Metabolomics analysis have been recently developed by utilizing complex omics platforms to investigate the effects of EO bioactives on microbiota and their host metabolism. However, specific interactions of fruit EOs-microbiota-host are difficult to investigate, since most of the analyzed biological samples are stool microbiota with free bacteria or blood samples, but not mucosal biopsies with adherent bacteria which should be ideally processed to cover the whole microbiota (31). Various animal models are currently investigated in order to monitor the microbiota-EOs interferences not only in feces, but also in the different regions of the gut (i.e., adhered to the GI mucosal proteins and epithelial cells) and outside the gut (i.e., central nervous system, systemic immune system, metabolites in the bloodstream etc.) (22).

Although rodents have been most widely used as model animals for studying in vivo interactions of gut microbiota with various molecules and particles, pigs are considered a more reliable alternative, since they display more anatomical, physiological and metabolic similarities to humans (22).

Studies showed that EOs could increase the weight gain and improve resistance to infection in swine and poultry (32). Besides, fruit EOs showed beneficial effects on antioxidant status, intestinal morphology, and barrier in animals (33).

Terpenes are a class of aromatic compounds found in EOs of many plants as well as fruit peels. These compounds define the distinctive smell of such fruits and plants as pine, citrus, thyme, cannabis, hops, etc. (34). Several studies show that terpenes display pharmacological and therapeutical activities such as anti-inflammatory, antioxidant, antibacterial and gastroprotective (35). The active component d-Limonene, which is present in the majority of the citrus family of fruits (such as sweet oranges, grapefruits and lemons) (36), improve metabolic parameters and modulate the intestinal microbiota.

An in vivo study on mouse model proved a significant decrease in weight gain which corelate with microbiota changes. Obese mice fed with d-Limonene based food supplements for 86 days showed lower cholesterolemia and reduced systemic inflammation parameters. These changes were compared with d-Limonene induced microbiota modulation, by decreasing the number of potentially pro-inflammatory taxa, such as Desulfovibrionaceae (known sulfate-reducing pathobionts), Peptostreptococcaceae and Erysipelotrichaceae, which are typically increased in metabolic inflammatory disorders, and increasing the number of Bacillaceae, Planococcaceae and Clostridiaceae bacteria. Thus, the study showed a positive modulation in gut microbiota and also demonstrated that even at higher doses, d-Limonene did not exert toxic manifestations (37). Similar effects were demonstrated in obese rat models, feed with microcapsules containing d-limonene extracted from sweet orange EOs. Dahu et al., demonstrated that d-limonene change the composition and abundance of microbiota at phylum- and genus-levels, correlated with body weight loss and improved fat metabolism (38).

Carvacrol is part of phenolic monoterpenes that poses pharmacological effects as anti-inflammatory, antioxidant, antibacterial and gastroprotective activity. Carvacrol was administered in rat models with intrarectal induced colitis to evaluate in vivo interference with the gut. After 3 days of treatment, it was observed a reduction in macroscopic and microscopic damage of the inner lining of the colon, and also an increase in antioxidant molecules and enzymes [catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx)]. These carvacrol-induced molecular changes at the gut level helped decrease the oxidative stress and could keep a balanced, healthy microbiota. The study concluded that the applied Carvacrol resulted in reducing inflammatory, nociceptive, and oxidative damage in rats (35)^.

Previous studies found that carvacrol and thymol could reduce the populations of Escherichia coli and Clostridium perfringens, while increasing the number of lactobacilli cells in the intestine of broilers (15). However, the effects of such compounds on the intestinal microbiota and microbial metabolites are limited. Table 1 offer a recent overview regarding bioactive compounds from fruit EOs which were investigated for gut interference and microbiota modulation.

AgNPs are used in hundreds of commercial products and studies demonstrated their microbiota modulatory potential in vitro and in vivo.

NPs can penetrate into human body by ingestion, inhalation, or dermal contact, the dietary ingestion being considered the primary penetrance route of silver nanoparticles (AgNPs) (58).

