Assembly of ProteinâPolysaccharide Complexes for Delivery of

Perspective Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Assembly of Protein−Polysaccharide Complexes for Delivery of Bioactive Ingredients: A Perspective Paper Zihao Wei and Qingrong Huang*

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Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, New Jersey 08901, United States ABSTRACT: Protein−polysaccharide complexes can be created in various ways (physical mixing, enzymatic cross-linking, chemical cross-linking, and Maillard reaction), and diverse protein−polysaccharide complexes are generally grouped into noncovalent and covalent complexes. Delivery systems constructed through assembly of protein−polysaccharide complexes (DSAPC) consist of emulsion-based delivery systems, capsule-based delivery systems, molecular complexes, nanogels, core− shell particles, composite nanoparticles, and micelles. DSAPC are effective delivery vehicles in enhancing the overall efficacy of bioactive ingredients, and DSAPC may possess multiple advantages over other delivery vehicles in bioactive ingredient delivery. However, designing and applying DSAPC are still faced with some challenges, such as low loading of bioactive ingredients. Efforts are required to reconsider and improve efficiency of DSAPC in many aspects, such as controlled release and targeted delivery. On the basis of more comprehensive and deeper understandings, DSAPC can be designed more rationally for delivery of bioactive ingredients. KEYWORDS: protein−polysaccharide complexations, assembly, bioactive ingredients, delivery vehicles, release properties, digestion, bioaccessibility, bioavailability, gut microflora

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INTRODUCTION Bioactive ingredients are components that can bring about beneficial health effects via modulating physiological or cellular activities.1 Common bioactive ingredients include polyphenols, essential oils, carotenoids, vitamins, minerals, phytosterols, bioactive peptides, and probiotic bacteria.1−6 A small quantity of these active compounds may exhibit various beneficial effects, such as anticancer, neuroprotective, anti-inflammatory, antioxidant, and pain-relieving activities.7−13 The major bioactive ingredients and corresponding biological functions are summarized in Table 1. Food is a major source of bioactive ingredients, and developing functional foods with incorporation of bioactive components is a promising means to increase intake of bioactive compounds. Nevertheless, there are plenty of challenges associated with simply incorporating bioactive ingredients into food systems in pure form: (i) poor solubility,8 (ii) weakened physical stability,14 (iii) inferior chemical stability during food processing and storage,13,15 (iv) offflavor,16 and (v) poor bioaccessibility and bioavailability.17 Designing suitable delivery systems is essential to overcome these shortcomings. The surfactant-based delivery system is a major class of oral delivery vehicles for bioactive ingredients, which includes liposome, solid lipid particle, and selfemulsifying system.17−19 However, application of a surfactant-based delivery system is severely limited by low loading capacities, irritation caused by a large quantity of surfactants, and leakage during storage.18−20 More efficient delivery systems are urgently required to encapsulate, protect, and release bioactive ingredients. Proteins and polysaccharides are natural biopolymers with abundant sources, and assembly of protein−polysaccharide complexes may have potential to construct economically viable © XXXX American Chemical Society

delivery systems with high biocompatibility and biosafety. Considering that assembly of protein−polysaccharide complexes may build versatile delivery vehicles and there are a variety of proteins and polysaccharides with distinct structural and functional properties,8,12,13,15 it can be expected that assembly of protein−polysaccharide complexes can be tailored for specific applications in delivery of bioactive ingredients. Delivery systems constructed through assembly of protein− polysaccharide complexes (DSAPC) may possess multiple advantages over other delivery vehicles. First, bioactive ingredients, such as polyphenols, may possess astringency, and either proteins or polysaccharides can reduce astringency of phenolic compounds.16,21 It may be speculated that protein−polysaccharide complexes can mask astringency of bioactive ingredients. Second, protein−polysaccharide complexation may enhance stability of delivery systems, which facilitate efficient encapsulation, protection, and release of bioactive compounds. Specifically, in terms of protein-based delivery vehicles, proteins may precipitate in large quantity around isoelectric points and involvement of polysaccharides may provide steric hindrance to restrain protein aggregation.14,15,22 Meanwhile, the presence of many bioactives, such as polyphenols, can accelerate protein precipitation, and protein−polysaccharide complexes may reduce co-precipitation of proteins and polyphenols. In addition, DSAPC may have better stability when compared to polysaccharide-based delivery vehicles. In a previous study, emulsions coated by chitosan alone are not stable and whey protein isolate− Received: Revised: Accepted: Published: A

