Nanostructured food proteins as efficient systems for the encapsulation of bioactive compounds

Mehdi Mohmmdin,Mostf I.Wly,Mrym Moghdm,Zhr Emm-Djomeh,*,Mrym Slmi,Ali Akr Moosvi-Movhedi

a Department of Food Science and Engineering,University College of Agriculture&Natural Resources,University of Tehran,Karaj,Iran

b Department of Food Science and Nutrition,College of Agricultural and Marine Sciences,Sultan Qaboos University,Muscat,Oman

c Institute of Biochemistry and Biophysics,University of Tehran,Tehran,Iran

ABSTRACT Recently,nanoencapsulation was introduced as an efficient and promising approach for the protection,delivery, and site-specific liberation of the nutraceuticals and bioactive ingredients.Food proteins are attractive materials for developing nanocarriers to protect and deliver bioactives due to their unique functional and biological properties.Food proteins extracted from animals and plants have the ability to form different nanostructures including nanoparticles,hollow particles,nanogels,nanofibrillar aggregates, electrospun nanofibers, nanotubular structures, and nanocomplexes.These nanostructured food proteins have been widely used as nanocarriers for the biologically active compounds and drugs.The release of bioactive compounds from nanocarriers depends mainly on pH as well as swelling and the degradation behavior of nanostructure in the simulated physiological conditions.This review presents the applications of the nanostructured food proteins for the encapsulation of bioactive compounds.The major techniques for the fabrication of nanocarriers are described.The encapsulation, protection, and release of bioactive compounds in different nanostructured food proteins were also discussed.

Keywords:Food proteins Nanocarriers Bioactive compounds Nanoencapsulation Release properties

1.Introduction

Nowadays, there is an increasing demand of consumers in the field of functional foods supplemented with biologically active compounds with potential health-promoting properties due to the growth of consumer’s awareness about the relationship between nutrition and health [1,2].However, the direct incorporation of bioactive compounds and nutraceuticals into the food products is not simply possible based on their low solubility in some cases and also may cause defects in the quality of the products such as negative effects on color,texture,flavor,and appearance[3].Moreover,most of the bioactive compounds lose their inherent functionality during food processing operations and storage or degrade by digestive enzymes and unfavorable environmental conditions like oxygen, light, and temperature [4,5].Different technologies have been used to protect bioactive molecules from the external environment or deliver them to the suitable target.Accordingly,nanoencapsulation was introduced as an efficient and promising approach for the protection, delivery, and site-specific liberation of the multi-functional nutraceuticals[2].The nanocarrier systems have been quickly and popularly developed and attracted a lot of attention owing to their special advantages such as improving the bioavailability and solubility of bioactive materials as well as enhancing their residence time in the gastrointestinal tract, ability to enter and permeate into living tissues and cells due to their nanoscale sizes,and higher stability in the harsh conditions of the gastrointestinal tract regions[4,6].

Fig.1.Different food protein-based nanocarriers.

Table 1 Examples of food protein-based nanocarries for delivery of bioactive compounds.

Fig.2.Fabrication of protein nanoparticles by desolvation method.Adapted with permission from Tarhini et al.[13].

Different types of food biopolymers including polysaccharides, lipids, proteins, and their conjugates have been used to develop a wide range of nanocarriers for the protection,entrapment, encapsulation, and controlled delivery of bioactive compounds and nutraceuticals [5].Lipid-based carriers can be classified as nanoemulsions, solid lipid nanoparticles, nanostructured lipid carriers,nanoliposomes,micelles,and nanosuspensions.Polysaccharide-based nanocarriers also include polymer nanoparticles, polymeric micelles, and inclusion complexes [4].As displayed in Fig.1, food proteins-originated nanocarriers also can be divided to different structures including nanoparticles, hollow nanoparticles, nano-hydrogels, heat-induced nanofibrillar aggregates, electrospun nanofibers, and tubular nanostructures [7,8].Moreover, most of the proteins, even in their native state, have a great potential to act as nanocarriers for bioactive molecules through the formation of nanocomplexes.These nanostructured food proteins are interestingly and particularly welcomed from different sectors owing to the unique and special functional and biological properties of food proteins such as ability to form different structures, amphiphilic nature, surface activity,biodegradability, biocompatibility, abundant renewable sources,high nutritional value, antioxidant activity, and excellent technological functionalities like emulsification, film-forming ability,foaming,and gelation[5,9,10].Furthermore,the presence of different functional groups on the surfaces of protein molecules enables them to be modified easily which allows food proteins to efficiently interact with different bioactive ingredients, drugs, and nutraceuticals [8,10].It is well-known that the hydrolysis of food proteins by digestive enzymes results in the formation of biologically active peptides with different physiological properties such as antioxidant,antidiabetic,antihypertensive,anticancer,and antimicrobial activities[11].According to these superiorities,food proteins derived from animals and plants such as whey proteins,soy proteins, egg proteins, corn proteins, gelatin, bovine serum albumin (BSA), and many others have been successfully used to fabricate nanocarriers for the delivery of both hydrophobic and hydrophilic biologically active ingredients[8].In this regard,a brief overview of recent studies on the food protein-based nanocarriers with the aim of loading and delivering of bioactive molecules and drugs is presented in Table 1.

With respect to the outstanding functional and nutritional properties of food proteins, in the present study, we focused on the food protein-based nanocarriers which used to encapsulate and deliver bioactive materials and nutraceuticals.Accordingly, their fabrication methods, characteristics, and release properties were reviewed thoroughly.

2.Protein-based nanocarriers

2.1.Nanoparticles

The biopolymer-based nanoparticulate systems have attracted substantial attention as delivery vehicles of bioactive molecules[12].Among different types of biopolymers with the ability to form nanoparticles, food proteins are of paramount importance owing to their unique advantages such as non-toxicity and biodegradability [13].Different edible proteins derived from animal and plant sources such as intact whey protein isolate(WPI)[2,3,14,15],whey protein hydrolysates[16],BSA[17-19],soy proteins[20-22],egg albumin [23], and zein [24,25] have been used to fabricate nanoparticles.The resulting food protein-based nanoparticles were employed as nanocarriers for a wide range of bioactive compounds and drugs such as curcumin [18,20,23], caffeine [2], vitamins[14,21], gallic acid [16], lycopene [15], quercetin [17], resveratrol[24],and doxorubicin[22,25].

Different techniques including desolvation,coacervation,emulsification, nanoprecipitation, nano spray drying, self-assembly,electrospraying, salting out, and cross-linking have been used to produce nanoparticles made of food proteins[13,26].Choosing the method for the preparation of nanoparticles depends on different aspects such as the amino acid constituent and the physicochemical properties of the protein employing as a nanocarrier,as well as the characteristics of the bioactive molecules that to be nanocapsulated [13].The mechanism of the desolvation technique as a common method using for the preparation of food protein-based nanoparticles is shown in Fig.2.This technique is a rapid and simple method without the involvement of destroying factors like heating and high shear rate[3].Briefly,desolvation method is the addition of a desolvating agent such as acetone or alcohol drop by drop into an aqueous solution of protein under stirring condition leading to the dehydration of the protein and the change in its conformation from stretched to coil[13].Finally,the resulting nanoparticles can be rigidized and stabilized through the cross-linking by different agents such as citric acid, glutaraldehyde, genipin, and transglutaminase [9].The properties of nanoparticles produced by this method such as particle size and surface attributes depend on different factors such as the initial protein concentration, agitation speed, type of the desolvating agent and its addition rate, type of the cross-linking agent,pH,and temperature[18].Different possible mechanisms have been reported for the release of cargo from the protein nanoparticles including polymer degradation or erosion, liberation from the polymer surface, or release due to the application of a sonic or an oscillatory magnetic field[26].