In vivo studies were conducted in rats or mice using high concentrations (up to 36 mg/kg bw/d), as compared to human dietary levels (0.03 to 0.65 μg/kg bw/d). Recent studies reported contradictory results regarding microbiota modulation, most researchers concluding that AgNPs increase the proportions of Bacteroidetes and pathogenic gram-negative bacteria, while decreasing the proportions of Firmicutes, Lactobacillus and Bifidobacterium in the mammalian gut (59). AgNPs derived microbiota alterations depend on the shapes of NPs, suggesting that their structure (which affects the surface-to-volume ratio) could determine the reactivity with gut bacteria. Moreover, the shift in the Firmicutes/Bacteroidetes ratio has been confirmed in the fecal microbiota of rats and mice orally given Ag-NPs at 2.5 or 3.6 mg/kg bw/d for 7–14 days (60). There are also reports about different effects of AgNPs on gut microbiota, depending on their shape such as cubes and spheres; for instance, AgNPs as cubes decreased Clostridium spp., Bacteroides uniformis, Christensenellaceae, and Coprococcus eutactus whereas, NPs as spheres reduced the relative abundance of Oscillospira spp., Dehalobacterium spp., Peptococcaeceae, Corynebacterium spp., Aggregatibacter pneumotropica (61).

In a study of Lyu and coworkers (2021) (62) performed on mice, the results showed that relative abundance of several bacteria was greater in the exposed group to AgNP, by comparison with the control group. These abundant bacteria belong to Prevotella spp., Bacillus spp., Staphylococcus spp., Enterococcus spp., Ruminococcus spp. and, Planococcaceae. Other few bacteria were less abundant in mice developmentally exposed to AgNPs, such as Coprobacillus spp., Mucispirillum spp., and Bifidobacterium spp. Or, it is demonstrated that in humans an increased relative abundance of Prevotella spp. is correlated with greater body mass index (BMI) (63), with colitis and an increased intestinal inflammation (64).

Exposure to low amounts of AgNPs induce in vivo toxic symptoms in Daphnia magna, which are corelated with diversity changes in gut microbiota. AgNPs caused concentration dependent down-regulation of genes encoding proteasome, ATP synthesis, fatty acid biosynthesis, and β-oxidation in the host when utilized in lower amounts, while higher NPs concentrations showed a more pronounced gene-regulation effect, ed with shorter body length, decreased digestion and reproduction ability in exposed D. magna. Microbial sulfidation of Ag⁺ ions, via the aggregation of nanoparticles and possibly the recovered synthesis of SCFAs relieved the observed AgNPs induced changes in the host. Symptomatic palliation observed in the AgNPs-tolerant microbiota inoculated D. magna further confirmed the detoxifying role of gut microbiota (65).

The impact of AgNPs on human microbiota is less investigated. In vitro study was done to investigate the short-term impacts of Ag-NPs on a defined human bacterial community called microbial ecosystem therapeutic-1 (MET-1, consisting of 33 bacterial strains, established from stool obtained from a healthy donor). Exposure to high concentrations of AgNPs (1–200 mg/L) for 48 h resulted in culture-generated gas production and changes in fatty acid methyl ester profiles. Bacterial composition modulation was characterized by decreased abundances of Bacteroides ovatus, Faecalibacterium prausnitzii, Roseburia faecalis, Roseburia intestinalis, Eubacterium rectale and Ruminococcus torques, and increased proportions of Raoultella sp. and of E. coli, suggesting a negative shift in the microbial community that favors the growth of pathogenic bacteria (65).

MET-1 model was also utilized in a custom colon reactor to determine the impact of TiO₂NPs on the human microbiota. In vitro studies reported a slight decrease in B. ovatus in favor of Clostridium cocleatum, after 48 h of treatment. However, longer exposure times (days) give significant changes in bacterial metabolites observed in the human colon microbiome, including in SCFAs production (66). In vivo studies performed in rodents proved very limited systemic absorption of TiO₂NPs, therefore relevant dose for humans (2.5 mg/kg bw/d) reveal no significant microbiota changes in mice after 1 week of exposure (67). However, increasing the exposure time to 1 month and the NPs concentration to 100 mg/kg bw/d, has translated to an increased proportion of potentially harmful Actinobacteria and Proteobacteria and a decrease in the abundance of beneficial Firmicutes and Bacteroidetes in mice (68).