November 2, 2018 December 21, 2018 January 14, 2019 January 14, 2019 DOI: 10.1021/acs.jafc.8b06063 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. List of Major Bioactive Ingredients and Their Biological Functions bioactive ingredient polyphenols

essential oils carotenoids vitamins minerals phytosterols bioactive peptides probiotic bacteria

representative compound

function

reference

curcumin resveratrol EGCG anthocyanins jasmine essential oil lavender essential oil lutein β-carotene folic acid vitamin D zinc β-sitosterol caseinophosphopeptides β-lactoglobulin fragments Lactobacillus Bifidobacterium

antioxidant and anticancer activities anticarcinogenic and anti-inflammatory effects anticancer, antioxidant, and neuroprotective activities prevent diabetes and inflammation and antioxidants lift up mood and reduce skin inflammation relieve pain and anxiety and improve sleep scavenge free radicals and enhance body immunity maintain healthy skin and vision and antioxidants nucleic acid synthesis and hemoglobin formation promote calcium absorption and bone formation wound healing and hormone metabolism lower cholesterol and regulate cardiovascular disease improve muscle recovery and athletic performance reduce blood pressure and aid sleep promote gastrointestinal health and immune function antitumor, hypolipidemic, and immunoenhancing activities

8 9 12 13 6 7 11 4 10 10 10 2 3 3 5 5

Figure 1. Schematic diagram showing interactions between proteins and polysaccharides.

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chitosan complexes can stabilize emulsions well as a result of synergistic stabilization.23 Third, because protein−polysaccharide complexes can function as a physical barrier of the polymer matrix, delivery vehicles based on protein−polysaccharide complexation may protect labile bioactive compounds against environmental stresses (light, oxygen, etc.).24 Finally, the colloidal network constructed by protein−polysaccharide complexes may retard burst release of bioactive ingredients.12 In the following sections, various types of protein− polysaccharide complexations will be summarized and current understandings toward DSAPC for bioactive ingredients will be elaborated. Later, challenges and future directions about assembly of protein−polysaccharide complexes for delivery of bioactive ingredients will also be included.

PROTEIN−POLYSACCHARIDE COMPLEXATIONS

Interactions between proteins and polysaccharides play a significant role in designing novel food systems, and major interactions between proteins and polysaccharides are summarized in Figure 1. It is worthwhile to point out that proteins in Figure 1 are depicted as spheres without reference to their specific shapes. Generally speaking, protein− polysaccharide interactions may result in non-covalent protein−polysaccharide complexes, incompatibility, or covalent protein−polysaccharide complexes (conjugates).22,25−27 The specific protein−polysaccharide interactions will be discussed in detail. Physical Mixing. Physical mixing of proteins and polysaccharides may lead to non-covalent protein−polysacB

DOI: 10.1021/acs.jafc.8b06063 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 2. Colloidal DSAPC for delivery of bioactive ingredients (not drawn to scale).

