An Updated Overview on Nanonutraceuticals: Focus on Nanoprebiotics and Nanoprobiotics

Over the last few years, the application of nanotechnology to nutraceuticals has been rapidly growing due to its ability to enhance the bioavailability of the loaded active ingredients, resulting in improved therapeutic/nutraceutical outcomes. The focus of this work is nanoprebiotics and nanoprobiotics, terms which stand for the loading of a set of compounds (e.g., prebiotics, probiotics, and synbiotics) in nanoparticles that work as absorption enhancers in the gastrointestinal tract. In this manuscript, the main features of prebiotics and probiotics are highlighted, together with the discussion of emerging applications of nanotechnologies in their formulation. Current research strategies are also discussed, in particular the promising use of nanofibers for the delivery of probiotics. Synbiotic-based nanoparticles represent an innovative trend within this area of interest. As only few experimental studies on nanoprebiotics and nanoprobiotics are available in the scientific literature, research on this prominent field is needed, covering effectiveness, bioavailability, and safety aspects.


Nutraceuticals
Beside the emerging need for natural origin alternatives to pharmaceuticals, the interest is focusing more and more on possible applications of food derived products that can be used as tools to prevent (and in some cases also cure) or delay the onset of a health issue [1][2][3]. Nutraceuticals, are a novel toolbox not completely explored so far for its full potential in medicine [4][5][6][7]. Nutraceuticals, a portmanteau of the words 'nutrition' and 'pharmaceutical' [2], have been defined as "the phytocomplex if they derive from a food of vegetal origin, and as the pool of the secondary metabolites if they derive from a food of animal origin, concentrated and administered in the more suitable pharmaceutical form" [8]. Examples of substances that have nutritional and nutraceutical interest are antioxidants, vitamins, polyunsaturated fatty acids, dietary fibres, prebiotics, and probiotics [9]. Nutraceuticals reside nowadays in a gray area between pharmaceuticals and food; their safety and efficacy in health conditions and safety must be substantiated by clinical data; moreover, there is lack of a shared regulatory system for them [7,10].

Characteristics of Nanoparticles and General Classification
Within the different definitions of nanomaterials, these can be described as the products of nanotechnology, characterized by at least one dimension within the size range below 100 nanometers [11][12][13]. Due to their remarkable properties and versatility, nanomaterials are being exploited in different fields, e.g., agriculture, health, electronics, cosmetics [14][15][16][17][18], representing a great challenge, in particular, in food science and technology, environment, and human health [19]. The progress in pharmaceutical nanotechnology has led to a new class of products, the so-called nanopharmaceuticals [20,21], defined as pharmaceutical drug molecules formulated in nanomaterials. Different types of nanoformulations are being exploited for the treatment of neurodegenerative diseases, cancer, infectious diseases, and others [22][23][24][25][26]. Besides, nanomaterials are also succeeding in offering new advanced tools for imaging and diagnosis [27] which, combined with therapy, have been proposed as nanotheranostics. These formulations are also being tailored for personalized medicine.
Nanoparticles can be produced from natural (e.g., proteins, polysaccharides, lipids) and from synthetic (e.g., polymers) sources. Ideally, materials should be biocompatible, biodegradable, and biotolerable, namely the way by which designed materials are tolerated by the body, and of generally recognized as safe (GRAS) status, in order to be used in pharmaceutical and nutraceutical products. Among the available options, and if the nanoparticles are intended for oral administration (as happens with nanonutraceuticals), lipid nanoparticles are of special interest [28][29][30][31]. Lipids are known for their role as absorption enhancers in the gut, which contribute to improving the oral bioavailability of several drugs and biomolecules. Besides this, the loading of poorly soluble drugs into lipid nanoparticles overcome the limitations encountered in their formulation into final products. Lipid nanoparticles can be produced from well-known lipids existing both in the human body and in foodstuff (e.g., fatty acids, triglycerides, phospholipids, waxes, cholesterol) thereby enhancing their biodegradability, and biocompatibility profiles [32].
Among polysaccharides, chitosan [33][34][35][36][37] and alginate [33,38,39], have been frequently used in the production of nanoparticles for oral delivery. Being a mucoadhesive polysaccharide, chitosan is able to increase cellular permeability and improves the bioavailability of orally administered drugs and proteins. Moreover, the molecule itself exhibits antimicrobial properties, and has a low toxicity. The molecule has chemical functional groups that can be modified for site specific targeting. Alginate is also a versatile mucoadhesive natural polymer with very low toxicity in vivo. Alginate nanoparticles have a hydrophilic character with improved loading capacity for hydrophilic drugs, being able to modify their release profile. Alginate nanoparticles are reported as adjuvants in vaccinations and can be produced conjugated with dextran to modify the release profile of proteins and other macromolecules intended for oral administration [40].
Nanopharmaceuticals and nanonutraceuticals are obtained, respectively, when a pharmaceutical or a nutraceutical is formulated in nanoparticles. The rationale for their development is mainly addressed to improve the physicochemical properties (e.g., solubility) and pharmacokinetic parameters