TiO₂NPs administered in drinking water for 3 weeks at concentrations of 0, 2, 10, or 50 mg TiO₂/kg BW/day revealed significant microbiota modulatory effects in a dose dependent manner. Proportions of Lactobacillus and Allobaculum were significantly elevated at all tested doses, while Parabacteroides were significantly elevated in mice treated with 50 mg TiO₂/kg BW/day. Higher TiO₂ concentrations (of 10 and 50 mg TiO₂/kg BW/day) also decreased the proportion of Adlercreutzia and Unclassified Clostridiaceae, as compared to the untreated mice group. Moreover, co-occurrence analysis by examining microbial interactions from mice treated with various amounts of TiO₂NPs revealed that certain genera (such as Ruminococcus, Desulfovibrio, and Oscillospira) are consistently associated with each other regardless of TiO₂ treatment. Increased TiO₂NPs intake (i.e. at the dose of 10 and 50 mg/kg BW/day) resulted in more significant connections within the network, as well as increased number of genera with significant contributions. Authors report that, while Akkermansia was not significantly involved in the microbial network of mice administered 0, 2, or 10 mg TiO2/kg BW/day, it is involved at a dose of 50 mg/kg involving numerous co-exclusion relationships (69). This recent evidence suggest that TiO₂NPs could impair gut homeostasis which may in turn prime the host for disease development.

ZnO is currently listed as a generally recognized as safe (GRAS) material by the Food and Drug Administration (USA) and is commonly used as food supplement or in food packaging. However, some in vivo studies report that ingested ZnO NPs could alter the intestinal microbiota and inflammation response. Shen and coworkers revealed that dietary exposure to coated ZnO in piglets results in a significant improvement in intestinal morphology and immunity, including increased villi length, elevated immunoglobulin A (IgA) levels, increased gene expression of IGF-1, occluding, zonula occludens 1, IL-10 and transforming growth factor β1 (TGF-β1), as well as reduced gut microbiota diversity (70). Studies made in piglets feed with 600 mg ZnO NPs/kg revealed an increase in the bacterial microbiota richness and diversity in ileum, while they decrease in cecum and colon. The relative abundances of Streptococcus in ileum, and Lactobacillus in colon were increased, while the relative abundances of Lactobacillus in ileum, Oscillospira and Prevotella in colon were decreased. These microbiota changes were ed with ZnO induced diarrhea in piglets, the effects of ingested 600 mg ZnO NPs/kg being similar to the ingestion of high dose of traditional ZnO (2,000 mg Zn/kg) (71).

Feng et al. investigated the impact of ZnO NPs on gut microbiota in ileal digesta and its relationship with blood metabolites in hens. Various concentrations of ZnO NPs, ranging 25, 50, and 100 mg/kg, were fed to hens for 9 weeks. Their study revealed that the bacterial community richness was negatively correlated with increasing amounts of ZnO NPs. The microbiota diversity was considerably reduced at ZnO NPs concentration of 100 mg/kg. Specifically, the proportions of Proteobacteria, Bacilli, and Fusobacteria were altered, while the amount of Lactobacillus was decreased. Moreover, microbiota modulation was correlated with changes in the plasma metabolism; for instance, methionine, lactate, and choline levels are positively associated with microbiota richness. These results suggest that ZnO NPs indirectly regulate blood metabolism via gut microbiota modifications (58). Dietary exposure of ZnO NPs was studied also on fish intestinal microbiota. Administration of 500 mg/kg ZnO NPs to Cyprinus carpio for 6 weeks indicated no significant difference in the gut microbiota of treated and untreated fish (61, 72).

