD. In general, ESD preserved more LGG cells during processing compared to SD, and provided better stability than FD and SD during storage, regardless of the applied voltage. The ESD product analysis demonstrated an efficient LGG preservation, close to FD; therefore, ESD presented to be a promising and scalable substitute for SD and FD.

Phase Diagram of Pickering Emulsions Stabilized by Cellulose Nanocrystals.

Cellulose is a promising renewable and biocompatible biopolymer for stabilizing Pickering emulsions (PEs). In the present study, PEs were produced by low-frequency ultrasounds with cellulose nanocrystals (CNCs) and caprylic/capric triglycerides. Phase diagrams allowed to understand mechanisms of formation and long-term stabilization of PEs. Emulsion type, continuous phase viscosity, and yield of oil incorporation were studied after PEs formation. Droplet size, oil release, and stability were measured weekly up to 56 days of storage. Results showed that oil mass fraction above 70% w/w led to unstable W/O PEs. Lower oil mass fraction formed O/W PEs of stability depending on CNC content and oil mass fraction. Droplet size stability increased with CNCs/oil ratio. A very low CNCs/oil ratio led to phase separation and oil release. High CNC content stabilized oil droplets surface, increased aqueous phase viscosity, and prevented creaming. Highly stable PEs were produced for CNC content above 3% (w/w) and oil mass fraction below 50% (w/w). Mechanisms for PEs formation and stabilization were proposed for various CNC contents and oil mass fractions.

Development of Chitosan Green Composites Reinforced with Hemp Fibers: Study of Mechanical and Barrier Properties for Packaging Application.

The use of bioresourced packaging materials is an interesting solution for ecological issues. This work aimed to develop novel chitosan-based packaging materials reinforced with hemp fibers (HF). For this purpose, chitosan (CH) films were filled with 15%, 30%, and 50% (w/w) of two kinds of HF: Untreated fibers cut to 1 mm (UHF) and steam exploded fibers (SEHF). The effect of HF addition and HF treatments on chitosan composites was studied in terms of mechanical properties (tensile strength (TS), elongation at break (EB), and Young's modulus (YM)), barrier properties (water vapor (WVP) and oxygen permeabilities), and thermal properties (glass transition (Tg) and melting temperatures (Tm)). The addition of HF, whether untreated or steam exploded, increased the TS of chitosan composites by 34-65%. WVP was significantly reduced by the addition of HF but no significant change was observed for O2 barrier property, which was in the range between 0.44 and 0.68 cm3·mm/m2·d. Tm of the composite films increased from 133 °C for CH films to 171 °C for films filled with 15% SEHF. However, no significant modification was observed for Tg (105-107 °C). The present study showed that the developed biocomposites had improved properties, mainly the mechanical resistance. Their use in food packaging will help industrials the move toward a sustainable development and circular economy.

Chitosan-Based Particulate Carriers: Structure, Production and Corresponding Controlled Release.

The state of the art in the use of chitosan (CS) for preparing particulate carriers for drug delivery applications is reviewed. After evidencing the scientific and commercial potentials of CS, the links between targeted controlled activity, the preparation process and the kinetics of release are detailed, focusing on two types of particulate carriers: matrix particles and capsules. More precisely, the relationship between the size/structure of CS-based particles as multifunctional delivery systems and drug release kinetics (models) is emphasized. The preparation method and conditions greatly influence particle structure and size, which affect release properties. Various techniques available for characterizing particle structural properties and size distribution are reviewed. CS particulate carriers with different structures can achieve various release patterns, including zero-order, multi-pulsed, and pulse-triggered. Mathematical models have an unavoidable role in understanding release mechanisms and their interrelationships. Moreover, models help identify the key structural characteristics, thus saving experimental time. Furthermore, by investigating the close relation between preparation process parameters and particulate structural characteristics as well as their effect on release properties, a novel "on-demand" strategy for the design of drug delivery devices may be developed. This reverse strategy involves designing the production process and the related particles' structure based on the targeted release pattern.

Alginate Based Core-Shell Capsules Production through Coextrusion Methods: Recent Applications.

Encapsulation is used in various industries to protect active molecules and control the release of the encapsulated materials. One of the structures that can be obtained using coextrusion encapsulation methods is the core-shell capsule. This review focuses on coextrusion encapsulation applications for the preservation of oils and essential oils, probiotics, and other bioactives. This technology isolates actives from the external environment, enhances their stability, and allows their controlled release. Coextrusion offers a valuable means of preserving active molecules by reducing oxidation processes, limiting the evaporation of volatile compounds, isolating some nutrients or drugs with undesired taste, or stabilizing probiotics to increase their shelf life. Being environmentally friendly, coextrusion offers significant application opportunities for the pharmaceutical, food, and agriculture sectors.