charides.27 It is noteworthy that, although the biopolymer complexes during complex coacervation are mostly made up of protein−polysaccharide complexes, a negligible amount of selfassociated proteins may coexist.30 Precise control of the protein/polysaccharide ratio and ionic strength is essential in assembling proteins and polysaccharides, because these variables may determine the network structure of protein− polysaccharide complexes and salts can weaken complex formation.32 Incompatibility of proteins and polysaccharides includes cosoluble polymers in a one-phase system and thermodynamic incompatibility in a two-phase system. With regard to cosoluble polymers, electrostatic repulsions between proteins and polysaccharides may exist, and another possible case is that proteins or polysaccharides do not carry any charges, because proteins are neutrally charged at isoelectric points and some polysaccharides (dextran, glucomannan, etc.) are uncharged in nature.27,31 Thermodynamic incompatibility often occurs at high ionic strengths and polymer concentrations,31 and segregative phase separations are not desirable in assembly of proteins and polysaccharides. Enzymatic Conjugation. Protein−polysaccharide conjugation may occur with the aid of enzymes via distinct mechanisms.25,33 A cross-linking technique using oxidases is

charide complexes or incompatibility, which greatly depend upon the intrinsic nature of polymers, pH, protein/ polysaccharide ratio, and ionic strength.27 In terms of noncovalent protein−polysaccharide complexes, non-covalent interactions between proteins and polysaccharides may include electrostatic interactions, hydrogen bondings, and hydrophobic interactions, among which electrostatic interactions are the dominant driving forces.28−31 Considering that most natural polysaccharides, except chitosan, are negatively charged, positively charged proteins at pH values below the isoelectric point may be expected for stronger (more difficult to reverse) protein−polysaccharide complexations. It is apparent that pH is a major determinant of protein−polysaccharide complexations, and three critical pH boundaries are closely linked with soluble and insoluble protein−polysaccharide complexes.28 The three critical pH values, pHc, pHφ1, and pHφ2, are associated with the formation of soluble protein−polysaccharide complexes, initiation of insoluble protein−polysaccharide complexes, and dissociation into uninteracted biomacromolecules (proteins or polysaccharides), respectively.28 Specifically, at pHc, the formation of soluble protein−polysaccharide complexes is initiated, which precedes association into insoluble complexes at pHφ1. At pHφ2, insoluble complexes dissociate completely into co-soluble proteins and polysacC


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Table 2. Overview of Delivery Systems Based on Assembly of Protein−Polysaccharide Complexes for Bioactive Ingredient Delivery type of delivery system nanoemulsion multiple emulsion multilayer emulsion Pickering emulsion nanocapsule double emulsion followed by coacervation molecular complex nanogel core−shell particle composite nanoparticle micelle

involved protein and polysaccharide

delivered bioactive ingredient

β-lactoglobulin and ι-carrageenan sodium caseinate and maltodextrin sodium caseinate and chitosan−EGCG conjugate gliadin and chitosan caseinophosphopeptide and chitosan gelatin and gum arabic

EGCG

whey protein isolate and apple pectin ovalbumin and dextran zein and citrus pectin

tannic acid and catechin curcumin resveratrol

zein and propylene glycol alginate ovalbumin and dextran

β-carotene

vitamin B12 β-carotene curcumin EGCG anthocyanins

EGCG

enhancing efficacy of bioactive ingredient

reference

improve anticancer activity, enhance permeability and cell absorption, and controlled release enhance loading capacity and encapsulation stability

12

inhibit degradation in harsh environments and improve chemical stability promote chemical stability and reduce loss improve intestinal permeability and absorption and enhance cellular antioxidant activity enhance chemical stability during storage and increase loading capacity improve solubility in the complex food matrix and inhibit co-precipitation with biomacromolecules increase bioaccessibility and enhance chemical stability enhance antioxidant and anticancer activity and improve loading capacity improve physicochemical stability and sustained release

40

improve permeability and absorption and increase loading efficiency

45

42

46 41 and 44 13 14 43 9 62

polysaccharides, such as dextran; otherwise, complexations of proteins with uncharged polysaccharides may be impossible.39 In addition to promoting the covalent bond formation between proteins and polysaccharides, the Maillard reaction may aid to develop attractive aromas, colors, and flavors. In many cases, considering that it is essential to endow proteins or polysaccharides with extra superiority and there are inherent inferiorities of biopolymers, native proteins or polysaccharides will be modified before application.15,40 Introduction of substantial reactive functional groups may accompany modification of proteins or polysaccharides with active reactants, and the newly introduced functional groups may participate in complexations with proteins or polysaccharides. The more complicated interaction modes may provide the possibility of developing more assembly methods of protein−polysaccharide complexes.