Similar to ZnO, SiO₂ NPs are intensively used food-grade particles, being approved by the European Union to serve as a food additive. SiO₂ acts as anti-caking agent and carrier for flavors or fragrances in food; however, it may cause adverse effects in humans, such as silicosis (an occupational pulmonary disorder induced by inhalation of many free SiO₂ particles) (73). Microbiota interactions were also observed in vivo after SiO₂ ingestion. SiO₂ NPs ingestion for 28 days determine disfunction of murine intestinal barrier, which might occur as a result of disordered gut microbiota (74). Significant decrease of intestinal bacterial phyla Verrucomicrobia, and bacterial genus Akkermansia, were observed after exposure. These bacteria are closely associated with the mucosal barrier and inflammatory responses in the intestine (72). Alterations in the gut microbiota composition were observed in patients who suffered from silicosis. The alteration of gut microbiota in these patients could be explained by the important amounts of inhaled SiO₂ NPs reaching the gastrointestinal tract. The results showed that the levels of Actinobacteria and Firmicutes in patients with silicosis were lower than those in healthy subjects. In addition, lower levels of Alloprevotella, Clostridiales, Devosia, and Rikenellaceae_RC9, as well as higher Lachnoclostridium and Lachnospiraceae levels, were highlighted in silicosis patients (75). Mesoporous silica nanomaterials were continuously administered for 14 days by gavage in a mouse model. The exposure to silica nanomaterials resulted in significant damage to the intestinal mucosal epithelial structure, microbiota imbalance and enhanced intestinal inflammation, ed with oxidative stress and apoptosis of intestinal cells (76). Specifically, the gut microbiota diversity was obviously changed, especially the harmful bacteria. Intriguingly, the administration of SiO₂ NPs dose similar to estimated human dietary intake (2.5 mg/kg bw/day) for seven consecutive days in mice increased gut microbial species richness as well as diversity. Researchers observed that the abundance of population belonging to Lactobacillus sp was correlated with the significantly increased levels of pro-inflammatory cytokines in the colon, including TNF-α, IL-1β, and IL-6. Nevertheless, no overall toxicity was observed in the mice exposed to SiO₂ NPs at this dose (67).

Collectively, when analyzing the results of the above examples regarding the evaluated inorganic NPs, we could conclude that, although a few studies indicate they have no influence on gut microbiota or do not cause histological lesions, they mostly occur in the case of low doses. However, most of the reports with higher doses and long-term exposure, such as occupational exposure, still show that negative bio-effects can be found when inorganic NPs meet gut microbiota, particularly inflammation, diversity reduction and phyla imbalance (72). These effects are usually associated with inorganic NPs–derived free radicals that cause oxidative damage, but also direct contact to the microbiota bacteria cells, causing membrane damage or cell lysis caused by NPs agglomeration.

EOs are recognized for their wide anti-inflammatory activity. This corelates with their significant reduction of relevant proinflammatory cytokines levels, such as necrosis factor alpha (TNFα), IL-1α, IL-1β, IL-6, IL-13, and NO (108, 109); as well as induction of anti-inflammatory markers, such as: IL-5, IL-13, expression of HO-1 (Nrf2-HO-1 pathway), increased production of CD8⁺, CD16⁺ cells, and IgA by activation of anti-inflammatory M2 phenotype of macrophages (110, 111).

On the other hand, inorganic NPs usually induce gut inflammation of various intensity, from mild to severe inflammatory processes, depending on the NPs type, exposure time and concentration (112). They usually increase the production of pro-inflammatory molecules, TNF-α, interferon-gamma (IFN-γ), IL-12 and of the Th2 cytokine IL-4 in the intestine (53, 113). These are opposite effects of EOs and NPs in gut inflammation and could lead to antagonistic interactions with unknown manifestation on the gut microbiota. Numerous studies demonstrate particular associations of some microbiota phyla with inflammatory processes in the gut (114, 115), however, researchers are still arguing if microbiota composition may induce gut inflammation, or inflammatory factors found in high amounts in the gut determine the modulation of microbiota composition.