Alginate Core-Shell Capsules Production through Coextrusion Methods: Principles and Technologies.

This paper provides an overview of coextrusion methods for encapsulation. Encapsulation involves the coating or entrapment of a core material such as food ingredients, enzymes, cells, or bioactives. Encapsulation can help compounds add to other matrices, stabilize compounds during storage, or enable controlled delivery. This review explores the principal l coextrusion methods available that can be used to produce core-shell capsules through the use of coaxial nozzles. Four methods for encapsulation by coextrusion are examined in detail, including dripping, jet cutting, centrifugal, and electrohydrodynamic systems. The targeted capsule size determines the appropriate parameters for each method. Coextrusion technology is a promising encapsulation technique able to generate core-shell capsules in a controlled manner, which can be applied to cosmetic, food, pharmaceutical, agriculture, and textile industries. Coextrusion is an excellent way to preserve active molecules and present a significant economic interest.

Influence of fatty acid-esterified waxy maize starch type and concentration on stability and properties of oil-in-water emulsions.

In the current study, native and different fatty acid-esterified waxy maize starches (octanoate, myristoate, and stearoate), followed by an OSA-potato starch (as an industrial emulsifier) were used to prepare sunflower oil-in-water (O/W) emulsion. The effect of emulsifier type and concentration were evaluated on properties of emulsions in terms of mean droplet size, droplet size distribution, and creaming index. To prepare the emulsion, the emulsifier to oil ratios of 1.25 and 0.5 for octanoate and industrial emulsifier (control) were considered as the selected formulations based on the lowest creaming index (2.63 and 0 %, respectively). The influence of various pHs and ionic strengths on droplet size, span and zeta potential value was similar for both produced emulsions. Therefore, the fatty acid-esterified starch could be suggested as a promising environmentally friendly alternative to industrial emulsifiers for fabrication of emulsions with similar stability.

Study and optimization of oil-in-water emulsions formulated by low- and high-frequency ultrasounds.

OBJECTIVE: A combined treatment using both low-frequency (20 kHz) and high-frequency ultrasounds (1.63 MHz) is a promising new process to stabilize emulsions with minimalist formulation. In order to optimize process parameters, a Doehlert experimental design was performed with oil-in-water emulsions, presently used for cosmetic products, composed of water, caprylic/capric triglycerides and oleic acid. METHODS: Effects of treatment time, oil content and oleic acid content were studied on emulsion properties (droplet size, polydispersity index, ζ-potential and yield of oil incorporation) and on emulsion stability after a 28-day storage (creaming index, Turbiscan stability index (TSI) and oil release). RESULTS: From experimental data, a model was established that allowed to study effects of each parameter and their interactions on emulsion formation and stability. Oleic acid content had a great impact on emulsion formation: It reduced droplet size, PDI and ζ-potential and increased yield of oil incorporation. However, a critical value could be highlighted, beyond which oleic acid effects reversed. Treatment time had an important beneficial effect on emulsion stability as it decreased creaming index, TSI and oil release after 28 days of storage. Oil content had a negative effect on emulsion formation and on emulsion stability. However, treatment time and oil content often had a beneficial synergistic effect. CONCLUSION: The optimized conditions for emulsion processing were obtained through a desirability approach. They were experimentally validated.

OBJECTIF: Un traitement combiné utilisant à la fois des ultrasons à basse fréquence(20 kHz) et à haute fréquence (1,63 MHz) est un nouveau procédé prometteur pour stabiliser les émulsions avec une formulation minimaliste. Afin d'optimiser les paramètres du procédé, un plan d'expériences de Doehlert a été réalisé avec des émulsions huile-dans- eau, actuellement utilisées pour des produits cosmétiques, composées d'eau, de triglycérides capryliques/capriques et d'acide oléique. METHODES: Les effets du temps de traitement, de la teneur en huile et de la teneur en acide oléique ont été étudiés sur les propriétés des émulsions (taille des gouttelettes, indice de polydispersité, potentiel-ζ et rendement d'incorporation de l'huile) et sur la stabilité des émulsions après un stockage de 28 jours (indice de crémage, indice de stabilité au Turbiscan (TSI) et relargage de l'huile). RESULTATS: A partir des données expérimentales, un modèle a été établi et a permis d'étudier les effets de chaque paramètre et leurs interactions sur la formation et la stabilité des émulsions. La teneur en acide oléique a eu un impact important sur la formation des émulsions : elle a réduit la taille des gouttelettes, le PDI et le potentiel-ζ, et a augmenté le rendement d'incorporation de l'huile. Cependant, une valeur critique a pu être mise en évidence, au-delà de laquelle les effets de l'acide oléique s'inversent. Le temps de traitement a eu un effet bénéfique important sur la stabilité des émulsions car il a diminué l'indice de crémage, le TSI et le relargage d'huile après 28 jours de stockage. La teneur en huile a eu un effet négatif sur la formation des émulsions et sur leur stabilité. Cependant, le temps de traitement et la teneur en huile ont souvent eu un effet synergique bénéfique. CONCLUSION: Les conditions optimisées pour le traitement des émulsions ont été obtenues par une approche de désirabilité. Elles ont été validées expérimentalement.

