Clinical interest in the human gut microbiota has increased considerably, because of the increasing number of studies linking the human intestinal microbiota and microbiome to an ever increasing number of non-communicable diseases. Many attempts at modulating the gut microbiota have been made using probiotics and prebiotics. However, there are other avenues that are still little explored from a clinical point of view that appear promising to obtain modifications of the microbial ecology and biological activities connected to the microbiome. This chapter summarizes all in vitro, in vivo and clinical studies demonstrating the possibility to positively modulate the intestinal microbiota by using probiotics, foods (and prebiotics), essential oils, fungus and officinal plants. For the future, clinical studies investigating the ability to modify the intestinal microbiota especially by using foods, officinal and aromatic plants or their extracts are required. More knowledge in this field is likely to be of clinical benefit since modulation of the microbiome might support the therapy of most non-communicable diseases in the future.
- essential oils
1. Introduction: The pivotal role of gut microorganisms in human health
Our knowledge of the relationship between human beings and the microorganisms we harbor in our gut has greatly increased in the past years, even if we are still far from having understood all their functions. We no longer consider these living entities as simply commensal, and we start to realize that humans are “super organisms” governed also by the microorganisms living inside us. There are approximately 100 trillion cells in the human body, and more than 90% of them are microbes. They make up the human microbiota, consisting of bacteria, fungi and even viruses, mainly located in the intestine where they are referred to as the intestinal microbiota.
The terms currently employed in this field are the following:
The first consideration that we have to do is that the microbial ecosystem of the intestine called gut microbiota, is one of the most dense communities that we know, surpassing for complexity those present in soil, subsoil and also oceans .
The second consideration is that the microbiota does not represent an inheritance dependent on our species or genes, but rather an environmental inheritance, mainly due to the type of environment to which we have been exposed in the first 3–4 years of our life . This also implies that we can act during life with the aim of improving our microbiota (Figure 1).
The last one is that our gut microbiota and microbiome are strictly connected with our state of health or illness and, together with genetics and environment, certainly represent a discriminating point in predisposing us to the onset of some particular diseases rather than that of others. The gut microbiota is closely related to our metabolic balance as well as to the development and functioning of our immune system, as studies on germ-free animals have clearly shown. It is also closely connected with the intestinal and systemic endocrine system, and indirectly with the central nervous system, via the enteric nervous system, within what is commonly called the gut-brain axis .
These considerations must not make us think of the microbiota and microbiome as something fixed and stable in the course of our life. The aging of our organism physiologically leads to a change in the gut microbiota with a decrease in some specific populations, such as the short-chain fatty acid (SCFA)-producing families
In addition to the physiological and irreversible increase in our biological age, there are other conditions that have a decisive impact on the composition and function of the intestinal microbiota. The first for importance and for the daily life with which it is implemented, is certainly our diet, which can cause, as we will see in the next paragraph, positive or negative changes in the microbiota. Another, often overlooked, condition is our lifestyle. Smoking and alcohol, for example, can negatively alter the microbiome , while regular physical activity seems to be capable of significantly improving it .
Finally, as we will analyze in the following paragraphs, there are many different pathologies, and consequent therapies, that can alter our intestinal microbiota, sometimes irreversibly. The most illuminating example concerns the transmissible pathologies of bacterial origin, encountered at an early age. The antibiotic therapies that often become necessary can, in the first 3 years of life, irreversibly alter the developmental trajectory of the intestinal microbiota leading, in the adult age, to a microbiota substantially different from that which would have developed in the absence of broad-spectrum antibiotic therapies . On the contrary, antibiotic therapy in adults only reversibly alters the intestinal microbiota, which returns exactly to the starting point after the end of the therapy  Other intestinal pathogens, such as
However, we must not think that the pathologies correlated to alterations of the microbiota are essentially limited to the gastro-intestinal or metabolic ones. In recent years, many studies have linked alterations in the gut microbiome with a plethora of various diseases, including the neurodegenerative ones, such as Alzheimer’s or Parkinson’s . Despite our limited mechanistic understanding of how the microbiota can predispose to neurodegenerative diseases, efforts to manipulate the microbiota through fecal microbiota transplantation, probiotic treatment, or other nutritional strategies, highlight the potential for microbial improvement in successfully preventing or decreasing the symptoms of these diseases, at least in laboratory animals . It is therefore not surprising that some studies today are explicitly aimed at microbiome-targeted interventions for the prevention or treatment of neurodegenerative diseases.