an enzymatic route to create protein−polysaccharide conjugates. Under the action of oxidases, such as tyrosinase and laccase, accessible tyrosine residues of proteins are oxidized into highly reactive quinone residues, which further react with nucleophilic substituents of polysaccharides.25 Transglutaminase (TGase) can also be used to covalently cross-link proteins and polysaccharides, and its working mechanism is to catalyze acyl transfer reactions between the γ-carboxamide groups of glutamine residues and free amino groups.33 The advantages of enzymatic conjugation are strong specificity and mild reaction conditions. Chemical Cross-Linking. Chemical cross-linking between proteins and polysaccharides may take place upon the addition of short- or long-range cross-linkers. Short-range cross-linkers include genipin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), glutaraldehyde, etc., which have low toxicity and high biocompatibility.34−36 Long-range chemical cross-linkers contain poly(ethylene glycol) dibutyraldehyde (PEG−DBA), and various cross-linking agents may lead to protein−polysaccharide conjugates with distinctive functional properties.26 Typically, chemical cross-linking brings about a higher conjugation yield with multiple cross-linkages between proteins and polysaccharides, which facilitates combining advantageous features of both proteins and polysaccharides. In comparison to enzymatic cross-linking, chemical crosslinking has its own advantages and disadvantages. When glutaraldehyde (one of the most popular cross-linking agents) is taken as an example, glutaraldehyde has strong reactivity, low cost, and high cross-linking efficiency in protein−polysaccharide cross-linking37 but glutaraldehyde exposure may bring about adverse health consequences, such as an irritating effect.38 Maillard Reaction. The Maillard reaction, a chemical reaction between proteins and reducing sugars on the polysaccharide backbone, is a common method to acquire protein−polysaccharide conjugates.22,39 Because a variety of polysaccharides contain reducing sugars and simply heating may trigger the Maillard reaction, the Maillard reaction finds broad practical applications. The Maillard reaction is a feasible means to covalently cross-link proteins with neutrally charged

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EXISTING DELIVERY SYSTEMS CONSTRUCTED THROUGH ASSEMBLY OF PROTEIN−POLYSACCHARIDE COMPLEXES (DSAPC) Protein−polysaccharide complexes can be employed as building blocks to create more complex structures as delivery vehicles for bioactive ingredients.12,14,40−42 As illustrated schematically in Figure 2, thus far DSAPC consist of emulsion-based delivery systems, capsule-based delivery systems, molecular complexes, nanogels, core−shell particles, composite nanoparticles, and micelles. As shown in Table 2, both hydrophilic [e.g., epigallocatechin gallate (EGCG) and anthocyanins] and hydrophobic (e.g., curcumin and resveratrol) bioactive ingredients can be effectively incorporated into DSAPC, which may enhance the overall efficacy of these bioactive ingredients.9,13,43−46 Tailor-made delivery systems should be properly designed and constructed on the basis of specific needs, and properties of existing DSAPC will be elaborated. Emulsion-Based Delivery Systems. In comparison to proteins or polysaccharides alone, protein−polysaccharide complexes are often capable of providing improved stabilization to emulsion interfaces and better protection for bioactive D