Modifying the Stability and Surface Characteristic of Anthocyanin Compounds Incorporated in the Nanoliposome by Chitosan Biopolymer.

In this study, a novel approach was investigated to improve the stability of anthocyanin compounds (AC) by encapsulating them in nanoliposomes resulting from rapeseed lecithin alongside chitosan coating. The results indicate that the particle size, electrophoretic mobility, encapsulation efficiency, and membrane fluidity of nanoliposomes containing anthocyanin compounds were 132.41 nm, -3.26 µm·cm/V·S, 42.57%, and 3.41, respectively, which changed into 188.95 nm, +4.80 µm·cm/V·S, 61.15%, and 2.39 after coating with chitosan, respectively. The results also suggest improved physical and chemical stability of nanoliposomes after coating with chitosan. TEM images demonstrate the produced particles were spherical and had a nanoscale, where the existence of a chitosan layer around the nanoparticles was visible. Shear rheological tests illustrate that the flow behavior of nanoliposomes was altered from Newtonian to shear thinning following chitosan incorporation. Further, chitosan diminished the surface area of the hysteresis loop (thixotropic behavior). The oscillatory rheological tests also show the presence of chitosan led to the improved mechanical stability of nanoliposomes. The results of the present study demonstrate that chitosan coating remarkably improved encapsulation efficiency, as well as the physical and mechanical stability of nanoliposomes. Thus, coating AC-nanoliposomes with chitosan is a promising approach for effective loading of AC and enhancing their stability to apply in the pharmaceutic and food industries.

Review of High-Frequency Ultrasounds Emulsification Methods and Oil/Water Interfacial Organization in Absence of any Kind of Stabilizer.

Emulsions are multiphasic systems composed of at least two immiscible phases. Emulsion formulation can be made by numerous processes such as low-frequency ultrasounds, high-pressure homogenization, microfluidization, as well as membrane emulsification. These processes often need emulsifiers' presence to help formulate emulsions and to stabilize them over time. However, certain emulsifiers, especially chemical stabilizers, are less and less desired in products because of their negative environment and health impacts. Thus, to avoid them, promising processes using high-frequency ultrasounds were developed to formulate and stabilize emulsifier-free emulsions. High-frequency ultrasounds are ultrasounds having frequency greater than 100 kHz. Until now, emulsifier-free emulsions' stability is not fully understood. Some authors suppose that stability is obtained through hydroxide ions' organization at the hydrophobic/water interfaces, which have been mainly demonstrated by macroscopic studies. Whereas other authors, using microscopic studies, or simulation studies, suppose that the hydrophobic/water interfaces would be rather stabilized thanks to hydronium ions. These theories are discussed in this review.

Chemical modification of waxy maize starch by esterification with saturated fatty acid chlorides: Synthesis, physicochemical and emulsifying properties.

In the current study, the physicochemical and emulsifying properties of modified waxy maize starch obtained through a new environmentally friendly method of esterification were evaluated. The starch modification was carried out in NaOH solution with different levels of octanoyl, myristoyl, and stearoyl chlorides. Increasing the fatty acid chlorides concentration led to the degree of substitution increment, while reaction efficiency and yield decreased. Based on fourier transform infrared spectroscopy results, the presence of two new bands of carbonyl (1740-1750 cm-1) and carboxyl (1570 cm-1) groups in the ester bond confirmed the successful starch esterification process. The level of 0.1 mL fatty acid chlorides/g of starch demonstrated the highest emulsifying properties. Upon esterification, the crystalline structure of amylopectin was destroyed, indicating no gelatinization features. Therefore, using the fatty acid chlorides in an alkaline condition could be suggested as a feasible way to modify waxy maize starch toward hydrophobicity increment with desirable properties.

Phytochemical Composition, Antimicrobial, Anticancer Properties, and Antioxidant Potential of Green Husk from Several Walnut Varieties (Juglans regia L.).