To conclude this paragraph of premises, we can state that while conventional medicine aimed at maximum specialization, with branches such as organ and cellular medicine, on the other side of the pond the role of the intestinal microbiota has gradually assumed more and more importance, to remind us that our “super organism” is unique and that alterations of our gut microbial component, that is not even part of our cellular pool, can have a broad-spectrum negative impact on many if not all the organs and apparatuses that make up our organism. The microbiota well represents the complex relationships that exist between our health and the environment in which we are born and spend the first years of our life. A compromised environment, due to excessive sterilization or pollution, certainly has a strong impact on the structure of our microbiota in adulthood and, consequently, also on our state of health and well-being. Although fecal microbiota transplantation has opened new frontiers on the prevention and treatment of many pathologies, it is indisputably true that this community of microorganisms represents a central node in the functioning of all our organs and systems, and at the same time it denotes a fundamental point of interaction between us and the environment in which we spend our lives.
2. Intestinal dysbiosis, immune system and related human pathologies
Intestinal dysbiosis is mainly characterized by lower bacterial diversity and it is often associated with an increase in bacterial species with pathogenic potential (
Moreover, together with the dysbiosis-related inflammation, the depletion of specific bacterial taxa involved in endocrine signaling may directly affect the function of different organs, and for these reasons dysbiosis has also been linked to metabolic, endocrine (e.g. thyroid-related) and also psychiatric disorders .
2.1 Dysbiosis in gastrointestinal disorders
A marked dysbiosis has been found to be associated with the main intestinal disorders, such as Inflammatory Bowel Diseases (IBD), Irritable Bowel Syndrome (IBS) and coeliac disease (CD). IBD are chronic inflammatory disorders characterized by the chronic activation of the immune system with an unbalanced production of inflammatory cytokines. Despite the pathogenesis of these diseases is unclear, there is evidence that, other than genetic and environmental factors, an abnormal immune response against the microbial component of the gut may be involved in inflammation development and maintenance. It has been supposed that dysbiosis could trigger an aberrant activation of immune system in IBD patients, resulting in an unbalanced inflammatory cytokine production. In particular, compared to controls, the anti-inflammatory butyrate-producing species
IBS is characterized by recurrent abdominal pain associated with a change in the bowel habits. IBS patients are divided into four subtypes: diarrhea-predominant (IBS-D), constipation-predominant (IBS-C), mixed diarrhea and constipation (IBS-M), and patients with non classifiable IBS symptoms (IBS-U) . These patients are characterized by a lower microbial diversity compared to the healthy population, and also by increased proportions of Proteobacteria and Firmicutes members, such as
Coeliac disease (CD) is a well-characterized gut autoimmune disorder triggered by the interaction between the gut-associated lymphoid immune system and the undigested gluten peptides that translocate through the epithelial barrier into the lamina propria. About 30% of the world population is genetically predisposed to develop CD, but only a small amount (about 1% in developed countries) develops the disease, so a multifactorial etiology is supposed for this disorder. CD patient microbiota is characterized by an increased relative abundance of
2.2 Dysbiosis in thyroid and autoimmune disorders