Fig.3.Schematic representation of hollow zein nanoparticles preparation procedure.Adapted with permission from Xu et al.[31].

The effect of different desolvating agents(ethanol and acetone)on the nanoparticles of BSA was studied and the resulting particles were used to encapsulate curcumin as a natural bioactive compound [18].Using ethanol, as a solvent, resulted in the formation of the smallest and most spherical nanoparticles, while applying acetone as a desolvating agent led to the generation of a mixture of spherical and rod shaped particles.Moreover, the curcumin encapsulation efficiency of nanoparticles prepared by ethanol was lower than those formed by acetone due to the higher solubility of curcumin in ethanol which prevents its loading in the protein particles.The release of curcumin from the nanoparticles was also governed by different mechanisms including diffusion and the polymer degradation.These authors claimed that the nanoparticles made of BSA have a high ability to control the release of curcumin in the physiological conditions.The BSA nanoparticles were also used as carriers for the quercetin as a flavonoid with strong antioxidant activity[17].The results showed that the association of protein and quercetin was mainly accrued through the hydrophobic interaction and hydrogen bonding which resulted in a promotion of the quercetin stability.In addition to the stabilizing effect,the antioxidant capacity of quercetin was also preserved through the encapsulation in the protein nanoparticles.Egg albumin nanoparticles generated by acetone as a desolvating agent was used to encapsulate curcumin.The optimum condition for the preparation of nanoparticles with high encapsulation efficiency(55.23%)and curcumin loading(4.125%)was included the protein concentration of 8.85%m/V and pH value of 5.0.The particle yield and size at these conditions were 72.85%and 232.6 nm,respectively[23].

Whey proteins were also used widely as proteins with excellent techno-functional properties and high nutritional value for the preparation of nanoparticles aimed to capsulate the bioactive ingredients.In this regard, Abbasi et al.[14] employed WPI nanoparticles for the encapsulation of vitamin D3.They reported that the nanoencapsulation within nanoparticles improved the stability of vitamin D3and the vitamin-loaded whey protein nanoparticles can be used in food drinks and beverages as enriching agents.Nourbakhsh et al.[16] also used potentially bioactive peptides produced by the enzymatic hydrolysis of WPI to fabricate nanoparticles through the microemulsification-cold gelation.The resulting nanoparticles were used as nanocarriers for gallic acid.According to the results of release experiments,these authors suggested that the whey peptide-based nanoparticles can be employed as systems for ileum and/or colon delivery purposes.WPI nanoparticles were also used to enhance the bioavailability of lycopene as a potential antioxidant, anti-carcinogenic and antiatherogenic carotenoid[15].

In addition to the animal proteins, plant-originated proteins were also employed to fabricate nanoparticulate systems.Teng et al.[20] produced soy protein nanoparticles using a desolvation method and used the resulting nanoparticles as encapsulation systems for curcumin.The curcumin-loaded particles showed a size of 220.1-286.7 nm and their highest encapsulation efficiency was 97.2%.The release properties of nanoparticles were also characterized at pH of 7.5 and the results showed a biphasic pattern(primary burst release followed by a sustained release behavior)for the release of curcumin from the nanoparticles.These features made soy protein-based nanoparticles a promising vehicle for bioactive molecules or drugs [15].Soy protein nanoparticles formed by a cold-gelation method were also employed to load vitamin B12to improve its intestinal transport and absorption [21].The results showed that the intestinal transport of vitamin B12was increased up to 4-fold after being nanocapsulated into soy protein nanoparticles with a size of 30 nm attributed to the taking up of the nanoparticles by different pathways including clathrin-mediated endocytosis and micropinocytosis.Soy protein nanoparticle was also used as a nanocarrier for doxorubicin as an anticancer drug[22].The biodegradable zein nanoparticles prepared by phase separation technique were also employed to load doxorubicin [25].These nanoparticles showed a pH-dependent release behavior;lower release was observed at pH value of 7.4 compared to the pH values of 5 and 6.5.This property of zein nanoparticles can be used to enhance the maintenance of drug molecules within the nanoparticle during blood circulation and also can decrease its cytotoxicity against the normal cells.

2.2.Hollow nanoparticles

Hollow nano/microparticles as novel classes of delivery systems are architectures containing interior void space which have some distinct advantages compared to the solid counterparts including higher encapsulating capacity and efficiency,lower density,better heat insulation, and larger specific surface area [27].These striking features boosted their applications in different research fields especially in nutraceutical delivery and bio-encapsulation.Hollow particles are usually prepared using sacrificing templates which are removed to create empty spaces inside of the particles [28].Generally, the synthesis procedure of biopolymeric hollow particles involves the following steps: (1) preparation of template which can be a soft or hard one, (2) coating of template surface with the biopolymeric shell through different approaches such as layer-by-layer assembly and chemical adsorption,and(3)selective removal of the template[27,28].Different food biopolymers including polysaccharides,lipids,and proteins have been used to fabricate hollow micro-and nanoparticles.Among the various food proteins,zein [6,29-31], casein [32], and collagen [33] were employed to design hollow nanoparticles and hollow spheres.

In a study conducted by Xu et al.[30], zein hollow nanoparticles with average diameter of 65 nm were prepared using sodium carbonate crystals as sacrificial templates (Fig.3).The resulting hollow particles were used as a carrier for metformin (an antidiabetic drug) and compared with the solid zein nanoparticles in terms of loading capacity and release properties.The loading capacity of hollow zein nanoparticles was significantly higher than solid particles attributing to their larger surface area and also the formation of cavities in the outer shell of hollow particles during the removal of templates.Moreover, these authors reported a more controlled and sustained drug release for hollow particles in comparison with solid counterparts which makes them appropriate vehicles for carrying bioactive molecules and drugs.In addition, above-mentioned hollow bodies showed a high ability to enter the fibroblast cells which boosts their application for delivery of protein and peptide drugs which could not passively enter into the target cells.The citric acid cross-linked zein hollow nanoparticles were employed to encapsulate 5-fluorouracil,an anti-cancer drug [6].With respect to the toxicity of this anticancer drug for healthy organs and cells and also its poor water stability and high susceptibility to enzymatic degradation,zein hollow particles were used to improve its stability against enzymatic degradation as well as to reduce its systematic toxicity.The cargo loading capacity of solid zein particles was tremendously lower than the hollow particles.The hollow particles cross-linked by citric acid also showed higher water stability in physiological conditions compared to the non-cross-linked hollow particles.The results of this study revealed that the chemically cross-linked zein protein hollow particles can be suitably used as a vehicle for carrying of anti-cancer therapeutics in a controllable manner and prolonged time.Tannic acid cross-linked zein hollow nanoparticles with a small dimension of 87.93 nm were also developed as potential oral delivery system for curcumin, a hydrophobic bioactive substance with a wide range of health-promoting properties[29].Hollow particles possessed higher curcumin encapsulation efficiency as well as better re-dispersibility after freeze-drying compared to the solid nanoparticles.Tannic acid-mediated cross-linking also enhanced the stability of zein hollow particles in the simulated gastrointestinal conditions.Moreover, the authors reported a controlled curcumin release at pH 4.0 and 0.7 for hollow zein nanoparticles which enables them to be employed as ideal carriers for hydrophobic drugs and bioactive compounds.Zein-based hollow nanoparticles with diameters less than 100 nm and high biodegradability were also successfully employed for removal of reactive dyes from the wastewater[31].Accordingly,the adsorption capacity of hollow zein particles for Reactive Blue 19 was much higher than the solid ones.So,it was concluded that the biodegradable zein hollow structures have the capacity to use as a highly efficient agent for the removal of dyes from the industrial effluents especially the textile dyeing industries which their wastewaters are significant sources of environmental pollution.