Focus on Nanotechnologies Applied to Prebiotics, Probiotics, and Synbiotics
Focus of this perspective is the application of nanotechnologies to food supplements containing prebiotics, probiotics, and synbiotics. This section consists of (i) shot on prebiotics, probiotics, and synbiotics; (ii) definition and delineation of nano-prebiotics, nano-probiotics, and nano-synbiotics.

Prebiotics
Prebiotics [62][63][64][65][66] are a special form of dietary fiber with health benefits, which invoke alterations in the host microbial ecosystem, not only in the gut, via their selective administration by live host microbes [67]. Food ingredients like prebiotics are classified on the basis of some principles, such as resistance to digestion in upper alimentary tract, selective stimulation of probiotic growth, beneficial health effects in the host, stability in different conditions of food/feed processing, and fermentation process through intestinal microbiota. They are found in various sources, including some non-digestible oligosaccharides, non-digestible carbohydrates, yacon, unrefined wheat, unrefined barley, soybeans, raw oats, breast milk, and inulin sources (e.g., chicory roots and Jerusalem artichoke) [68]. Some compounds found in prebiotics are soya-oligosaccharide, xylo-oligosaccharide, pyrodextrins, gluco-oligosaccharide, lactulose, malto-oligosaccharide, galactans (galacto-oligosaccharide (GOS)), oligofructose, isomalto-oligosaccharide (IOS), fructans (FOS and inulin), mannan-oligosaccharide (MOS), lactitol, and non-starch polysaccharides (NSP). Figure 1 gives an overview of prebiotics. xylo-oligosaccharide, pyrodextrins, gluco-oligosaccharide, lactulose, malto-oligosaccharide, galactans (galacto-oligosaccharide (GOS)), oligofructose, isomalto-oligosaccharide (IOS), fructans (FOS and inulin), mannan-oligosaccharide (MOS), lactitol, and non-starch polysaccharides (NSP). Figure 1 gives an overview of prebiotics. Several mechanisms are involved in the bioactivity of prebiotics and probiotics [69,70], as described in Figure 2. The metabolic products of such microorganisms can drop the gastrointestinal (GI) pH by carbohydrate fermentation via Bifidobacteria and Lactobacillus thereby influencing mineral uptake, growth, and spread of gut microbiota, epithelial integrity, and hormonal regulation. They also are able to enhance the absorption of trace elements and especially of iron and act on the regulation of body immune function. The prebiotics can use the short-chain fatty acids (SCFAs) as an energy source.