There is rising evidence that the intestinal microbiota compositional structure may impact on thyroid function, since microbial components can regulate iodine, selenium, iron and zinc uptake, and also enterohepatic cycling of thyroid hormones. Moreover, the microbiota may also impact on the bioavailability and metabolism of L-thyroxine and the anti-hyperthyroid drug propylthiouracil (PTU) . The gut microbiota influences the synthesis of neurotransmitters, such as dopamine, which can inhibit thyroid-stimulating hormone (TSH) and modulate hypothalamus-pituary axis. It is therefore reasonable to affirm that intestinal dysbiosis may contribute to the abnormal immune activation in Hashimoto’s thyroiditis (HT)  but also in Grave’s disease (GD), which is the second leading autoimmune thyroid disease. Studies on animals showed that microbiota transplant may increase the susceptibility to HT in rats. A proposed mechanism of action, is that
HT and GD evolve, respectively, in hypothyroidism and hyperthyroidism, with two distinct immunological patterns. HT is characterized by antibodies against thyreoperoxidase and thyroglobulin while GD is characterized by the presence of antibodies against TSH receptor. Nevertheless, in both disorders, anti-gliadin, anti-transglutaminase and anti-
2.3 Dysbiosis in metabolic disorders
Obesity, type-2 diabetes, metabolic syndrome and nonalcoholic fatty liver disease (NAFLD) are all metabolic disorders that manifest in comorbidity, and lead to an exacerbation of atherosclerosis and cardiovascular diseases . These disorders are characterized by different microbial signatures, which may contribute to their chronicization. The intestinal microbiota has an active role in regulating host metabolism, indeed experiments on mice showed that conventionally raised mice had more total body fat than mice raised in germ-free condition, and that a fecal transplant in these mice was able to restore nutrient adsorption, metabolic function and body fat .
In obese subjects, a lower bacterial richness was detected, along with a predominance of “pro-inflammatory” taxa, such as
Intestinal dysbiosis has also been found in subjects with a high risk for cardiovascular diseases compared to subjects with low risk. In particular, some bacterial genera, such as
2.4 Dysbiosis in cancer
Intestinal microbiota disruption has been linked to the development of cancer, and different specific strains have been linked to the development of different tumors. In colorectal cancer (CRC) a particular strain of
In hepatocellular cancer, the translocation of gut microbiota and its products via the portal vein seems to be a condition able to trigger inflammation and chronic liver disease that predisposes patients to the development of cancer .
Leukemia patients showed a marked dysbiosis. In acute lymphoblastic leukemia (ALL) patients, a lower microbial diversity has been found, along with an enrichment in
In non-small cell lung cancer (NSCLC) patients, a depletion of butyrate producers such as
2.5 Psychiatric disorders
There is evidence that psychiatric disorders such as schizophrenia (SCZ), autism spectrum disorders, mood disorders, and anxiety are linked to gut inflammation and that inflammatory status could be sustained by gut microbiota eubiosis breakdown . Epidemiological studies link autoimmune and atopic disorders such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and ankylosing spondylitis (AS) to affective, personality, and neurotic disorders .
A study conducted on Danish population demonstrated that individuals with SCZ have a 50% lifetime prevalence of autoimmune disorders. On the other hand, given a history of autoimmune disorders, the relative risk for SCZ increased by 45% .
An association between SCZ and RA, autoimmune thyroiditis, type 1 diabetes mellitus (T1DM), SLE, Guillain-Barre´ syndrome, psoriasis, multiple sclerosis (MS) and autoimmune hepatitis has been described . Interestingly, all these diseases have been associated with CD and non–celiac gluten sensitivity, with a higher prevalence of immunological markers of CD among these patients .
Clinical and animal preclinical studies support the relationship between gut inflammation and mental disorders. Indeed, high levels of pro-inflammatory circulating cytokines such as IL-1b, IL-6, and TNF-α, have been found in patients suffering from SCZ. Moreover, immunomodulatory drugs have been used to effectively treat psychosis . In patients with a high risk of psychosis, Clostridiales, Lactobacillales and Bacteroidales were found to be significantly higher than in healthy controls .