In addition to the plant-based proteins, animal proteins have been also used to fabricate hollow nanostructures.Kraskiewicz et al.[33] used collagen to produce hollow spheres using polystyrene beads as templates.The resulting hollow reservoirs were employed for sustained delivery of neurotrophins which belong to a family of proteins that induce survival, development,and function of neurons.The drug loading capacity and efficiency of these hollow structures were 10 μg of payload per mg of collagen and 90%-99%, respectively.Studying in cells and no-cells systems showed a slow therapeutic release kinetic (over a period of 13 days)for collagen hollow spheres.Moreover,the authors indicated that the bioactivity of encapsulated therapeutic was not influenced by loading into the collagen-based hollow spheres.Generally, the results proposed that the collagen-based hollow spheres containing a nerve growth factor can be considered as an efficient delivery system for multiple neuro-therapeutic applications.Milk caseinbased hollow spheres were also successfully fabricated by Liu et al.[32].These food protein-based hollow structures were introduced as promising delivery systems for gens and drugs with high biocompatibility and extraordinary cell-penetrating ability.

2.3.Nano-hydrogels

The nano-hydrogels or nanogels are nano-sized threedimensional hydrogel particles made of cross-linked polymeric networks with a high ability to retain water or biological fluids as well as high swelling ratio [34,35].Nanogels can be fabricated by different natural and synthetic polymers such as edible proteins.Some striking properties such as high loading capacity due to having enough internal space for entrapping guest materials,ability to permeate into living tissues, high capacity for multivalent bio-conjugation,high biological affinity,and biocompatibility were reported for edible protein-based nanogels which triggered the scholars to use them in designing delivery systems for nutraceuticals and bioactive compounds which can be used in food and drug formulations [5,34-36].Furthermore, naturally accruing biopolymers like food proteins are able to be functionalized due to having many functional groups.This superiority compared to the synthetic polymers, makes food proteins as promising candidates for designing nanogels with the specific application and tailorable functionalities [37].Generally, preparation approaches of protein nanogels are based on the self-assembly of proteins through the physical (amphiphilic- and electrostatic-association) or chemical(covalent)interactions[34].

Different food proteins such as soy proteins[37-39],casein[40],ovalbumin[41],lysozyme[42-44],lactoferrin-glycomacropeptide[45-47],BSA[48],and gelatin[36,49]were employed to fabricate nano-hydrogels.The resulting protein-based nanogels were used as encapsulation bodies for guest bioactive molecules and drugs like riboflavin [37], curcumin [39,45], caffeine [45], methotrexate[42,44], and doxorubicin [48,49].Most of these food proteinbased nanogels have been fabricated through the self-assembly approach.This approach is a facile, versatile, and cost-effective green bottom-up technique that the molecules spontaneously form ordered aggregates through non-covalent binding such as electrostatic, hydrogen, and hydrophobic interactions [39,41].The glycated proteins as the amphiphilic copolymers are usually used to produce nanogels by the self-assembly method which the resulting nanogels have a protein core and a polysaccharide sell.This type of nanogels formed by self-assembly method can be used as promising nanocarriers for hydrophobic bioactive molecules in physiological conditions due to their high stability at a broad range of pH and salt.In fact, in the nanogels made of glycated protein formed by self-assembly approach,the protein moiety can be adsorbed onto the hydrophobic surface of nutraceuticals and drugs,whereas the polysaccharide provides strong steric repulsion at the particle exterior which improves the stability of nanogels[41,42].In fact, in the self-assembly method, the protein is firstly conjugated with a polysaccharide(commonly high-molecular mass non-ionic polysaccharides such as dextran) and the resulting protein-polysaccharide complexes are heated at the isoelectric point of protein to form stable nanogels through the heat-gelation process[37,39,41].

Fig.4.Fabrication procedure of nanogels made of β-conglycinin and dextran.Adapted with permission from Feng et al.[39].

The stable and pH-sensitive single component soy proteinbased nanogels were fabricated through the self-assembly of heat denatured (95°C) protein dispersion at pH value of 5.9[38].Hydrophobic interactions and disulfide bonds with a minor contribution of hydrogen bonding were proposed as the main forces involving in the formation of the soy protein-based nano-hydrogels.Feng et al.[39]also fabricated soy β-conglycinindextran nanogels with a hydrodynamic diameter of about 90 nm using a self-assembly technique at the protein isoelectric point(Fig.4).For the preparation of these nanogels, at first stage, the protein was conjugated covalently with dextran through the Maillard reaction at a dry condition to produce amphiphilic graft copolymers.After that, the resulting hybrid system was heated(95°C for 50 min) at the isoelectric point of protein (i.e.pH 4.8)to form nanogels.The resulting nanogels were fairly stable and were introduced as promising systems to deliver of hydrophobic bioactive compounds [39].Accordingly, the self-assembled modified soy protein-dextran nanogels with sizes of 32-40 nm were used as vehicles to encapsulate the riboflavin.The electrostatic interaction between the amino groups of protein and carboxyl groups of riboflavin was proposed as the main driving force for the entrapment of riboflavin within the nanogels.The maximum encapsulation efficiency of riboflavin in these soy protein-dextran nanogels was 65.9%depending on the formulation of the nanogels.The evaluation of riboflavin in vitro release from the nanogels under simulated gastrointestinal tract also showed a notably sustained release behavior for nanogels which in turn can decrease the unwelcome side effects of riboflavin.The drug diffusion and the swelling/erosion of the soy proteins and dextran were reported as the mechanisms responsible for the release of riboflavin from the soy protein/dextran nano-hydrogels[37].

In addition to the soy proteins, egg white proteins including ovalbumin and lysozyme were also successfully used to design nanogels aimed to entrap different biologically active compounds.Lysozyme as a globular protein derived from egg white proteins in combination with sodium carboxymethyl cellulose as a biodegradable polysaccharide with a good biocompatibility and low immunogenicity was employed to fabricate nanogels [43].Nanogels were prepared by a green and simple method without employing any chemical treatment, only with heating the binary biopolymeric solution at the denaturation temperature (95°C) of protein.The resulting hydrogels were loaded with 5-fluorouracil as a model drug which is widely used to treat cancer and their release ability was investigated.The nanogels with smallest size(241 nm)and highest drug loading efficiency (10.16%) were obtained after 60 min heating of biopolymeric solution with a lysozyme to sodium carboxymethyl cellulose weight ratio of 2:1.The results of in vitro release experiments revealed a slight and steady release for the model drug from nanogels in simulated gastric and intestinal fluids.Moreover,a slower drug release was found in simulated gastric condition compared to the intestinal fluid which can warranty the site-specific delivery of 5-fluorouracil to the intestines.Selfassembled lysozyme/carboxymethyl cellulose nanogels were also used as bodies to deliver another anticancer and antitumor agent named methotrexate[42].Their results indicated that the loading of methotrexate in nanogels made of lysozyme and carboxymethyl cellulose increased its bioavailability and anticancer activity.The slow release of drug from the above-mentioned nanogels also can be considered as a unique feature which is very beneficial for chemotherapy applications.The nano-hydrogel made of lysozyme and pectin through self-assembly approach was also efficiently used to encapsulate the methotrexate [44].The loading capacity of lysozyme/pectin nanogels in the case of methotrexate was higher compared to the lysozyme/carboxymethyl cellulose counterparts (17.58% vs 14.2%).The methotrexate which was loaded in lysozyme/pectin nanogels possessed a higher cancer cell apoptosis in comparison with the free methotrexate.The nanogels also showed a pH-triggered release behavior; fast release of drug in mildly acidic environments.In another study which was conducted by Feng et al.[41], ovalbumin-dextran nano-hydrogels prepared by the self-assembly of ovalbumin-dextran Maillard conjugates followed by heat-gelation at the isoelectric point of protein were used to improve the bioavailability of curcumin.According to the results, these authors reported that the curcumin-loaded ovalbumin-dextran stable nanogels can be potentially employed to fortify food products to improve their health-promoting properties.