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Journal of Agricultural and Food Chemistry ingredients in internal phases of emulsions.40,47,48 Therefore, protein−polysaccharide complexes are widely applied to create emulsion-based delivery systems, and these emulsions are divided into four categories: nanoemulsions, multiple emulsions, multilayer emulsions, and Pickering emulsions. Electrostatic protein−polysaccharide complexes and protein−polysaccharide conjugates may stabilize nanoemulsions with droplet size in the nanometric scale (ordinarily in the range of 50−1000 nm).12,47 To reduce oil oxidation in nanoemulsion products, Maillard reaction products can be used to coat oil−water interfaces. That is because Maillard reaction products with antioxidant activity can form a thick interfacial layer around oil droplets, which can hinder the free radical chain reactions.49 Because the hydrophilic character of most protein−polysaccharide complexes outweighs their lipophilic characteristic, protein−polysaccharide complexes generally stabilize oil-in-water emulsions. Therefore, nanoemulsions based on protein−polysaccharide complexes mainly deliver hydrophobic bioactive ingredients, and a small quantity of hydrophilic bioactive ingredients can also be delivered via nanoemulsion vehicles. Our research showed that EGCG was successfully encapsulated in nanoemulsion stabilized by βlactoglobulin and ι-carrageenan, and the nanoemulsion vehicle could enhance controlled release and cell absorption of EGCG.12 Multiple emulsions formed with participation of noncovalent or covalent protein−polysaccharide complexes may act as promising delivery vehicles.42,50 Because there are both water and oil compartments in multiple emulsions, multiple emulsions can simultaneously deliver hydrophilic and lipophilic bioactive ingredients, although multiple emulsions are generally applied in delivery of hydrophilic bioactive ingredients. The most common multiple emulsion is waterin-oil-in-water emulsion, and double emulsions stabilized by protein−polysaccharide complexes can improve loading capacity, chemical stability, and controlled release properties of bioactive ingredients.42,50 Multilayer emulsions with thicker multilayered interfacial membranes are another category of emulsion vehicles for bioactive ingredient delivery. On the basis of layer-by-layer electrostatic deposition technology, protein−polysaccharide complexation occurs between protein and polysaccharide layers at emulsion interfaces.40 Multilayer emulsions have great potential to exhibit superior physical stability to environmental stresses and protect loaded bioactive ingredients from damage. It has been reported that bilayer emulsions coated by sodium caseinate and the chitosan− EGCG conjugate could inhibit β-carotene degradation in harsh environments.40 In addition, because the layer compositions in multilayer emulsions can be modified with environmental triggers (e.g., temperature, pH, etc.), multilayer emulsions can act as stimuli-responsive controlled-release delivery systems. A previous study reveals that multilayer emulsions coated with βlactoglobulin and pectin layers can control release of volatile compounds with pH and salt triggers.51 Pickering emulsions are emulsions that are stabilized by solid particles, and the key factors affecting properties of Pickering emulsions include the nature of the Pickering emulsifier, oil−water ratio, pH value, emulsifier concentration, etc. Pickering emulsions stabilized by protein−polysaccharide particles are recently developed to deliver bioactive compounds, owing to advantages such as outstanding stability against coalescence. Protein−polysaccharide particles as