Husk powder was prepared from seven varieties of walnut fruit and their hulling rate, chemical compounds, and total phenolic contents were evaluated. The apolar and polar extracts were prepared, respectively, from hexane and a hydroethanolic solvent, while qualitative and semi-quantitative analyses were performed by GC/MS and UHPLC-PDA-HRMS/MS. The antioxidant, antimicrobial, and antitumor properties of green walnut husk were also evaluated. The total content of phenolic compounds varied between the varieties, ranging from 35.2 ± 0.9 to 58.0 ± 0.0 mg/g gallic acid equivalent of dry husk weight (dw). The apolar extract was found to contain alkanes, tocopherols, sterols, and fatty acids, including oleic, linoleic, and linolenic, while the polar extract showed the presence of phenolics including salicylate glucuronide, taxifolin, catechin, and quercetin isomers. The antioxidant power obtained by the PAOT (total antioxidant power) method for the husk powders ranged from 256.5 ± 5.9 to 746.8 ± 6.9 score/g dw, and seemed consistent with the total phenolic content and the results obtained by the classic antioxidant test with DPPH. The walnut husk also showed an antibacterial effect against Gram-negative and Gram-positive bacteria and cytotoxic potential against HepG2. Among the selected varieties, the green Saman had the highest antioxidant properties, while the Saman with a brown color had the lowest.

Encapsulation of Berberis vulgaris Anthocyanins into Nanoliposome Composed of Rapeseed Lecithin: A Comprehensive Study on Physicochemical Characteristics and Biocompatibility.

In the present study, nanoliposomes composed of rapeseed lecithin were used for the encapsulation of anthocyanin compounds (AC). The nanoliposomes were prepared using hydration and ultrasound combined method, and the effect of AC concentration (4.5, 6.75, 9% w/w) on the characteristics of nanoliposomes including particle size, polydispersity index (PDI), zeta potential, and the encapsulation efficiency (EE) of nanoliposomes with and without AC were studied. The results suggested the fabricated nanoliposomes had a size range of 141-196 nm, negative zeta potential and narrow particle size distribution. Further, the samples containing 9% extract had the maximum EE (43%). The results showed elevation of AC concentration resulted in increased particle size, PDI, EE, and surface charge of nanoparticles. The presence of AC extract led to diminished membrane fluidity through the hydrophobic interactions with the hydrocarbon chain of fatty acids. TEM images suggested that the nanoliposomes were nearly spherical and the AC caused their improved sphericity. Further, in vitro biocompatibility tests for human mesenchymal (MSC) and fibroblast (FBL) cells indicated nanoparticles were not toxic. Specifically, the best formulations with the maximum compatibility and bioavailability for MSC and FBL cells were AC-loaded nanoliposomes with concentrations of 0.5 mL/mg and 10.3 mL/µg and, respectively.

Metabolomics approach based on LC-HRMS for the fast screening of iron(II)-chelating peptides in protein hydrolysates.

Production of iron-chelating peptides from protein hydrolysates requires robust and adequate screening methods to optimize their purification and subsequently valorize their potential antioxidant properties. An original methodology was developed for direct and sensitive screening of iron(II)-chelating peptides based on ion-pair reverse phase liquid chromatography (IP-RPLC) coupled to high-resolution mass spectrometry (HRMS). Peptide mixture was first added to iron(II) solution to form iron(II)-peptide complexes. Then IP-RPLC-HRMS analysis was conducted on this iron-peptide mixture and on the iron-free peptide solution for comparative mass spectra analysis. This protocol, initially applied to a range of low molecular weight standard peptides, allowed detection of [(Peptide-H)+56FeII]+ complex ion for iron(II)-chelating peptides (GGH, EAH, DAH, ßAH, DMH, DTH, DSH). GGH was added in complex peptide mixtures and targeted analysis of [(GGH-H)+56FeII]+ complex showed a limit of detection (LOD) below 0.77 mg L-1 of GGH. This protocol was finally tested in combination with metabolomics software and additional digital processing for non-targeted search for iron(II)-chelating peptides. Applicability of this new screening methodology has been validated by detection of GGH as iron(II)-chelating peptide when added at 0.77 mg L-1 in casein hydrolysate. Graphical abstract.

Interest of Pickering Emulsions for Sustainable Micro/Nanocellulose in Food and Cosmetic Applications.