The milk-derived proteins including lactoferrin, glycomacropeptide,and BSA were also used to fabricate nano-hydrogels with various bioactive attributes such as antioxidant and antimicrobial activity [47,48].In this regard, it was reported that the system consisting of lactoferrin and glycomacropeptide has the ability to form nanogels through a 2-stage procedure involving electrostatic interaction and heat-induced gelation [47].The morphological properties and hydrodynamic diameter of these nano-hydrogels were dependent on different parameters including protein concentration, pH, heating temperature and duration,and molar ratio of proteins.These protein-based nanogels were introduced as a new type of smart carriers for the encapsulation of bioactive compounds and nutraceuticals.In this regard,the nanohydrogels made of lactoferrin and glycomacropeptide were used as carriers for lipophilic(curcumin)and hydrophilic(caffeine)model bioactive compounds [45].The nanogels showed high encapsulation efficiency (more than 90%) for these bioactive compounds.The size of curcumin-loaded nanogels was 112 nm, whereas the caffeine-loaded counterpart had a size of 126 nm.However, both of them showed a spherical shape with a low polydispersity index(0.2).The release behavior of encapsulated materials from the nano-hydrogels was investigated in different pHs(i.e.2.0 and 7.0)and the results indicated a pH-dependent release behavior for the nanogels.At pH 2.0 the release of the cargoes from the nanogels matrices was mainly governed by relaxation phenomenon.At pH value of 7.0 the Fickian diffusion was also the main mechanism for the release of caffeine, whereas the curcumin did not show any release from the lactoferrin-glycomacropeptide nanogels at this pH attributing to its low solubility at physiological pH.The effect of coating with chitosan on the release properties of lactoferringlycomacropeptide nanogels was also studied by Bourbon et al.[46].Accordingly, the protein-based nano-hydrogels were coated with chitosan through a layer-by-layer approach and caffeine was loaded as a model cargo within the resulting nano-hydrogels.The coating of nanogels with chitosan significantly affected the release behavior of caffeine at a pH related to the stomach (i.e.pH value of 2.0) and human body temperature (37°C).In fact,the amount of caffeine release from the chitosan-coated nanogels(27%) was drastically lower than the non-coated counterpart(62%) attributing to the slower moving of cargo through the nanogels matrix in the presence of chitosan which mitigates the burst release phenomenon.The release of curcumin from nanogels was governed by Fickian and relaxation mechanisms.Applying of chitosan coating also increased the contribution of Fickian mechanism in the release of bioactive molecules from the nanogels.Therefore, these coated nanostructures could be appropriately used to control the delivery of biologically active molecules which are susceptible to the harsh condition of the stomach.Nanogels based on BSA and chitosan were also fabricated to entrap doxorubicin hydrochloride as the most effective agent for tumor therapy[48].The results indicated that the drug entrapment ratio of these nanogels was 43.6%.Encapsulation of doxorubicin in BSA/chitosan nanogels reduced its cytotoxicity and the loaded drug also showed a slow and sustained release profile within 24 h.Moreover,according to the results of cellular uptake assays,it was investigated that the drug which was loaded into nano-hydrogels had a faster diffusion into the cancerous cells compared to the bare doxorubicin.Generally,the observations of the above studies proposed that the food protein-based nanogels can be effectively used to deliver drugs and bioactive molecules.

2.4.Nanofibrillar aggregates

Many of globular food proteins are able to form nanofibrillar structures with a nanometric thickness(1-10 nm)and micrometric length (1-10 μm) through the prolonged heating of protein solutions under acidic condition (typically pH approximately 2.0) and low ionic strength.In fact,the heat-induced formation of nanofibrillar aggregates can be considered to be a self-assembly process which the denatured proteins or peptides formed at pH 2.0 are the building unites of the final ordered nanostructures [50,51].Selfassembling of food proteins to fibrillar aggregates has attracted a lot of interests from different research areas such as food science,medicine, and nanotechnology owing to the outstanding technological functionalities and biological attributes of the resulting nanofibrils [52].A wide range of food proteins with plant or animal origin such as milk,soy,pea,rice,meat,and egg white proteins have been used to prepare nanofibrils.Two main pathways including monomeric model and polypeptide model were proposed for the formation of protein nanofibrils through the prolonged heating at acidic conditions [51].The monomeric model as the first model which was introduced for describing the nanofibrillation process of portions involves different steps including the partial denaturation of proteins for the production of active monomers,the formation of nuclei by assembling of active monomers,growth step, and lastly a termination phase [53].In this model, the lack of enough active monomers resulted from the acid hydrolysis of proteins was accounted for the incomplete conversion of proteins into the nanofibrils [54].In contrast, in a research conducted by Akkermans et al.[55], it was revealed that the hydrolysis step plays a crucial role in the formation of protein-based fibrillar aggregates and the peptides generated by hydrolysis of proteins during the fibrillation process are the building units of nanofibrillar aggregates instead of the intact monomers.As a consequence,the polypeptide model with more acceptability was developed which contains a hydrolyzing step to produce fibril-forming peptides[56].In both above-mentioned models, nanofibrillation process occurs through a sigmoidal pattern, initiated by a lag phase, followed by an elongation and growth phase,and finished by a steady or maturation phase[53].Structural,morphological,and functional aspects of food protein nanofibrillar structures are affected by various parameters such as primary protein concentration,incubation temperature and time,pH value of the protein solution,ionic strength,seeding,and stirring[51].

Numerous outstanding features of food protein nanofibrils such as excellent emulsifying and film-forming abilities, high resistance to heat and a wide range of pH values,desirable mechanical attributes, having a highly organized supramolecular structure,high aspect ratio,easy and fast production,high ability for binding to other molecules,and higher antioxidant activity compared to the non-fibrillated proteins triggered the scholars to employ them for the fabrication of delivery systems[51,56-58].In this regard,food protein nanofibrils were used as nanocarriers for bioactive substances in the structure of various encapsulation systems, which some examples of these applications are explained.

The surface hydrophobicity of proteins increases during the nanofibrillation process,so it was proposed that the resulting fibrils have higher ability for binding to the hydrophobic bioactive molecules.In this regard, Mohammadian et al.[59] employed the whey protein nanofibrils as carriers for enhancing the aqueous solubility and antioxidant activity of curcumin at pH 3.2 to simulate the conditions of common food drinks and beverages(Fig.5).They reported that the aqueous solubility of curcumin was increased by about 1200-folds through binding to the nanofibrils, whereas binding to non-fibrillated whey proteins enhanced its water dispersibility by 180-folds.The fibril-curcumin nanocomplexes also showed a higher antioxidant activity and lower release in the simulated gastrointestinal conditions compared to the complexes of non-fibrillated protein and curcumin.This research group[60] also employed the electrostatic-driven complexes of whey protein nanofibrils and gum Arabic as an encapsulation system for curcumin and reported high encapsulation efficiency (more than 98%)for this system suggesting the high affinity of curcumin toward the carrier.Moreover, they reported a high antioxidant activity, sustained release, and significant photo-stability for the curcumin-loaded complexes made of nanofibrils and gum Arabic.