Pickering emulsifiers can be assembled using insoluble (e.g., gliadin and chitosan) or soluble (e.g., gelatin, glucomannan, etc.) biopolymers.46,52 Protein−polysaccharide particles often have good Pickering emulsibility. Our research has demonstrated that stable Pickering emulsions with an oil fraction of 0.7 can be stabilized by 0.1 wt % ovotransferrin−gum arabic particles, indicating that Pickering emulsions with high oil fractions can be stabilized well by a small amount of protein− polysaccharide particles.53 Pickering emulsions can be applied to protect bioactive compounds, because particles adsorbed at emulsion interfaces may provide a physical barrier against environmental stresses. Developing Pickering emulsions with antioxidant benefits is an attractive strategy to protect bioactive compounds, and a recent study confirmed that curcumin loss was diminished in antioxidant Pickering emulsions stabilized by gliadin/chitosan hybrid particles.46 Considering that Pickering emulsions have excellent stability and double emulsions can be applied in producing reduced fat products, the reserach trend is to fabricate Pickering double emulsions, which may combine advantages of both Pickering emulsions and double emulsions. Pickering double emulsions are double emulsions stabilized by layers of colloidal particles. Food-grade Pickering double emulsions have seldom been studied, and a recent study shows that Pickering double emulsions with long-term stability can be stabilized by whey protein concentrate−gum arabic particles.54 Capsule-Based Delivery Systems. Developing capsulebased delivery systems can overcome drawbacks (poor dispersibility, lability, etc.) of bioactive ingredients and improve bioaccessibility of these compounds.55 Capsules constructed by protein−polysaccharide complexes can be divided into two main groups depending upon size, namely, nanocapsules (50−1000 nm) and microcapsules (>1000 nm).56 Driving forces of nanocapsule formulation include hydrophobic interactions, hydrogen bondings, and electrostatic attractions.24 Because nanocapsules may enhance intracellular uptake of bioactive ingredients as a result of subcellular size, nanocapsules may enhance bioavailability of bioactive compounds.41,44 Microcapsules are often developed to protect bioactive ingredients from the influence of the external environment, and the wall materials may inhibit leakage of bioactive compounds.11 Common microcapsules are designed for delivery of hydrophobic ingredients, and double emulsion followed by complex coacervation is a promising technique to develop microcapsules for hydrophilic compounds. Our group recently developed microcapsules using double emulsions followed by the complex coacervation method, and the microcapsules provided improved protection for anthocyanins.13 Molecular Complexes. Molecular complexes in this paper refer to molecular assembly of bioactive ingredients and biopolymers (proteins or polysaccharides) held together by interactions between bioactive ingredients and biopolymers. Polyphenols are the most studied bioactive ingredients in molecular complexes, and interactions between polyphenols and biopolymers (proteins or polysaccharides) consist of noncovalent or covalent interactions.40,57,58 In molecular complexes, proteins and polysaccharides can be electrostatically assembled or covalently cross-linked and polyphenols are bound to either proteins or polysaccharides. In some cases, polyphenols may bind to both proteins and polysaccharides. Because free polyphenols may form insoluble aggregates with biomacromolecules, molecular complexes can enhance polyE


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To achieve the best delivery performance, suitable proteins and polysaccharides should be selected as wall materials to design DSAPC based on the specific needs of bioactive delivery. For example, proteins with hydrophobic cavities are more suitable to deliver hydrophobic bioactive compounds. To improve intestinal permeability of bioactive compounds, design of DSAPC with chitosan as the outer layer is a good choice, because chitosan can adhere and infiltrate into the mucus layer easily when compared to other polysaccharides.64 After preliminary selection of protein and polysachharide candidates in constructing DSAPC, DSAPC constructed by different combinations of proteins and polysachharides should be evaluated by an in vitro model or animal model. The optimal DSAPC may be designed after an overall characterization of parameters, such as sensory properties, allergy, physicochemical stability, bioaccessibility, loading amount, controlled release, targeted delivery, and bioactive protection. Currently, the loading amount of bioactive ingredients in DSAPC is sometimes not satisfying, and structural engineering should be performed to increase the loading capacity of DSAPC. For lipophilic bioactive ingredients, replacing oils with organogels in emulsions stabilized by protein−polysaccharide complexes may be feasible to improve the loading of lipophilic compounds.65,66 Double Pickering emulsions stabilized by protein−polysaccharide particles have potential to increase loaded hydrophilic bioactive ingredients, because double Pickering emulsions have a higher degree of stability than conventional double emulsions and the volume ratio of the internal water phase can be elevated. Another concern is the loss of bioactive ingredients during long-term storage, and DSAPC with antioxidant strengths may provide enhanced protection for bioactive components. Introduction of antioxidants into the emulsion interfaces, particle surfaces, or capsule wall materials may inhibit damage of incorporated bioactive ingredients. In many cases, bioactive ingredients in DSAPC are released immediately after oral ingestion, leading to fast but temporary health benefits. Obviously, burst release of bioactive ingredients is not desirable, and sustained as well as controlled release of bioactive ingredients over a long period of time is our goal. Constructing DSAPC with cross-linkers, such as genipin, glutaraldehyde, and TGase, may enhance stability of delivery vehicles by suppressing particle dissociation in the gastrointestinal tract, fast swelling, etc. The improved stability of DSAPC facilitates the controlled release profile during the gastrointestinal tract until bioactive ingredients are completely released. The current research trend is to develop targeted DSAPC for bioactive ingredients, because some bioactive ingredients may be degraded or deactivated at some parts along the gastrointestinal tract and these bioactive components should be transported to specific sites of the gastrointestinal tract to function well. Using specific polysaccharides or proteins while constructing DSAPC may help to achieve targeted delivery. For instance, pectin is seldom degraded in the upper gastrointestinal tract and mostly digested by colonic microflora in the colon.67 DSAPC with pectin as outer layers may have potential to be applied as colon-specific delivery vehicles. In addition, pH- and enzyme-responsive DSAPC can be applied in target delivery of bioactive ingredients. Because there are distinct pH and enzyme conditions in different compartments of the gastrointestinal tract, DSAPC can be designed to remain intact at most sites of the gastrointestinal tract and break down