It has been hypnotized that the excessive rise of SCFA synthesis could be one of the causes of microglia activation. Studies on SCZ patients showed heterogeneous results on the microbiota dysbiosis so, despite such a dysbiosis was always confirmed in these patients, it is difficult to link specific taxa to this disorder . Anyway, fecal transplantation from SCZ patients to germ-free mice resulted in the development of SCZ-like behaviors in receiving mice, providing final evidence of the gut microbiota involvement in SCZ. An unbalanced microbiota was also detected in bipolar disorders and autism spectrum disorders, to underline that our gut microbiome may contribute, probably with varying importance, to most mental and stress-related disorders .
2.6 Neurodegenerative disorders
The implication of gut microbiota in neurodegenerative disorders has been widely investigated. Several clinical studies in Parkinson’s disease (PD) patients showed modifications in the gut microbiota, characterized by a rise in the relative abundance of
For what concerns Alzheimer’s disease (AD), animal experiments on mice with induced dysbiosis and on germ-free mice showed that microbiota manipulation can impact on disease severity and cognitive impairments. LPS seems to be involved in fibrillogenesis of β-Amiloid (Aβ), and some bacterial species, such as
3. Probiotics for the modulation of human microbiota: Effectiveness and limits
Since the second half of the 19th century, with Metchnikoff’s studies on the possibility of using lactic acid bacteria to decelerate the process of self-intoxication and infection by intestinal microbes  probiotics have been recognized as a tool to modulate the gut microbiota while conferring benefits to health. Their economic value was recognized shortly thereafter, and their global market is estimated to reach USD 69.3 billion by 2023 [https://www.marketsandmarkets.com/PressReleases/probiotics.asp]. Nowadays, probiotics represent one of the most commonly consumed food supplements worldwide, being present in yogurt, cheese, ice cream, snacks and nutritional bars, breakfast cereals, infant formulas and more recently also added to cosmetic products. They are also marketed as lyophilized pills, and their consumption is widely supported by physicians, particularly gastroenterologists . The administration of probiotics is indeed a more than feasible approach in clinical practice, compared for example to diet, despite its recognized role as a pivotal determinant of the structure and function of the gut microbiota, able to support homeostasis or
According to the International Scientific Association for Probiotics and Prebiotics consensus meeting in October 2013 , the framework “probiotics” must include microbial species that have been shown in properly controlled studies to confer health benefits. Probiotics are also new commensals and consortia that include defined strains from human samples, for which adequate evidence of safety and efficacy exists. On the other hand, live cultures, traditionally associated with fermented foods (with no evidence of health benefits), and undefined, fecal microbiota transplants must be kept outside this framework.
Probiotics may have several effects on the host, including certainly the modulation of the gut microbiota but also the metabolism of lactose with improved digestion or bile salts with various systemic effects, vitamin synthesis, direct and indirect pathogen antagonism, regulation of intestinal transit and alleviation of visceral pain, strengthening of the gut barrier, production of specific bioactives and neurological, immunological and endocrinological effects. As expected, some underlying mechanisms are observed across taxonomic groups, such as the inhibition of potential enteropathogens or the production of useful metabolites or enzymes, while others, especially those at the extra-intestinal level, are more likely to be strain specific. These effects can be contact-dependent and/or mediated by surface molecules, e.g. lipoteichoic acid, peptidoglycan, cell surface proteins, exopolysaccharide, pili or other appendages, or by secreted molecules, e.g. SCFAs and bacteriocins . In light of this, it is not surprising that paraprobiotics and postbiotics have recently been proposed as an alternative with a longer shelf-life and enhanced safety, especially for compromised individuals, with the former being non-viable (intact or broken) microbial cells or crude cell extracts  and the latter microbial cell constituents and metabolites, which act as bioactive compounds with local and systemic effects .