In a recent study carried out by Shen et al.[61],β-lactoglobulin nanofibrils were successfully employed as efficient and safe nanocarriers for the iron fortification with striking features such as high in vivo iron bioavailability and desirable sensorial attributes without abnormal iron accumulation in organs.For the fabrication of nanofibrils-Fe hybrid systems, iron nanoparticles were decorated on the nanofibrils by mixing nanofibrillar structures with iron chloride and sodium borohydride as a reducing agent.The resulting hybrids showed a fast dissolution and rapid ion release during simulated enzymatic and acidic digestion which avoids the aggregation of iron particles.Moreover, the nanofibril-iron system showed a higher colloidal stability,lower cost,and improved sensory performance in comparison with the water-soluble FeSO4as a standard form of nanosized iron enhancing their applications for the fortification of food products with iron which can reduce the risks of iron-deficiency anemia as a major global public health issue.Bolisetty et al.[62] also applied the long and semi-flexible β-lactoglobulin nanofibrils formed by 5 h heating of protein solution at pH 2.0 and 90°C as shuttles for transporting of metal nanoparticles in living cells.In this study, nanofibrils were decorated with different nanoparticles including gold,silver,and palladium nanoparticles.The results demonstrated that the decorating of particles on the fibrillar aggregates increased their transport properties into the bone marrow-derived dendritic cell and human MCF7 breast cancer cells in comparison with the pristine nanoparticles.These findings might be used to extend the applications of food protein nanofibrils in the treatment of diseases such as cancer by providing new drug delivery systems with improved functions.

Fig.5.Whey protein nanofibrils as carriers for curcumin(designed with respect to Mohammadian et al.[59]).

Food protein nanofibrils alone or in combination with other biopolymers were used to produce microcapsules for entrapping of bioactive compounds with respect to their excellent emulsifying properties and good film-forming ability.Humblet-Hua et al.[63]used ovalbumin nanofibrils and high methoxyl pectin to form microcapsules using layer-by-layer adsorption method.Ovalbumin fibrils with a contour length of a few hundred nanometers were produced by 3 h heating of protein solution (5% m/m) at pH value of 2.0 and 80°C.The resulting multilayer capsules were loaded with limonene and their release behavior was characterized.The release of limonene form microcapsules happened by diffusion mechanism through the pores of the shell.The results also showed that the cargo release depended on the number of layers in the shell of the microcapsules;the amount of release was decreased by increasing of the number of layers.Ansarifar et al.[64] also used soy protein isolate nanofibrils with the diameter of 1-10 nm and high methoxyl pectin to fabricate multilayer capsules with high stability to sedimentation and flocculation for the encapsulation of limonene as a bioactive compound.Their results showed a good agreement with those of Humblet-Hua et al.[63].Nanofibrils from whey protein isolate also were employed to encapsulate fish oil using spray drying[50].The encapsulation efficiency of microcapsules made of nanofibrils was higher than the native whey proteins(95% vs 90%) attributing to the higher stability of interfacial films consisting of nanofibrillar aggregates.In addition, it was investigated that the fish oil which was capsulated in the fibril-based capsules experienced a lower oxidative deterioration compared to the non-fibrillated counterpart.This can be attributed to the greater antioxidant capacity of the fibrillated protein solution in comparison with the parental native proteins which was confirmed by the results of Mohammadian and Madadlou [56] who studied the antioxidant activity of fibrillated whey protein solution by free radical scavenging test and reducing power assay.Song et al.[65]also used hen egg white lysozyme nanofibrils to fabricate fibrillosomes;an emerging type of colloidosomes.They claimed that these fibrillosomes can be potentially employed for the encapsulation and controlled delivery of biologically active molecules including enzymes and antibodies in a green and facile method.In fact,they produced fibrillosomes by stabilizing of all-aqueous interfaces by protein nanofibrils without using any organic solvent with ability to denature the proteins.

Food protein-originated nanofibrils were also used as building units of hydrogels which can be exploited as delivery vehicles for hydrophilic bioactive ingredients such as minerals, antioxidants,and probiotics.In fact,hydrogels are three-dimensional structures consisting of cross-linked hydrophilic polymer networks with high ability to retain water or physiological fluid[66-68].In this regard,Mohammadian and Madadlou [69] fabricated hydrogels made of whey protein nanofibrils using different divalent cations (CaCl2,MnCl2, and ZnCl2).Zinc and manganese were used owing to their essential roles for human health and body metabolism.In contrast to the nanofibrils,heat-denatured whey proteins did not form self-standing hydrogels at the protein concentration of 54 mg/mL attributing to the higher ability of nanofibrillar aggregates to produce entangled networks.Hydrogels formed by ZnCl2were firmer than those prepared by CaCl2and MnCl2and also degraded to a much more extent in the simulated gastric condition.The authors suggested that the above-mentioned hydrogels can be used as delivery systems for minerals and also are good candidate for carrying of drugs and bioactive molecules intended for the treatment of stomach adenomas.Farjami et al.[52] produced ash-free hydrogels made of whey protein nanofibrils using a deacidification process.These authors claimed that the resulting hydrogels can be considered as suitable candidates for conveying of nutraceuticals which are susceptible to ions such as those drugs which binding to ions reduces their therapeutic effects.Recently,protein microgels were also developed by lysozyme nanofibril networks through a high-temperature long-period incubation of emulsified lysozyme drops seeded with pre-formed lysozyme nanofibrils[70].The resulting mono-disperse microgels were used to load four different drug-like small molecules including thioflavin T (selected according to its high affinity to amyloid fibrils), penicillin V (a hydrophilic antibiotic),tetracycline(a hydrophobic antibiotic),and remazol brilliant blue R (an aromatic water-soluble reactive dye).The release experiments showed that the release of payloads from nanofibril-based microgels governed by two mechanisms including the diffusion of drugs into the solution in combination with the dissolution of the microgels.Moreover, it was investigated that the loading of antibiotics into the microgels improved their antibacterial activity against Staphylococcus aureus.Generally, the observations of studies which mentioned before indicated that the protein nanofibrils are promising candidates for employing as nanocarries of bioactive molecules owing to their advantageous features.

2.5.Electrospun nanofibers

Electrospinning is a simple and cost-effective approach to fabricate nanofibers from polymer solutions using electrostatic forces[71].The resulting biopolymer-based nanofibers have been widely employed as nanocarriers for different bioactive compounds owing to their unique and special attributes such as having controlled cargo release profiles, high surface-to-volume ratio, high porosity,and tunable mechanical and wetting properties.Moreover,the electrospun nanofibers were introduced as promising encapsulation systems for heat-sensitive bioactive molecules due to the lack of heating during the electrospinning process [72-74].The properties of the electrospun nanofibers are dependent on different electrospinning parameters including process factors such as flow rate,spinning distance,and applied potential as well as the parameters related to the fluid using for the formation of nanofibers like viscosity, surface tension, dielectric constant, and electrical conductivity[73].