In the present review, natural and non-toxic particles made of micro/nanocellulose were specifically targeted as stabilizers of emulsions located at dispersed and continuous phases interfaces (called Pickering Emulsions, PEs). PEs are biphasic systems stabilized by solid particles with a recent interest in food and cosmetic domains. PEs have been more and more studied in the last ten years due to their advantages compared to conventional emulsions with surfactants. PEs have already been stabilized with various types of particles and particularly cellulose. Even if some studies showed that PEs were more stable when cellulose was chemically modified, numerous other recent studies showed that unmodified micro/nanocellulose is also promising biomaterial to stabilize PEs. Micro/nanocelluloses can be extracted by various green processes from numerous agricultural wastes and co-products, as banana peels, corncob, ginkgo seed shells, lime residues, mangosteen rind, oil palm empty fruit bunches, pistachio shells, as well as wheat straw. Main green processes used to treat cellulose are grinding, high pressure homogenization, microfluidization, enzymatic hydrolysis, subcritical water, extrusion, electron beam irradiation, cryocrushing, microwaves or sonication. PEs formulated with cellulose clearly participate to a global sustainable development but, additional studies will be necessary to better understand PEs stability and improve properties.

Elaboration of hemicellulose-based films: Impact of the extraction process from spruce wood on the film properties.

In this work, steam explosion (STEX), microwave assisted extraction (MAE) and high voltage electrical discharges (HVED) pretreatments have been evaluated for their impact on the physicochemical characteristics of extracted hemicellulosic polymers and on the resulting hemicellulose-based films. Extraction was carried out on spruce sawdust pre-soaked in water (WPS) or 1 M NaOH solution (SPS). The results have shown that STEX pretreatment gave the highest hemicellulose yields (64 and 66 mg g-1 of dry wood from WPS and SPS respectively) followed by MAE and HVED whilst MAE pretreatment produced the highest molecular mass (Mw~66 kDa of arabinoglucoronoxylans from SPS and 56 kDa for galactoglucomannans from WPS). A relatively high acetylation degree was found for STEX WPS (acetylation degree ≈ 0.35) and a high lignin content for STEX SPS (≈12%). Films have been produced by casting using sorbitol as plasticizer. Low oxygen barrier and light transmittance properties were observed for the films obtained from hemicelluloses extracted from SPS due to their high molecular mass and to intermolecular bonding of hemicelluloses and lignin.

Bioactive Films Containing Alginate-Pectin Composite Microbeads with Lactococcus lactis subsp. lactis: Physicochemical Characterization and Antilisterial Activity.

Novel bioactive films were developed from the incorporation of Lactococcus lactis into polysaccharide films. Two different biopolymers were tested: cellulose derivative (hydroxylpropylmethylcellulose (HPMC)) and corn starch. Lactic acid bacteria (LAB) free or previously encapsulated in alginate-pectin composite hydrogel microbeads were added directly to the film forming solution and films were obtained by casting. In order to study the impact of the incorporation of the protective culture into the biopolymer matrix, the water vapour permeability, oxygen permeability, optical and mechanical properties of the dry films were evaluated. Furthermore, the antimicrobial effect of bioactive films against Listeria monocytogenes was studied in synthetic medium. Results showed that the addition of LAB or alginate-pectin microbeads modified slightly films optical properties. In comparison with HPMC films, starch matrix proves to be more sensitive to the addition of bacterial cells or beads. Indeed, mechanical resistance of corn starch films was lower but barrier properties were improved, certainly related to the possible establishment of interactions between alginate-pectin beads and starch. HPMC and starch films containing encapsulated bioactive culture showed a complete inhibition of listerial growth during the first five days of storage at 5 °C and a reduction of 5 logs after 12 days.

Fabrication of whey proteins aggregates by controlled heat treatment and pH: Factors affecting aggregate size.

Protein aggregates can be formed by heating globular proteins above the thermal denaturation of the proteins. These aggregates can be used as delivery systems or to modulate the physicochemical and sensory properties of food products. In this study, the effects of heat treatment (15 min at 80 °C), pH adjustment (between 4 and 9), aging at different temperatures (between 10 and 60 °C) and the rate of acidification (pH decreased from 6.25 to 4 at 5 different rates) on whey proteins aggregates were studied. The results indicated that these treatments led to creation of aggregates with different sizes (0.72-5.1 µm). Heat treatment and reducing the pH down to nearly 4 led to an increase in the size of aggregates. At pH < 7, by increasing the aging temperature, the size of aggregates increased (without heat treatment). In the heat treated samples with pH < 9, increasing the aging temperature led to an increase in the size of aggregates. Also larger aggregates were formed by increasing acidification rate. The amount of insoluble whey proteins aggregates increased as a result of applying heat treatment, decreasing pH and increasing aging temperature. The results of this study can be used for fabrication of whey proteins aggregates with specific size and functionality.