With specific regard to the gut microbiota, probiotics may impact resident communities through at least three different mechanisms: trophic interactions (
Among the main (although sometimes only suggested) prophylactic and therapeutic indications and claims of probiotics, we can certainly mention gastrointestinal diseases, including the prevention or treatment of acute, antibiotic-associated and
In this regard, the awareness that one size does not fit all is rapidly gaining ground. It is now a fact that distinct baseline features of the host (e.g. age and underlying medical condition) and its microbiota (taxa represented and functions performed), including varying environmental exposure (mainly diet), can actually lead to differing outcomes even with the same probiotic preparation. As discussed recently, this could for example be due to the fact that the individual configuration of the gut microbiota may be permissive or resistant to even transient colonization of probiotics . Moreover, it has been shown that probiotics could even perturb rather than aid in the recovery process of the gut microbiota after antibiotic treatment . It is therefore now clear not only that their validity is not to be considered absolute but also that, if not tailored, probiotic-based interventions could not be entirely risk-free.
Future directions will be the adoption of a mechanism-based approach, in which probiotic strategies are designed
Alongside traditional probiotics, it should be mentioned that novel candidate microorganisms with potential health benefits have been discovered thanks to recent research on the composition and function of the gut microbiota, deeply accelerated by massive sequencing. These microorganisms are referred to as next-generation probiotics or live biotherapeutics , as they fit well within the US Food and Drug Administration definition of live biotherapeutic as “a biological product that contains live organisms, such as bacteria, is applicable to the prevention, treatment or cure of a disease or condition of human being and is not a vaccine”. Unlike currently used probiotics, they are generally strict anaerobes and therefore present a number of manufacturing challenges, and they should undergo a formal regulatory approval process similar to drugs or any other medical intervention. Among them, we can list SCFA producers, e.g.
Alternatively, it has been thought to engineer GRAS organisms or commensals as a delivery vehicle for bioactive molecules or to express certain functionality. In this approach, the bacterial vehicle is known not to produce any virulence factors, it will be tolerated by the host and, if chosen carefully, may not even colonize the host. As an example, some researchers have used
However, in addition to the limitations discussed above, it should be emphasized that for most of these next-generation probiotic candidates, the available evidence is currently mostly preclinical,
In the future it is expected that overcoming all these challenges in the probiotics field will improve the state of evidence, regulation of use and, finally yet importantly, public awareness, for a precision, informed use. The current limitations in the field and future strategies to be undertaken to overcome them are summarized in Figure 3.
4. Foods and their prebiotic activities for the modulation of the gut microbiota
Food is a primordial need for our survival and well-being. However, diet is not only essential to maintain human growth, reproduction and health, but it also modulates and supports the symbiotic microbial communities that colonize the digestive tract, the gut microbiota. Type, quality and origin of our food shape our gut microbes and affect their composition and function, impacting on host–microbe interactions. Macronutrients (fat, protein, carbohydrate) and micronutrients (vitamins, minerals, polyphenols) directly interact with gut microbes and are involved in the production of key metabolites such as SCFAs and vitamins. Moreover, dietary fiber impacts on gut microbial ecology, host physiology, and health.
During or shortly after birth, the human gut is colonized by microbes. The fact that babies born spontaneously have higher bacterial counts in the gut at 1 month of age than those born by the cesarean section indicates that colonization of the gut by microbes starts and is improved during natural birth . The growth and maintenance of a healthy gut microbiota is essential for the development of the immune system and continues during breastfeeding, a stage that seems essential to the individual’s long-term health. Oligosaccharides found in breast milk encourage the growth of
A standard Western style diet offers about 50 g daily of potentially fermentable substrate, primarily dietary fiber, to the colonic microbiota. Non-starch polysaccharides are major components of dietary fiber and constitute 20–45% of the dry matter supplied to the colon. Simple sugars and oligosaccharides also account for another 10%, whereas starch (and starch hydrolysis products) supply less than 8% of dry matter. Some sugar alcohols also avoid the absorption of the small intestine and are minor dietary substrates for colonic microbiota . Approximately 90% of dietary polyphenols (approximately 1 g/day) avoid digestion and absorption in the small bowel and can have a major effect on microbial composition and activities.