Probiotics
The FAO (Food and Agriculture Organization) and WHO (World Health Organization) have defined probiotics as non-pathogenic living microorganisms that ensure host health if used properly in foods or as dietary supplements [71,72]. Probiotics come from different sources, such as various natural environments, human gut microbiota, and foods. The main properties of probiotics like the ability to survive through the gastrointestinal tract, the resistance against bile and gastric acidity, and the stimulation of the activity of bile salt hydrolase, promote health benefits to the host [68,[73][74][75][76][77][78][79][80][81]. The count of probiotic bacteria (colony-forming units (CFU)/g) in probiotic-containing products differ among the countries; for example, 10 7 CFU/g in the USA and 10 9 CFU/g in Canada. The effective dose generally contains >10 6 -10 8 CFU/g or >10 8 -10 10 CFU/d of live probiotic bacteria [82,83]. Most probiotics are found in Gram-positive bacteria, including Streptococcus, Bacillus, Lactobacillus, Enterococcus, and Pediococcus. The probiotics can also include fungal and yeast species such as Saccharomyces cerevisiae and Kluyveromyces. Only some microorganisms such as Lactobacillus spp., Bifidobacterium spp., and Lactococcus are known as generally recognized as safe (GRAS) despite the existence of diverse microorganisms which can act as probiotics with health benefits [84][85][86]. Figure 3 gives an overview of probiotics. The metabolic products of such microorganisms can drop the gastrointestinal (GI) pH by carbohydrate fermentation via Bifidobacteria and Lactobacillus thereby influencing mineral uptake, growth, and spread of gut microbiota, epithelial integrity, and hormonal regulation. They also are able to enhance the absorption of trace elements and especially of iron and act on the regulation of body immune function. The prebiotics can use the short-chain fatty acids (SCFAs) as an energy source.  [82,83]. Most probiotics are found in Gram-positive bacteria, including Streptococcus, Bacillus, Lactobacillus, Enterococcus, and Pediococcus. The probiotics can also include fungal and yeast species such as Saccharomyces cerevisiae and Kluyveromyces. Only some microorganisms such as Lactobacillus spp., Bifidobacterium spp., and Lactococcus are known as generally recognized as safe (GRAS) despite the existence of diverse microorganisms which can act as probiotics with health benefits [84][85][86]. Figure 3 gives an overview of probiotics. The reported key mechanisms of action of probiotics [87] have been mentioned as follows (see Figure 2): enhancement of epithelial barrier, modulation of insulin-sensitive tissues, synthesis of antimicrobial substances, multi-pathogen competition, and induction of mucin secretion. The probiotics are able to adhere to epithelium, resulting in microbial elimination. They also modulate the immune function via the stimulation of signaling pathways to upregulate anti-inflammatory cytokines and growth factors, to differentiate T-regulatory cells (Tregs), and to interact with the gut-brain axis (GBA) by endocrine regulation and neurologic functions.

Synbiotics
The synbiotic agents are a combination of prebiotics and probiotics with beneficial effects on host through the enhancement of activity and survival of beneficial microorganisms in the gastrointestinal tract, so that they can selectively provoke the growth and stimulate the metabolism of one or more health-promoting bacteria, thereby enhancing the host welfare [88][89][90][91][92][93][94][95][96][97]. The most The reported key mechanisms of action of probiotics [87] have been mentioned as follows (see Figure 2): enhancement of epithelial barrier, modulation of insulin-sensitive tissues, synthesis of antimicrobial substances, multi-pathogen competition, and induction of mucin secretion. The probiotics are able to adhere to epithelium, resulting in microbial elimination. They also modulate the immune function via the stimulation of signaling pathways to upregulate anti-inflammatory cytokines and growth factors, to differentiate T-regulatory cells (Tregs), and to interact with the gut-brain axis (GBA) by endocrine regulation and neurologic functions.

Synbiotics
The synbiotic agents are a combination of prebiotics and probiotics with beneficial effects on host through the enhancement of activity and survival of beneficial microorganisms in the gastrointestinal tract, so that they can selectively provoke the growth and stimulate the metabolism of one or more health-promoting bacteria, thereby enhancing the host welfare [88][89][90][91][92][93][94][95][96][97]. The most important issue in the design of synbiotics resides in the prebiotic and probiotic selection criteria and requirements, which should be clearly described.