Food proteins are interesting biopolymers for the production of nanofibers by this method.However, in the most cases, they need a spinnable carrier such as poly(ethylene oxide) to facilitate electrospinning as well, to prevent the formation of protein capsules [73].In fact, the addition of electrospinning carrier into a biopolymer solution improves their ability to form nanofibers through the modification of the biopolymers physical attributes,especially the degree of the chains entanglements/associations[75].Different food proteins such as whey protein concentrate and isolate,soy proteins,egg proteins,zein,and gelatin have been used to produce nanofibers by electrospinning and the resulting electrospun nanofibers were used as nanocarriers for bioactive components[76,77].In the case of encapsulation purposes,various electrospinning techniques such as blend electrospinning,co-axial electrospinning,emulsion electrospinning,and the surface modification of electrospun fiber mats have been employed[74].

Whey proteins in conjugation with poly(ethylene oxide) as a spinnable polymer was used to produce nanofibers by electrospinning[71].The resulting electrospun nanofibers with diameters ranging from 312 nm to 690 nm were employed as nanocarriers for rhodamine B as a model flavonoid.A more uniform distribution of rhodamine B was observed for nanofibers made of protein and poly(ethylene oxide) compared to the singlecomponent nanofibers made of poly(ethylene oxide).However,no significant difference was investigated in their release properties and about 90% of the model flavonoid was released into water within 10 min.Li et al.[78] incorporated vitamin A and vitamin E into gelatin by electrospinning aimed to use the electrospun nanofibers as antibacterial wound dressing materials.These authors reported a sustained release profile over more than 60 h for vitamin A and vitamin E from the nanofibers.Moreover, the vitamin-loaded nanofibers showed an effective antibacterial activity against S.and Escherichia coli.In the study of Tavassoli-Kafrani et al.[74], the gelatin electrospun nanofibers were employed to encapsulate the orange essential oil as a natural flavoring agent using in orange juice.The release behavior of the essential oilloaded gelatin nanofibers in ethanol contained three major steps including initial burst release at the first 4 h of the experiment resulting from the surface oil release, followed by a subsequent decrease, and lastly a gradual release behavior.Moreover, these authors reported that the storage stability of the orange essential oil was improved through the encapsulation in the gelatin electrospun nanofibers.Therefore,these scholars claimed that the gelatin nanofibers prepared by electrospinning could be used for the incorporation of nutraceuticals into food products and beverages.

Zein as a plant protein with numerous commercial applications was also used to form nanofibers by electrospinning method to design nanocarriers for the bioactive compounds.In this regard,ultrafine fibers of zein prolamine were employed to stabilize βcarotene as a light-sensitive colorant and antioxidant ingredient[79].The results indicated that the light stability of the β-carotene was significantly improved by encapsulation in zein nanofibers.Regarding the excellent film-forming ability of zein proteins,these authors suggested that the antioxidant-loaded zein fibers can be used as a bioactive coating for food products.Ibuprofen as a model drug was also loaded in the zein nanofibers through a co-axial electrospinning process [72].The in vitro release behavior of the resulting drug-loaded fibers was studied in a physiological saline at a body temperature and the results showed that the release of ibuprofen form the nanofibers was mainly happened through the Fickian diffusion mechanism.In another study conducted by Lu et al.[80], zein was co-electrospun with hydrophobic ethyl cellulose and the resulting composite nanofibers were loaded with indomethacin as a model drug.Theses nanofibers showed a sustained drug release behavior which mainly was driven by the concentration gradient between the release medium and the cargoloaded composite nanofibers.Accordingly, indomethacin-loaded zein/ ethyl cellulose electrospun nanofibers can be potentially applied as wound dressing materials and scaffolds for tissue engineering owing to their controlled release behavior and high stability.

2.6.Nanotubes

The hollow tubular nanostructures or nanotubes made of food proteins are new classes of nanocarriers which have been used to load and deliver bioactive compounds.Moreover,these nanostructures were used for other food applications such as viscosifying and gelation owing to their specific characteristics such as high aspect ratio,relative stiffness,nanometric cavity,and ability to form transparent and strong gels[81,82].Two main methods were vigorously employed to fabricate protein-based nanotubes.The first one is the self-assembly of proteins and peptides into the tubular nanostructures and the second method is the layer-by-layer electrostatic deposition on a template[83-85].

With respect to the self-assembly method, it was reported that the α-lactalbumin as a milk whey protein is the only food protein which has the ability to form nanotubes after limited enzymatic hydrolysis [86].As illustrated in Fig.6, the formation of bio-nanotubes from α-lactalbumin under neutral pH contains three main steps including(1)partial hydrolysis of protein using a serine protease from Bacillus licheniformis or from S.aureus V8, (2) formation and exceeding of the concentration of the dimeric building blocks in the presence of an appropriate divalent cation such as Ca2+which forms bridges between the building blocks to generate stable nucleus consisted of five building units, and (3) elongation of tubular nanostructures [87].It was investigated that different factors can influence on the formation of α-lactalbumin nanotubes including the concentration of protein,type of the enzyme,type and concentration of divalent cations,and temperature[7].In addition to the hydrolysis by enzymes,Esmaeilzadeh et al.[88]used partial chemical hydrolysis method as a new technique for the fabrication of α-lactalbumin-based nanotubes.They used specific agents such as surfactants,Tris-HCl buffer,polar solvents,and pH reagents for the acid hydrolysis of protein for the formation of tubular structures instead of the enzymatic proteolysis.This method was introduced as a low cost,efficient,and controllable method without any need to high temperatures which are usually used for the fabrication of nanotubes.This method resulted in the formation of tubular structures with a range of 3-8 nm in outer diameter with high applicability in food science, medicine, nanotechnology, drug delivery,and surgery.

Fig.6.Schematic representation of the self-assembly of hydrolyzed α-lactalbumin into tubular nanostructures in the presence of Ca2+.Adapted with permission from Graveland-Bikker and de Kruif[87].

The formation and structural properties of α-lactalbumin-based nanotubes were investigated by different studies.Graveland-Bikker et al.[81] studied the growth rate of nanotubes.They produced tubular nanostructures with a cylindrical diameter of 19.9 nm and cavity diameter of 8.7 nm under conditions of the α-lactalbumin concentration of 28 g/L,pH 7.5,the enzyme to substrate molar ratio of 1:260, and calcium concentration of 3 mol/L of protein.At these conditions, the elongation rate of the tubular structures was about 10 nm/min.Moreover,they reported that about 40% of protein molecules did not assemble to nanotubes maybe due to the increasing of the solution viscosity and the formation of a gel-like structure which reduces the efficiency of assembly reaction.Circular dichroism spectroscopy was also employed to characterize the conformational properties of the nanotubes.The results revealed that the secondary structures of α-lactalbumin were not significantly changed upon the generation of nanotubes.Moreover,it was investigated that the α-lactalbumin hydrolysates with molar masses around 11 kDa are the main building blocks of the nanotubes [89].Fourier transform infra-red spectroscopy also suggested that the Ca2+ions form bridges between the negativelycharged carboxyl groups at Asp and Glu side chains in the peptides released by partial hydrolysis of α-lactalbumin which finally leads to the formation of tubular nanostructures[90].