About 5–15 g of proteins and 5–10 g of lipids, mainly of dietary origin, pass daily through the proximal colon. Various other minor dietary constituents, including catechins, lignin, tannins and others, also nourish colonic microbes . The action of all these macro and micronutrients is certainly synergistic and complex at the level of the intestinal microbiota, however in the following paragraphs we will analyze separately the effects of individual macro and micronutrients, trying not to lose the overall vision that is fundamental when it comes to microbial ecology.
4.1 Macronutrients and microbiota
These changes have also been observed in weight gain-resistant mice, which implies a direct effect of dietary lipids on the microbiota. It has recently been found that microbes in the small intestine are highly susceptible to fat load and are essential for lipid digestion and absorption. These data suggest that the regional microbiota composition may have significant functional implications, and highlight the need for distinct microbiota and microbiome analysis along the gastrointestinal tract . The lipid-mediated effects on the microbiota depend on the form and source of lipids. For example, mice fed with an isocaloric diet rich in long-chain saturated fats derived primarily from meat products showed greater insulin resistance and inflammation of the adipose tissue compared to mice fed with a high-fish oil diet. In addition, transgenic mice that constitutively generate n-3 polyunsaturated fatty acids (PUFAs) have higher phylogenetic diversity of the microbiome, which provides protection against the metabolic consequences of a high-saturated, high-sugar diet. One mechanism by which gut microbes can mediate the negative metabolic effects of high-fat intake could be by translocating LPS, a membrane toxin of gram-negative bacteria. An increase in circulating LPS after a high-fat meal has also been documented in humans, with amplified effects in obese people. Once in circulation, LPS induces a powerful inflammatory response by activating Toll-like 4 receptor signaling, which has been involved in cardiovascular and metabolic disease development .
Inflammation appears to be the common denominator among the seemingly unrelated biological negative effects of fats on the gut microbiome, involving the immune system and n-3 PUFAs. It is currently accepted that inflammation plays a key role in the progression of several chronic diseases, such as atherosclerosis, inflammatory bowel disease, cancer, diabetes, and neurodegenerative syndromes . Moreover, as described above, several evidence supports the role of n-3 PUFAs on the microbiota and on the regulation of inflammation and the immune system . In addition, dietary n-3 PUFAs have been shown to reduce clinical colitis in IBD patients . In clinical human studies, n-3 PUFA administration resulted in decreased Firmicutes/Bacteroidetes ratio, reduced relative abundance of Coprococcus and Facecalibacterium, and increased proportions of health-associated genera, i.e., Bifidobacterium, Lachnospira, Roseburia and Lactobacillus . These data were consistent with those obtained in a subsequent study in which the authors also found a significant correlation between the plasma levels of n-3 PUFAs and the relative abundance of SCFA producers . In addition, a diet supplemented with n-3 PUFAs has been able to prevent neuropsychiatric disorders and dysbiosis caused by social instability stress during adolescence, and these effects have been maintained through adulthood, supporting the concept that a healthy diet enriched in fish or n-3 PUFAs can have beneficial long-lasting effects and may help to prevent neuropsychiatric disorders . Taken together, all these data allow us to hypothesize the existence of a strong link between n-3 PUFA intake, gut microbiome shaping and modulation of the immune system, with the ultimate objective of hampering the existing loop between bowel inflammation and gut dysbiosis.
In the fat dietary component, n-3 PUFAs can rightly be considered prebiotics. Therefore, the consumption of an n-3-rich diet is currently thought to be beneficial for microbiota health, even if the gut microbiome changes induced in humans by n-3 PUFA supplementation deserve further clinical investigations.