phenol efficacy by improving the physical stability of delivery systems.14,22 Nanogels. Nanogels are nanosized hydrogel particles with advantages such as high loading capacity of bioactive ingredients and smart responses to environmental stimuli. Nanogels can be fabricated via self-assembly of proteins and polysaccharides, followed by the heat-gelation process or ultrasonication treatment.59,60 Although nanogels mainly encapsulate hydrophilic bioactive ingredients (e.g., folic acid and riboflavin), they can also deliver hydrophobic compounds (e.g., curcumin).43,59 Nanogels fabricated by multiple protein/ polysaccharide pairs prove effective in enhancing bioaccessibility, chemical stability, and controlled release properties of bioactive ingredients.43,59,60 Core−Shell Particles. Core−shell particles are often applied in delivery of hydrophobic bioactive ingredients. Generally, hydrophobic proteins (zein, soy protein, etc.) first assemble into protein particles as the inner core, and bioactive ingredients are encapsulated into the inner core mainly through hydrophobic interactions.9,61 Afterward, hydrophilic polysaccharides attach to the core spheres as the shell layer. The protein core facilitates encapsulation and protection of hydrophobic bioactive ingredients, and the polysaccharide shell improves particle stability via generating stronger steric and electrostatic repulsions. Properly designing shell layers in core−shell particles may be beneficial to bioactive ingredient delivery, and a study revealed that replacing part of pectin with alginate in the shell layer improved stability of zein-based core−shell particles, which facilitates better water dispersibility and controlled release properties.8 Composite Nanoparticles. Protein−polysaccharide composite nanoparticles are hybrid particles made from proteins and polysaccharides with significantly different physicochemical properties and may have different characteristics from the individual proteins and polysaccharides. Electrostatic interactions, hydrophobic interactions, and hydrogen bonding may be involved in the formation of protein−polysaccharide composite nanoparticles. It has been reported that zein− propylene glycol alginate (PGA) composite nanoparticles can improve physicochemical stability and sustained release of βcarotene, suggesting great application potential of protein− polysaccharide composite nanoparticles in the food system.62 Micelles. Micelle delivery vehicles refer to spherical supermolecular assemblies of amphiphilic block copolymers. For micelles based on proteins and polysaccharides, amphiphilic block copolymers are mostly Maillard-type protein−polysaccharide conjugates, where proteins and polysaccharides act as hydrophobic heads and hydrophilic tails, respectively.45 Both hydrophobic and hydrophilic bioactive ingredients can be delivered using micelles based on protein− polysaccharide conjugates.39,63 To improve the loading capacity of hydrophilic bioactive ingredients, the micelle vehicles based on protein−polysaccharide conjugates can be reinforced using cross-linkers, such as glutaraldehyde.45

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CHALLENGES AND PERSPECTIVES Despite the fact that assembly of protein−polysaccharide complexes may facilitate delivery of bioactive ingredients in many aspects, designing, evaluating, and applying DSAPC are still faced with plenty of challenges. Efforts are required to reconsider and improve overall efficiency of DSAPC for bioactive ingredient delivery. F