Health Promoting Effect of Prebiotics, Probiotics, and Synbiotics
The International Scientific Association for Probiotics and Prebiotics (ISAPP) introduced a wide range of products containing the probiotics with health promoting effects, including non-edible products (e.g., vaginal preparations), baby formulas (e.g., first milk), drugs, therapeutic supplements (e.g., for enteral nutrition), and foods (e.g., fermented milk with reportedly health beneficial effects) [98].
In an in vitro study, Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) as a quantitative analysis technique was applied to evaluate the proteomic profile of colon cancer cells in Lactobacillus kefiri SGL 13, and the results indicated antiproliferative and pro-apoptotic activities for this strain on human colon adenocarcinoma cell line HT29 [99]. In another study, the airway hyper reactivity was suppressed in ovalbumin-sensitized samples by Lactobacillus spp. (such as Lactobacillus and Pediococcus) via a reduction in the level of Th2 cytokines, OVA-specific IgE and IgG1 as well as an increase in the level of IgG2a [112]. Lactobacillus fermentum cell-free supernatant (LCFS) caused cancer cell death in 3D HCT-116 conditions through the induction of apoptosis in the colon cancer cell line and the antiproliferative activity by the inhibition of NF-κB signaling [129]. The use of lactoferrin and Bifidobacterium longum BB536 managed the enteropathy caused by diclofenac in rat samples by modulating the proinflammatory pathway of TLR-2/-4/NF-kB [130]. Othman et al. [131], studied the effect of inactivated Bifidobacterium longum intake on obese diabetes affected mice. They reported a significant decrease of body weight gain, adipose tissue mass and blood glucose levels, as well as a significant reduction in blood glucose after a 5 weeks treatment. The treatment also resulted in reduced levels of cholesterol and triglycerides [131].
The administration of three strains of Bifidobacteria in the adult rats improved neuronal plasticity and cognitive behavior [132].
Prebiotics have been reported to have different activities; for example, generation of bacteriocins, maintenance of gut health [133], possibility to be used as food additive and starter culture, clearance of cholesterol [134,135], potentiation of immune defense [136], inhibition of constipation and risk of obesity [137,138], inhibition of colitis [139], protection of colon and other organs against cancer [140][141][142], reduction of cardiovascular disease risk factors, antioxidant activity [143,144], over-bioavailability [145]. According to scientific published data, the administration of oligofructose-enriched inulin (OEI) promotes malondialdehyde content, lipid profile, glycemic indices, and antioxidant level in female patients suffering from type II diabetes [146]. The supplementation of inulin in shaken cultures was found to increase the growth rate of L. plantarum ST16 [147]. Based on the findings from Ramos et al. [148], the administration of fructooligosaccharides (FOS) was tolerated and decreased the total and free p-cresyl sulfate (PCS) in the serum samples of patients with non-diabetic chronic kidney disease (NDD-CKD).
The therapeutic potential of synbiotics has been comprehensively discussed in a recent review published by Flesch et al. [149]. According to their findings, the patients with irritable bowel syndrome (IBS) when receiving B. longum BB536 and L. rhamnosus HN001 plus vitamin B6 showed restoration of intestinal permeability and gut microbiota, as well as amelioration of the disease symptoms [150]. In the research of Mohan et al., the synbiotic AMF TM 15 + manuka honey yogurt showed antibacterial properties, followed by increasing probiotic bacteria and producing lactic and propionic acids [151]. A study reported gut health enhancement following the administration of seaweed-based synbiotic of Gracilaria coronopifolia which caused the reduction of inflammation, the generation of reactive oxygen species (ROS), and diminution of the oxidative stress-induced cell damage [152]. According to Sarwar et al., the textural properties, such as adhesiveness, cohesiveness, and hardness, were enhanced following the co-administration of inulin and Saccharomyces boulardii [153]. In Table 1 an updated overview of in vitro and in vivo studies on prebiotic, probiotic, and synbiotic products is given. Anti-inflammatory effect The inflammatory response was modulated in mucositis caused by 5-FU (fluorouracil) via the probiotic Saccharomyces boulardii CNCM I-745 through the control of TLR 2 and 4 as well as the reduction of pro-inflammatory and NF-κB cytokines [103] L. casei IMAU60214 Immunomodulatory effect The use of L. casei IMAU60214 killed by heat increased the activity of M1-like pro-inflammatory phenotype through the TLR2 signaling pathway [123] L. plantarum Antimicrobial effect L. plantarum ZLP001 impeded the ETEC adhesion and linked with IPEC-J2 cells via the competition and exclusion [122] Lactobacillus Anti-diabetic effect The lactobacillus strain alleviated the levels of blood sugar and HbA1c in diabetic rats [115] L. plantarum LMT1-48 Anti-obesity effect The body weight and abdominal fat content were decreased in mouse models fed a modified diet through the administration of L. plantarum LMT1-48 at a density of 10 6 CFU/mL [117] Hafnia alvei Anti-obesity effect Fat mass, food intake, and body weights were reduced in the mouse model of obesity and hyperphagia [118]