The α-lactalbumin tubular nanostructures can be considered as efficient delivery vehicles for the bioactive compounds and drugs due to their specific properties such as having nano-sized cavities,the controlled disassembly,and the presence of open ends on both sides of the nanotubes[87].Despite these interesting features,very limited studies have been done in this area (i.e.employing nanotubes as nanocarriers) and it seems that more studies are needed to expand the applications of self-assembled protein nanotubes as vehicles for bioactive compounds and nutraceuticals.Accordingly, in a study carried out by Fucinos et al.[91],α-lactalbumin nanotubes were used as encapsulation agents for caffeine as a biologically active ingredient.These authors reported that the encapsulation efficiency of nanotubes for the capsulation of caffeine was about 100% and the functionality and structural properties of the nanotubular structures were not significantly damaged after loading of the cargo.They also studied the retention of the caffeine in the nanotubes as a function of environmental conditions which may happen in foodstuffs.The results showed that the storage of caffeine-loaded nanotubes under refrigerating temperature at neutral or alkaline pHs resulted in the maximum maintenance of the caffeine encapsulated into the α-lactalbumin tubular nanostructures.Tarhan and Harsa [82] also reported that the α-lactalbumin nanotubular gels could be employed as carriers with a transparent appearance for natural food colorants.They used Congo red dye as a representative colorant agent and investigated that the nanotubular protein gels are promising structures in the entrapment of coloring agent which makes them suitable candidates for the incorporation of natural colorants into the food matrices especially those food products having a transparent appearance.

Some disadvantages were reported for the formation of nanotubes through the self-assembly method such as needing to extremely pure protein and high concentrations of protein as well as having very strict conditions for manufacturing of nanotubes[84].These limitations resulted in the introduction of templateassisted layer-by-layer deposition as a new and highly versatile alternative method for the fabrication of edible polyelectrolyte complex tubular nanostructures.This method relies on the electrostatic interactions which make it applicable for a wide range of proteins [83,92].Sadeghi et al.[84] fabricated biocompatible nanotubes using poly-D-lysine and BSA through layer-by-layer deposition technique.They used track-etched polycarbonate membrane as a template for the fabrication of nanotubes.They reported that the stable,strong,and uniform tubes with a thickness of 61 nm were formed when three bilayers were employed.In contrast,employing one or two bilayers resulted in the generation of malformed nanotubes.After that, the resulting nanotubes were used as carriers for curcumin.They reported that the curcumin loading capacity of nanotubes was dependent on the type of interior layer;higher entrapment(encapsulation efficiency of 45%)was observed when the interior layer of nanotubes was fabricated of BSA compared to those with an interior layer of poly-D-lysine.Zhang et al.[83] also employed the template-assisted layer-by-layer assembly for the fabrication of nanotubes of BSA.They produced single component protein nanotubes by sequential filtration of protein solution at different pHs (i.e.3.8 and 7.0) through the nano-pores of anodic aluminum oxide templates.The average wall thickness of the resulting nanotubes was about 19 nm.The results of the above-mentioned study suggested that the morphology of the single component protein nanotubes can be controlled by tuning the condition of the filtration.In another study which was recently conducted by Maldonado and Kokini[92],BSA as a positively charged biopolymer and sodium alginate as a negatively charged were used to fabricate edible polyelectrolyte complex nanotubes using layer-by-layer deposition method.They also used polycarbonate membrane as the template to produce nanotubes.The results of this study showed that the formation and properties of the resulting tubular nanostructures were influenced by different parameters including pH,the concentration of biopolymers,protein to polysaccharide ratio,pore size of the polycarbonate template,and the flow rates through the template.These authors claimed that the nanotubes made of BSA and sodium alginate can be used potentially as nanocarriers for drugs,DNA,and biologically active ingredients.

2.7.Proteins in their native state as natural nanocarriers for bioactives

In addition to the nanostructured that are discussed, most of the food proteins, even in their native state, have a great potential to act as nanocarriers for biologically active compounds and nutraceuticals through the formation of nanocomplexes.In fact,nanocomplexation with food proteins was introduced as a simple and efficient method to improve the solubility,stability,adsorption,and bioavailability of hydrophobic bioactive compounds[93].Most of the food proteins are naturally nano-sized and therefore can be used as nanocarriers for bioactive molecules [94].The bioactives can bind to the hydrophobic patches of native proteins especially their aromatic acid residues through the hydrophobic interaction to form nanocomplexes.With less importance, hydrogen bonds also contribute to the formation of proteins-bioactives nanocomplexes[95,96].The nanocomplexation is a very simple process in which the hydrophobic bioactive molecules are first dissolved in a suitable solvent such as ethanol and then added to the protein solution.The resulting mixture is then stirred to form the nanocomplexes.In some cases,the resulting nanocomplexes are centrifuged to remove the un-loaded and free bioactive molecules.The bioactive-protein nanocomplexes can be used as dispersions or can be converted into powders by freeze drying [97-100].As an important point,it should be noted that final concentration of the solvent which was used for the solubilization of the bioactive compound in the protein-bioactive binary solution should be very low to have no significant effect on the nutritional and structural properties of the food proteins as the nanocarrier[59].

Different plant and animal proteins such as egg proteins [93,96,101], milk proteins [94,98,99,102-104], soy proteins[95,97,100,105], and pea proteins [106] have been used in their native state as nanocarriers for bioactive molecules through the nanocomplexation process.These complexes were employed as nanocarriers for different bioactive compounds and nutraceuticals such as curcumin [93,95-97,99], resveratrol [98,105],vitamins [94,101,106], 1-Octacosanol [100], epigallocatechin-3-gallate [102], glabridin [103], and gallic acid [104].The results of these studies showed that these nutraceuticals were more bioactive in the nanocomplexes compared to their free forms regarding to the higher water solubility,chemical stability,antioxidant activity,and bioavailability of the complexed form.For example,it was reported that the aqueous solubility of curcumin was increased by about 370,812,and 2500 folds through the nanocomplexation with ovalbumin[93],soy proteins[95],and β-casein[99],respectively.Nanocomplexation with α-lactalbumin also increased the water solubility of resveratrol by 32 times compared to the free form[98].The soy protein-resveratrol nanocomplexes also showed higher and faster release than free drug in the physiological conditions attributing to the improved solubility of resveratrol resulted from the nanocomplexation with soy protein isolate[105].The water solubility of glabridin as an isoflavonoid found in the roots of licorice was enhanced by 21 times after binding to β-lactoglobulin [103].Nanocomplexation with β-lactoglobulin was also reported that significantly improved the stability of epigallocatechin-3-gallate against oxidation and degradation [102].Therefore, the results of the above studies showed that the food proteins in their native states can be considered as efficient nanocarriers for bioactive molecules through the nanocomplexation process.The resulting protein-bioactive nanocomplexes have good solubility, stability,antioxidant activity, and biological properties which can be used as multi-functional ingredients in the food and drug formulations.

Casein as the major protein of the cow milk also is widely used as a natural nano-vehicle for bioactive molecules.Casein exists in the micellar form in milk with particle size from 50 nm to 500 nm (150 nm in average) and can be considered as spherical colloids [94].These micellar nanostructures are formed by assembling of casein fractions through the hydrophobic interactions and calcium phosphate bridges[107].In fact,casein micelles are in effect nano-capsules created by nature to deliver nutrients such as protein, phosphate, and calcium to the neonates[94,107,108].As an interesting feature, casein micelles have the ability to be re-assembled in vitro, presenting similar characteristics to those of naturally occurring casein micelles [109].In the food field, the casein micelles are one of most investigated selfassembled nanostructures for the delivery of bioactives because the casein is a green, safe, cheap, and abundant material with a simple production process [110].Therefore, re-assembled casein micelles have been proposed as promising protein-based nanocarriers for bioactive compounds to improve their bioavailability and stability against the harsh conditions of the processing’s and gastric environment [111].In this regard, native or re-assembled casein micelles have been successfully used as nano-carriers for different bioactive molecules such as vitamins [94,111-113], quercetin[107], curcumin [107,110,114,115], β-carotene [109,116], rutin[117], doxorubicin [118], and celecoxib as an anti-inflammatory hydrophobic drug [119].Different methods such as spray drying,high pressure homogenization,pH-shifting,restoration of mineral composition and ultra-high pressure homogenization,solvent displacement,emulsification-evaporation,and ultrasound treatments can be used to form bioactive-loaded re-assembled casein micelles[120].