Perspective


ingredients.72,73 Overall, on the basis of more comprehensive and deeper understandings toward the impact of DSAPC on the efficacy of bioactive ingredients, delivery vehicles for bioactive ingredients can be designed rationally through assembly of protein−polysaccharide complexes.

at specific sites as a result of stimuli-responsive properties, which perform as ideal target-specific delivery platforms. Although DSAPC have greatly improved bioavailability of bioactive ingredients,43 multiple measures can be taken to further promote oral bioavailability of bioactive compounds. For example, because nanoscale DSAPC can be more efficient in nutraceutical delivery, using nanosized DSAPC instead of microsized DSAPC can further increase compound bioavailability. In addition, because digestive juices may contain various components, such as bile salts, calcium, and enzymes, interactions between these components and DASPC may significantly modify physicochemical properties of DSAPC and alter the fate of delivered bioactive compounds. Therefore, interactions between various components in digestive juices and DSAPC should be comprehensively considered while designing DSAPC with high oral bioavailability. In terms of predicting oral bioavailability of bioactive ingredients delivered via DSAPC, in vitro digestion models, such as bottle-based static systems, are often applied to assess the bioaccessibility of bioactive components.43 The oversimplified bottle-based digestion model does not consider factors such as peristalsis motion of the gastrointestinal tract and elimination from the absorption site, and a dynamic gastrointestinal simulation system, such as the TNO gastrointestinal model (TIM) should be used to predict oral bioavailability accurately.68 It should be noted that, although complex in vitro digestion models provide close simulation to the human gastrointestinal tract, an in vivo pharmacokinetic study that takes many biological factors into account is the most accurate way to predict human oral bioavailability. Thus far, even in vivo pharmacokinetic studies about how proteins or polysaccharides affect bioavailability of bioactive ingredients are still very limited,69 and in vivo animal studies about the impact of DSAPC on the bioavailability of bioactive components are urgently required. Although multiple studies reveal that protein−polysaccharide particles loaded with bioactive ingredients can enter intestinal epithelial cells, the exact cellular internalization pathways of these particles remain largely unknown. It is interesting to know whether loading of bioactive ingredients may affect internalization pathways of protein−polysaccharide particles, because it has been reported that particle design may influence cellular internalization pathways.70 In addition, the cellular uptake of protein−polysaccharide particles loaded with bioactive compounds can be enhanced by designing physicochemical properties of these particles.71 For example, positively charged protein−polysaccharide particles may facilitate higher cellular uptake than negatively charged particles, because electrostatic attractions between positively charged particles and negatively charged mucosa facilitate higher cell absorption. Lastly, because some bioactive ingredients (e.g., polyphenols) may contribute to a balanced gut microbiota composition mainly through stimulating beneficial bacteria and suppressing pathogenic bacteria, it is expected that these bioactive compounds may modulate the gut microbiota community.72 Nevertheless, benefits of bioactive ingredients on intestine health may be enhanced or weakened by DSAPC, and related research is seriously lacking. It is worth exploring whether DSAPC can improve intestinal beneficial effects of the bioactive components. Apart from in vivo animal models, an in vitro simulator of the human intestinal microbial ecosystem (SHIME) model can serve as a powerful tool to evaluate the influence of DSAPC on intestinal beneficial effects of bioactive

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +1-848-932-5514. Fax: +1-732-932-6776. E-mail: [email protected]. ORCID

Zihao Wei: 0000-0002-1942-7907 Qingrong Huang: 0000-0001-8637-0229 Funding

The authors acknowledge financial support from the China Scholarship Council for Zihao Wei. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED DSAPC, delivery systems constructed through assembly of protein−polysaccharide complexes; EDC, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide; EGCG, epigallocatechin gallate; PEG−DBA, poly(ethylene glycol) dibutyraldehyde; SHIME, simulator of the human intestinal microbial ecosystem; TGase, transglutaminase; TIM, TNO gastrointestinal model

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