Type Microorganisms/Prebiotics Activity Study References
Eurotium cristatum Anti-obesity effect The administration of Eurotium cristatum showed anti-obesity activity in mice fed a high-fat diet (HFD) through the modulation of gut microbiota [119] L. plantarum strain TCI378 Anti-obesity The expression of glucose transporter type 4 (GLUT-4) and adipocyte-specific genes perilipin 1 was suppressed by metabolism derivatives from L. plantarum strain TCI378 [120] Prebiotic Galacto-oligosaccharides and phycocyanin Anticancer effect The prebiotics co-administered by phycocyanin arrested the cell cycle at the G0/G1 phase, resulting in inhibited growth of HCT116 cells [141] Chondroitin Sulfate Disaccharide Anticancer effect The growth of HT-29, human colon cancer cell line, was controlled by Chondroitin sulfate (CS)-Keel disaccharide (CSD) generated by chondroitin AC lyase, estimating at 80% antiproliferative activity [140] Short-chain fatty acids Antiproliferative effects The administration of short-chain fatty acids (SCFAs) prevented the expression of genes involved in human colorectal cancer cells [142] Blueberry anthocyanins Antioxidant effect The density and composition of intestinal microbiota in human models were increased by consumption of high purity blueberry anthocyanins through the increase in the modulatory and prebiotic activities [143] Oligosaccharides Antioxidant effect The water-soluble oligosaccharide of EMOS-1a showed 1420% proliferation level [144] Lycium barbarum polysaccharide Immunomodulation effect The administration of polysaccharides derived from Lycium barbarum in mice showed immunomodulatory effects, and enhanced density of beneficial bacteria and gut microbiota [136] Synbiotic Djulis (Chenopodium formosanum) with L. acidophilus

Anticancer and antioxidant effect
Ten weeks of low-calorie diet program along with synbiotic supplementation enhanced the activity of superoxide dismutase (SOD) and reduced the serum level of malondialdehyde (MDA) in obese patients suffering from breast cancer-related lymphedema [89] Weissella cibaria FB069 with xylooligosaccharides Anticancer effect The use of synbiotic-fermented soymilk (containing xylooligosaccharides and Weissella cibaria FB069) inhibited the proliferation of HCT116 and Caco-2, colorectal cancer cell lines, through the reduction in the transcription of MD2/TLR4/MyD88/NF-κB [90]  The use of multispecies symbiotic showed immunoregulatory effects on the expression levels of CD4 and IgA in mice exposed to lipopolysaccharide (LPS) [92] L. plantarum with inulin Neuropsychological effect Concomitant administration of inulin and L. plantarum in diabetic rats improved CREB/BDNF/TrkB signaling pathway, serotonin secretion, brain parameters, intestinal microbial composition, and oxidative stress, thus leading to improved memory and learning disorders [93] β-glucan, Bacillus coagulans, and inulin, lactic acid Grape pomace extract with lactobacilli

Anti-inflammatory effect
The co-administration of lactobacilli and prebiotic grape pomace caused a downregulation of inflammatory genes, proteins, signaling molecules through the symbiotic effects [96] L. acidophilus, L. rhamnosus, B. longum and Bifidobacterium bifidum, Saccharomyces boulardii with fructo-oligosaccharides

Hepatoprotective effects
The administration of synbiotic soy yogurt controlled hypercholesterolemia in mice liver by reducing the levels of low-density lipoprotein cholesterol, triacylglycerols, blood cholesterol, and lipid peroxidation. [97]