In a study conducted by Semo et al.[94], the following procedure was used to fabricate vitamin D2-loaded re-assembled casein micelles.At first, the vitamin D2was added to the solution of sodium caseinate and then was mixed with tri-potassium citrate,K2HPO4and CaCl2at a pH range of 6.7-7.0 and temperature of 37°C.This was followed by stirring and the centrifugation.The resulting supernatant was collected, ultra-filtered, and then was treated with ultra-high pressure homogenization.These authors reported that the vitamin was about 5.5 times more concentrated within the micelles than in the serum and loading into re-assembled casein micelles improved the stability of vitamin against UV-light-induced degradation.Ghayour et al.[107] also used the same procedure to encapsulate curcumin and quercetin in casein-based delivery systems.They reported that the entrapment efficiency of this method for the both ligands was higher than 90%.Moreover,they observed that the encapsulation of these phenolic compounds in the re-assembled casein micelles significantly improved their chemical stability during an accelerated shelf-life test and also improved their anticancer activity against MCF-7 human breast cancer cells.The reassembly of casein micelles with this approach was also successfully employed to encapsulate β-carotene to improve its stability against different thermal processing technologies applied in food industry[109]and also to improve its bio-accessibility[116].The reassembled casein micelles prepared by this method were also used as nanocarriers for loading of other bioactive molecules such as vitamins A and D3[111],epigallocatechin gallate, and folic acid [113].Pan et al.[115] also used the pH-shifting method to prepare curcumin-loaded selfassembled casein nanoparticles.In this method, the pH of casein solution adjusts to high pH values(more than 11)and then returns to neutral pH values.During this process,alkaline dissociation followed by re-association of casein micelles through the subsequent acidification makes them able to encapsulate bioactive compounds in their structure.These authors reported that the encapsulation of curcumin in casein particles through the pH-shifting method significantly improved its anti-proliferation activity against human colorectal and pancreatic cancer cells.Somu and Paul [110] also used the self-assembled nanostructure of casein of spherical shape formed by desolvation method as a nanocarrier for loading of curcumin.They observed that the cytotoxicity of nano-capsulated curcumin was higher in four different cancer cells (breast cancer;MCF-7 and MDAMB231,cervical cancer;HeLa,osteosarcoma;MG 63)than that of free curcumin.

Another example of protein self-assembly for the delivery of bioactives is on the self-assembly of soy protein induced by alcohol or urea.In this regard, Liu et al.[121] used the soy βconglycinin nanoparticles formed by ethanol-assisted disassembly and reassembly as a nanocarrier for curcumin.In this method, at first the protein was treated with high concentrations of ethanol to unfold its structure which results in the protein denaturation and aggregation.After that, the ethanol was removed by dialysis to reassembly of protein to form nanostructured particles.These authors reported that the curcumin encapsulated in these reassembled nanoparticles showed a much greater chemical stability and bio-accessibility compared to the free curcumin.In another study conducted by Liu et al.[122], the urea-assisted disassembly and reassembly strategy was used to fabricate nanostructured soy βconglycinin.The resulting nanostructured protein was employed as a carrier for curcumin delivery.They reported a high loading amount for these nanostructures(about 18 g curcumin per 100 g of protein).Moreover, they reported that the water solubility, thermal stability, and bio-accessibility of curcumin were significantly improved by loading into nanostructured soy β-conglycinin prepared by urea-assisted disassembly and reassembly strategy.

3.Perspective on challenges and future trends

Despite the strong upsurge in the investigations of food proteins-based nano-delivery systems and their proven role in enhancing bioavailability, solubility, and protection of bioactive compounds, there are still challenges in this area.As an essential point, it is important to consider the effects of unique structure characteristics of different food proteins before discussing their potential to be applied in the nanoencapsulation of bioactives.Moreover, it should be considered that the properties of different bioactives vary considerably.Therefore,an ideal encapsulation efficiency and release behavior for a specific bioactive compound can be obtained by selecting a suitable nanostructured protein with fully considering the structural properties of the carrier and the cargo.As a problem, some toxic or hazardous materials are used to produce some types of protein-based nano-delivery systems which can be harmful to environment and human health.Therefore,the development of solvent-free and environmentally friendly methods such as green nanotechnology for the fabrication of food protein-based nano-delivery systems will require concerted effort from researchers, governments, and other stake holders.Most of the studies related to the food protein-based nanocarriers have been carried out on a laboratory scale and as a shortcoming, a few of these food protein-based nanocarriers which are discussed in the present review have been commercialized due to the lack of cost-effective methods of scale-up production.Therefore, the future studies should focus on the development of easily scaleup-able methods to produce bioactive-loaded nanostructured food proteins and determine these methods are economically feasible for large-scale production which can greatly help their commercialization.Moreover, the studies on the use of bioactive-loaded nanostructured food proteins in real food formulations are very limited and this area needs more attention due to this fact that other components present in the food matrices as well as the food processing conditions can affect the properties of nanostructured proteins and the bioactive compounds which are used to enrich the food products.Therefore, more in-depth scientific studies are required to study the applications and characteristics of bioactiveloaded nanostructured proteins in the real food systems to establish their function under the harsh conditions present in many food products.Furthermore, it seems that the lack of in vivo experiments is a limitation of many included reports and more detailed studies are needed for this area.Accordingly,in vivo trials on food proteins-based nano-delivery systems need further investigations to characterize the efficiency, safety, and reproducibility of these nanostructured proteins loaded with bioactive cargoes.Moreover,the safety and regulatory requirements should be appropriately considered.Generally, nanostructured food proteins are expected to play more central roles in future advanced drug delivery systems due to growing interest in recent years.Finally,we suggest readers to study other good review articles in the field such as those written by Fathi et al.[8],Elzoghby et al.[11],Chen et al.[12],Tarhini et al.[13], Acosta [123], Livney [124], Ezhilarasi et al.[125], and Tang[126]for a better and comprehensive understanding of food protein-based nanocarriers as well as the encapsulation methods for the biologically active compounds.

4.Conclusion

Nanoencapsulation is a well-established and effective technique for the entrapment, protection, and controlled delivery of bioactive molecules and nutraceutical which improves their stability and bioavailability.The incorporation of biologically active materials into the food products is more feasible in a nanocapsulated form.Different food biopolymers have been employed for the nanoencapsulation of biologically active components.Among them, protein-based nanocarriers offer advantages such as high nutritional value and versatile functional attributes which expanded their applications in the delivery of bioactives and drugs.Food proteins can form various nanostructures with different morphological and techno-functional properties including nanoparticles, hollow nanoparticles, nano-hydrogels, nanofibrillar aggregates, nanofibers, nanotubes, and nanocomplexes under different conditions and using various techniques.These nanostructured food proteins can be employed to carry and deliver a wide range of bioactive substances with respect to the properties of the cargo such as their hydrophilic and hydrophobic nature and sensitivity to heat and other external parameters.Furthermore, it was investigated that an ideal release behavior can be obtained by selecting a suitable nanostructured protein.Generally, this study showed that the nanostructured food proteins have high potentials to be used as nanocarriers for different bioactive ingredients.

Declaration of Competing Interest

The authors declare that there is no conflict of interest regarding the publication of this article except with our former colleague Dr.Ashkan Madadlou(Ashkan.madadlou@inra.fr)who left our department three years ago.

Acknowledgements

The support of University of Tehran and Iran National Science Foundation(INSF)is acknowledged.