Microbiota composition and diversity is modulated distinctly by EOs and NPs; while the first tend to reduce the occurrence of pro-inflammatory phyla (37), the latest show non-concluding results regarding specific NPs induced microbiota modulation toward the pro-inflammatory phyla (53). However, the currently available literature reveal that NPs exhibit a moderate to extensive impact on intestinal microbiota composition, highlighting a recurrent signature that favors gut colonization by pathobionts at the expense of beneficial bacterial strains. Such particular changes are frequently observed in metabolic and inflammatory conditions, for instance in inflammatory bowel diseases (IBD, which include Crohn's disease and ulcerative colitis), obesity and colorectal cancer (53, 116), which are highly influenced by the microbiota. Generally, the inorganic NPs-induced gut microbiota signature resembles that of dysbiosis-associated human diseases, characterized by alteration of the Firmicutes/ Bacteroidetes ratio together with depletion of Lactobacillus and enrichment of Proteobacteria (53, 60, 70, 117). Moreover, microbe-microbe interactions could also impact the microbiota-EOs-NPs triad. This idea is supported by a recent study demonstrating relevant microbiome modulation based on probiotic application. Moreover, along with their impact in microbiota diversity changes, probiotics are also studied as potent metabolic modulators, being proposed for the management of health conditions, such as obesity and cancer (118).

Both EOs and inorganic NPs showed unquestionable anti-pathogenic effects, expressed against numerous microorganisms, including gut and food pathogens. Also, synergistic interactions and enhanced antibacterial activity was frequently reported in recent studies when utilizing EOs-NPs combinations both in vitro and in vivo. It seems that, generally, EOs could target microbial pathogens (119), while protecting beneficial bacteria (i.e., supporting the growth of anti-inflammatory microbiota phyla), NPs seem to exert undifferentiated antimicrobial activity, impacting similarly in commensal and pathogenic microorganisms (120). Since some antimicrobial mechanisms of EOs and NPs are similar and show unspecific targets among different bacteria (i.e., oxidative stress in the bacterial cells, membrane damage, permeability changes), it is difficult to explain their potential different effects in antimicrobial effect against microbiota or pathogenic strains. However, impressive research developed in recent years propose innovative solutions to target specific microorganisms (i.e., pathogenic species, virulent or resistant bacterial strains) by using tailored NPs (121). Such nanosystems could act as delivery agents for EOs, ensuring their targeted effect against intended pathogens (122). Moreover, NPs-EOs combinations could make a significant impact in antibiotic resistant bacteria by: i) targeting multiresistant strains and ii) interfering with QS signaling, virulence genes expression and progress of the infectious process and iii) reducing the risk of selection for resistant mutants, which would be difficult to escape the antimicrobial mechanisms imposed by two agents in the same time.

Gut microbiota regulates intestinal mucosa permeability, nutrient uptake and is involved in digestion through the production of molecules with fermentative, saccharolytic (with the production of SCFAs) (123) and proteolytic properties. It is widely recognized that microbiota SCFAs production is highly altered by EOs and NPs, they being the most studied microbial metabolites.

SCFAs are monocarboxylic acids with maximum six carbons atoms, being the result of the microbial anaerobic fermentation process of indigestible carbohydrates such as dietary fibers and resistant starch, in the colon (124). Major SCFAs in GI are acetate, propionate, and butyrate (>95%), the quantity being dependent on the diet and microbiota pattern (125). These main three SCFAs are found normally in molar ratios varying from 3:1:1 to 10:2:1. Most SCFAs are metabolized to CO₂. Butyrate is used locally as an energy source for colonocytes, caliciform cells, or Paneth cells and regulates apoptosis, cell differentiation, and changes in the chemical structure of proteins and nucleic acids. In contrast, acetate and propionate pass into circulation, from where they are taken up by the liver and peripheral organs, where they are used as substrate for gluconeogenesis or lipogenesis (27, 126). Acetate and propionate are mainly produced by Bacteroidetes, while butyrate is produced mostly by Firmicutes (21).

Most SCFAs are absorbed in the proximal colon in exchange for bicarbonate, which neutralizes the luminal pH. As a result, the pH in the cecum is lower (5.5) than the rectum (6.5). This phenomenon can be explained by the lower capacity of Bacteroides compared to Firmicutes species to tolerate the presence of SCFAs at pH 5.5 than at pH 6.5 resulting in a microbial composition shift. This shift limits propionate and stimulates butyrate production (116).