Type Activity Study References
Probiotic Antimicrobial effect The polylysine-induced poly glutamic acid (PG) films caused protection of probiotics against food-borne pathogens [154] Anticancer effect The high levels of synthesized silver/Lactobacillus rhamnosus GG nanoparticles (Ag-LNPs) led to a decline in the rate of HT-29 live cells [155] Anticancer and antimicrobial effect The fabrication of copper oxide nanoparticles (CuO-NPs) using L. casei could control the proliferation of HT-29, a human colon carcinoma cell line, and human gastric carcinoma cell line, as well as could eliminate Pseudomonas aeruginosa and Staphylococcus aureus [156] Anticancer and antioxidant effect The L. casei capped-SeNPS suppressed the cytotoxicity caused by Diquat and oxidative damage, impeded the cell damage and apoptosis induced by H 2 O 2 , and induced the apoptosis mediated by the HepG2 cell line [157] Anticancer and antioxidant effect The findings from the administration of L. casei 393-SeNPs were the induction of HepG2 cell line apoptosis, the elevation of oxidative damage caused by Diquat in IECs, and the reduction in gut barrier dysfunction caused by ETEC K88 via the antioxidant functions, the regulation of inflammation, the establishment of gut epithelial barrier integrity, and the balance of gut microflora [158] Anticancer effect Dead nano-scale L. plantarum could impede the proliferation of a colorectal cancer cell line through an increase in the expression level of IgA, an induction of cancer cell cycle arrest and apoptosis, and a suppression of inflammatory response [159] Anticancer and antioxidant effect The synthesis gold nanoparticles (AuNps) having antioxidant activity and low cytotoxicity using L. kimchicus DCY51T strain exhibited the activity of a protective protein capping layer [160] Prebiotic Improve drug delivery High molecular weight (HMW) inulin nanoparticles were fabricated to achieve drug delivery system, whose concentration of <200 µg/mL had no toxicity for peripheral blood mononuclear cells (PBMCs) [161] Antimicrobial effect The probiotics were internalized by phthalyl dextran nanoparticles (PDNs) to construct pediocin, aiming at the alteration of gut microbiome composition, the suppression of pathogenic intestinal infections, and the elevation of beneficial bacteria species [162] Antimicrobial effect The higher pediocin generation following the administration of PIN-internalized probiotics with 0.171 polydispersity index (PDI) with a size of about 203 nm showed the maximum antimicrobial properties [163] Synbiotic Antimicrobial effect The activity of Listeria monocytogenes and Escherichia coli K99 was inhibited by L. plantarum exposed to phthalylpullulan nanoparticle (PPN) due to production of antimicrobial peptides via intracellular stimulation [164] The photo protective effect A cream containing L. rhamnosus plus Selenium nanoparticles could heal the side effects induced by sunburn and showed sun protection factor (SPF) of 29.77 in Wistar rat model [165] Improve delivery system A new formulation of nano-emulsion containing E. faecium plus inulin could increase probiotic bacterial viability and stability [166] Improve tolerance of probiotic bacteria Beads reinforced by inulin (5% w/v) had the highest effect on bacterial protection against bile salts [167] Caneus et al. [168] remarked how nanomedicine, together with the known practices of prebiotics, probiotics, and synbiotics, represents a valuable approach in creating an optimal environment within the gastrointestinal tract.
Exploring the nanonization strategies of probiotics and the utility of nanoprobiotics in the delivery of encapsulated bacteria is being carried out. For encapsulation of probiotic have been used mainly nanoparticles i.e., with of selenium and gold particles of a size in the range 10-1000 nm; nanolayers, consisting of at least three layers of a charged polyelectrolyte, a polymeric layer, and a functionalized polysaccharide or polyether; nanoemulsions consisting of a liquid phase dispersion in another liquid phase with droplet size less 200 nm; nanobeads (nanosized bacteria-enabled autonomous delivery system) and emerging product of nanofibers [169]. The best technique for probiotics encapsulation was mainly chosen for protecting the cells against an adverse environment in the gastrointestinal tract, in order to allow their release in a viable and metabolically active state in the intestine [170].
Kazmierczak et al. [171] describe an innovative engineering approach to load such nanoparticles onto a biological "mailman" (a novel, nontoxic, therapeutic strain of Salmonella typhimurium engineered to preferentially and precisely seek out, penetrate, and hinder prostate cancer cells as biological delivery system) that will deliver the therapeutics to a target site. Another example of probiotic bacteria encapsulated with nanoparticles was given by Hu et al. [172] that showed how coating live bacterial cells with synthetic nanoparticles represents a promising strategy to engineer efficient and versatile DNA vaccines. Feher et al. [173] have reported the use of nano-sized particles of probiotics for preventing and treating neuroinflammation.
Probiotics are indeed receiving special interest as an alternative to the classical antibiotics to overcome bacterial resistance. As prebiotics enhance the activity of probiotics, Kim et al. [162] proposed the development of a prebiotic formulation composed of Pediococcus acidilactidi loaded in phthalyl dextran nanoparticles by conjugating phthalic anhydride with dextran [162]. The authors evaluated the cellular effects of the produced nanomaterial and checked the antimicrobial properties of the probiotics. The loading of P. acidilactidi into phthalyl dextran nanoparticles was found to enhance the production of antimicrobial peptides by probiotics by a self-defense mechanism, with improved antimicrobial effect against Gram (+) and Gram (−) micro-organisms compared to the probiotics alone. The same authors previously reported that prebiotic phthalyl inulin nanoparticles could also enhance the antimicrobial activities of P. acidilactici [174].
Hong et al. also reported the enhanced antimicrobial activity of phthalyl pullulan nanoparticles treated with L. plantarum against Escherichia coli K99 and Listeria monocytogenes [164]. The nanoparticles were internalized into the L. plantarum by an energy-dependent and galactose transporter-dependent mechanism and a higher amount of plantaricin, a natural antibacterial peptide, was secreted from the developed nanoprobiotic than from probiotic alone.
The use of spores from probiotics have been recently proposed as a delivery system for chemotherapeutic drugs. Song et al. [175] produced deoxycholic acid-modified spores to be loaded with doxorubicin and sorafenib as an approach for autonomous production of nanoparticles in the gastrointestinal tract. Such approach envisions drug protection upon oral administration to improve bioavailability. Besides, the release is based on the disintegrated hydrophobic protein and the hydrophilic deoxycholic acid with enhanced uptake by the epithelial cells via the bile acid pathway, increasing basolateral drug release.
The anticancer activity of silver/Lactobacillus rhamnosus GG nanoparticles was described by Aziz et al. [155]. Using the MTT assay, the authors demonstrated that the viability of HT-29 cell lines has been significantly reduced when applying the highest tested nanoparticle concentration, leading to apoptosis. The method of synthesizing silver/Lactobacillus rhamnosus GG nanoparticles was also found to be cost-effective, offering a viable nanoprobiotic approach for biomedical applications.
It is worth mentioning the work of Fung et al. [176] where, by investigating the agrowaste-based nanofibers as a probiotic encapsulant, has proposed the use of nanofibers for the nanoencapsulation of L. acidophilus using 8% poly(vinyl alcohol) to produce nanofibers by electrospinning technology.
The authors suggested how thermal behavior of nanofibers suggested possible thermal protection of probiotics in heat-processed foods. Nagy et al. [177] by investigating the suitability of electrospinning for biodrugs delivery to produce vaginal drug delivery systems, concluded how nanofibers can provide long term stability for huge amounts of living bacteria if they are kept at (or below) 7 • C. The recent work of Zupancic et al. [178], who studied the incorporation of a range of safe lactic acid bacteria into poly(ethylene oxide)-based nanofibers, evidenced that all of the lactic acid bacteria were viable after incorporation into nanofibers, with 0-3 log CFU/mg loss in viability, depending on the species. Moreover, the authors reported that viability can be correlated with the hydrophobicity and to the extreme length of lactic acid bacteria, whereas a horizontal or vertical electrospinning set-up did not have any role. Development of nanofibers via electrospinning has a great potential and use in pharmaceutical and food industry for their properties i.e., sterile nature, biocompatibility, adhesiveness, efficiency, and as vehicle for controlled and sustained release in drug delivery [179][180][181][182]. Electrospinning and electrospraying represent innovative technologies for the delivery of nutraceuticals [183].
An example of nanolayers coated probiotics has been given by Franz et al. [184] who developed layer-by-layer nano self-assembly coating of Allochromatium vinosum with different polyelectrolyte combinations and investigated substrate uptake in bacteria: surface charge neither affected sulfide uptake nor the contact formation between the cells and solid sulfur, whereas increasing layers slowed or inhibited the uptake of sulfide and elemental sulfur.
The recent work of Ebrahimnejad et al. [185] described the use of chitosan for nanoencapsulation of L. acidophilus as probiotic bacteria, by concluding how nanoencapsulation of probiotic bacteria represents a promising strategy in enhancing the viability and survival of them against gastro-intestinal environmental conditions. Ranjan et al. [186] reported physicochemical characterization and potential prebiotic effect of whey protein isolate/inulin nano complex.
Atia et al. [167] developed an encapsulated oral-symbiotic supplement by studying the effect of adding inulin in alginate beads and observed their ability to protect three probiotic strains, namely, P. acidilactici, L. reuteri, and L. salivarius. The antimicrobial and probiotic properties of bacterial strains were found not to be affected by the encapsulation.
Krithika and Preetha [166] have developed a protein-based inulin incorporated symbiotic nanoemulsion for enhanced stability of probiotic; whey protein concentrate/inulin nano complex can be recommended as a delivery system for various probiotics in food products.
Salmerón et al. [187] reported the development fermented beverages with synbiotic properties, and the incorporation of nanoparticles with unique and specific bioactivity, to improve organoleptic characteristics, absorption, and delivery of nutrients and bioactive compounds which has opened a new horizon in this segment of food created to improve human health and well-being.
Formulation of protein-based inulin incorporated synbiotic nanoemulsion for enhanced stability of probiotic are currently studied extensively.
It is worth mentioning the work of Rezaee et al. [188] that investigated the antimicrobial activity of Ag and TiO 2 nano-particles on three species of Lactobacillus i.e., L. casei ATCC 39392, L. plantarum ATCC 8014, and L. fermentum ATCC 9338 in the presence and absence of raffinose, lactulose, and inulin, respectively. The results indicated that silver nanoparticles decreased 85%, 85%, and 71% of L. casei, L. plantarum, and L. fermentum, respectively, after 48 h and decreased percentages of L. casei, L. plantarum, and L. fermentum that were 16%, 64%, and 4% in the presence of the prebiotics. Nano TiO 2 particles decreased 59%, 85%, and 61% of L. casei, L. plantarum, and L. fermentum, respectively, after 48 h, and decreased percentages of L. casei, L. plantarum, and L. fermentum which were 16%, 2%, and 4% in the presence of these prebiotics.
The treatment of gastrointestinal disorders (e.g., diarrhea) using nanoprobiotics is also a relatively unexplored field. Khan et al. [189] aimed at quantifying the concentration of nanomaterials commercialized in chocolates and evaluated their effect on a commercial probiotic formulation (containing Bacillus coagulans, Enterococcus faecalis, and Enterococcus faecium) usually used to treat diarrhea in children [189]. The known probiotic activities, such as acid production, biofilm formation, growth, and antibiotic resistance were observed from isolated bacteria, while the isolated titanium oxide nanoparticles from chocolates were shown to inhibit the growth and activity of the probiotic formulation in a concentration range of 125-500 µg/mL in vitro [189]. The outcomes of this study concluded that TiO 2 in chocolate discourages the survival of probiotic bacteria in the gastrointestinal tract.
To trace target probiotics in situ and in real-time, Liu et al. [190] developed an in vivo probing strategy using persistent luminescence nanophosphors surface-modified by plasmid-like DNA as optical labelling and background-free fluorescence bioimaging as signal readout. The surface modification with DNA molecules was shown to promote the nanoparticles penetration into the bacteria and facilitated in vivo bioimaging. Such an approach opens new research perspectives in terms of food safety making use of nanotechnologies.