All gut commensals ferment pyruvate for butyrate production, whereas the pathogenic bacteria, for example Fusobacterium, utilizes different pathways like those for Glutamate (4-aminobutyrate) and Lysine, which are associated with a release of harmful byproducts like ammonia (85).

Reduced levels of commensal bacteria in dysbiosis, such as butyrate producers, and the consequent ecological gap, could promote the growth of some intestinal opportunistic pathogens (127). It was also observed an inverse correlation between the abundance of Faecalibacterium prausnitzii, a major butyrate producer, and the local and systemic inflammation (8). A decreased production of SCFAs is correlated with the extinction of the microbial taxa producing the glycoside hydrolases as a consequence of a decreased dietary fibers intake (128).

SCFAs could be also absorbed and transported into the portal circulation, being used as energy source by hepatocytes (129). SCFAs are now considered as potential therapeutics (116), having the ability to improve the gut health, contributing to intestinal barrier integrity, mucus production, protection against inflammation and colorectal cancer (4, 130). The influence of SCFAs modulation on the central nervous system (CNS) have lately attracted the attention of researchers, being a subject of intense debate in recent years.

Dietary changes, infections, but mostly antibiotherapy drastically act on the gut microbiota composition, leading to frequent opportunistic infections, but also long-term impact on brain and behavior (130, 131). The ability of NPs and EOs to modulate microbiota diversity could also lead to distal effects in the host, impacting on various organs and tissues. The connection between the microbiome, gut and brain is a new concept called microbiome-gut-brain axis (131–133). So, there is a real crosstalk between the gut microbiota and brain, with some intestinal bacteria able to modulate neurobehavioral responses, by direct effects of the bacterial cells, their metabolites that mimic some host molecules, such as neurotransmitters, and/or stimulation of host inflammation (62).

Despite their demonstrated antimicrobial activity and the plethora of NP applications as additives in various products, some studies are reporting long-term and long-distance noxious effects, depending on the route of pentetration into the body, such as dysbiotic and metabolic alterations, mucosal immunity modifications, and neurotoxicity (62, 134).

It seems that AgNPs can accumulate in the GI tract, thereafter enter in the blood and can reach the brain, being able to cross the blood–brain barrier (BBB) or via retrograde axonal transmission (135).

A short-term experimental study using radioactive NPs, has confirmed that maternal oral exposure to AgNPs of ~ 50 nm (polyvinyl pyrrolidine coating) leads to placental and milk transfer and subsequent accumulation in the neonate brains (136).

Although it is unclear how NPs and EO could affect gut microbiota, it is becoming obvious that some disorders in gut bacteria can affect host health outside the gut, especially neural development and consequent risk for neurobehavioral disorders, such as autism spectrum disorders (ASD). It seems that over-abundance of a Ruminococcus spp. occurs in human patients with generalized anxiety disorder (137).

Among the effects observed in animals developmentally exposed to NPs, the most concerning is the reduced abundance of beneficial bacteria Bifidobacterium spp., known for their probiotic properties. One study showed that supplementation with a probiotic containing Bifidobacterium spp. led to improved health status in children with ASD and gastrointestinal disorders (2). On the contrary, reductions of the bifidobacteria number may induce neurobehavioral and GI diseases in rodent models and susceptible humans too (62).

Another effect is the inverse association between abundant Prevotella spp. and steroid hormone biosynthesis. Steroid hormones, including testosterone and aromatization of testosterone to estrogens, is essential in reproduction, but also in regulation of many neural functions. Reductions in these steroid hormones, especially estrogens, is linked to later neurobehavioral disorders, such as Alzheimer's Disease (AD) (62, 138, 139). The increased abundance of Prevotella spp. and Ruminococcus spp. also disturbs the biosynthesis pathway of unsaturated fatty acids. These fatty acids, especially docosahexaenoic acid (DHA), are essential for normal cognitive functions such as learning and memory, stress management, regulating emotional responses, inhibiting inflammation in the central nervous system, reducing the impulsive behavior, AD, and other brain disorders (140–142). SCFAs are considered key candidate mediators of gut-brain communication, and altered SCFA production has been demonstrated in a variety of neuropathologies (130, 143, 144).