Conclusions
Only a few experimental studies are present in literature on nanoprebiotics and nanoprobiotics, while studies on this prominent issue are needed, covering effectiveness and safety aspects as it has been developed for pharmaceuticals. The potential of nanotechnologies in the food area is an emerging challenge as well as the nanonutraceuticals, which are an emerging field of study in the nutraceuticals area. Safety and regulatory aspects should be considered to depict the potentiality of nanoprobiotics and nanoprebiotics. Nanoformulation should be accompanied with regulatory requirements to ensure efficacy, safety, and authorization procedures. As a general guideline, the European Authority for Food Safety (EFSA) [191] has developed an approach for assessing the potential risks arising from the applications of nanoscience and nanotechnologies in the food and feed chain. Regarding prebiotics and probiotics, McClements and Xiao [192] developed a summary of the possible applications of inorganic and organic nanoparticles in foods, a description of the nanoparticle characteristics, and discussed the importance of the food matrix and gastrointestinal tract effects on nanoparticle properties as well as potential possible toxicity mechanisms of different food-grade nanoparticles. The same authors concluded, however, that many of these nanoparticles are unlikely to have adverse-side effects on human health in line with previously reported data [193]. Nonetheless, in order to assess the effective use of food-grade nanoparticles, further studies are expected to exploit and assess safety, improved bioavailability, and efficacy.