What we can conclude for the fat dietary component is that the lipid excess present in HFD diet is dangerous for the microbiota and, on the other hand, that a diet enriched in n-3 PUFAs protects the microbiota from possible alterations. However, n-3 PUFA sources, mainly fish, should not considered completely safe, considering the pollution of the sea and the growing presence of microplastics and xenobiotics in the trophic chain of marine animals. In particular, scientific data suggest that shellfish and other small marine organisms consumed with their intestine pose particular concern because they accumulate and retain microplastics. The biological effects of microplastics in human gut are poorly understood, but it has been supposed that in high amounts they could cause an alteration of the gut microbiome, with cascading effects on host physiology .
Digestible carbohydrates are enzymatically degraded in the small intestine and contain starches and sugars such as glucose, fructose, sucrose and lactose. All these compounds release glucose into the bloodstream upon degradation, triggering an insulin response. Human subjects fed high levels of glucose, fructose and sucrose in the form of fruit, had increased relative abundance of bifidobacteria, with reduced
4.2 Micronutrients and microbiota
The administration of retinoic acid (physiologically active vitamin A metabolite) in patients with norovirus infection significantly increased the abundance of
Vitamin C is the most important antioxidant agent, and it must be obtained from dietary sources (mainly fruits and vegetables). This vitamin regulates the redox state and can considerably modulate the gut microbiota. In weaned piglets, vitamin C levels correlated positively with Firmicutes and negatively with Bacteroidetes relative abundances . Vitamin D is thought to be a multifunctional vitamin involved in calcium homeostasis and in a list of systemic physiological functions that include the modulation of gut microbiota . A randomized controlled trial showed that weekly vitamin D supplementation (50,000 ergocalciferol IU) over 12 months increased SCFA fecal levels and the relative abundance of SCFA-producing genera such as
Some vitamins of the B group have been shown to promote bacterial colonization of the gut, modulate bacterial virulence and participate in pathogen clearance . However, they may also have a role in the growth of enteropathogens, such as
It is evident that there is a high and complex interaction between vitamins and the gut microbiota: some vitamins are produced by the microbiota itself and others, particularly liposoluble vitamins, are responsible for its modulation. On the other hand, some of these vitamins may also contribute to enhanced virulence and colonization of potential pathogenic microbes. These studies together suggest that vitamin supplementation could modulate the gut microbiota, but its effects depend on the level of vitamin in the host and the microbiota status. Further clinical trials should be carried out to understand the effects of multivitamin supplementation, in order to evaluate possible effects linked to over-supplementation.
Conceptually, it is difficult to isolate the activity of polyphenols from the overall activity of the foods that contain them. Nevertheless, overall we can conclude that a diet rich in foods with high polyphenol content, can have positive effects on the human intestinal microbiota.
Artificial sweeteners such as saccharin, sucralose and aspartame have been considered as options that might be used to replace natural sugar to prevent and control glucose dysmetabolism. However, recent evidence suggests that consumption of all types of artificial sweeteners may induce glucose intolerance. It is important to note that artificial sweeteners are thought to mediate this effect also by altering the gut microbiota. For example, it was noted that saccharin-fed mice had intestinal dysbiosis with increased relative abundance of
Even on the large category of xenobiotics it is very difficult if not impossible to generalize. Just as an example, analysis of the microbiome of children with Crohn’s disease developed at a very young age showed that the most altered metabolic patterns in the gut microbiome were those related to xenobiotic metabolism .
4.3 Dietary patterns
Several popular diets have been studied for their ability to modulate the intestinal microbiota, including Western, ketogenic, omnivore, vegetarian, vegan and Mediterranean diets. The Western diet (high in animal protein and fat, low in fiber) has led in several studies to a marked decrease in microbial diversity and in some beneficial genera, such as
Ketogenic diets are characterized by a very low consumption of carbohydrates (5 to 10 percent of total caloric intake), sufficient to increase the production of ketone bodies. They were originally developed as a treatment for refractory childhood epilepsy, and the gut microbiota responses to a ketogenic diet seem to play a role in the effectiveness of this intervention in epileptic infants [41, 42]. In recent years, these diets are commonly adopted in order to obtain rapid weight loss and in some studies, they have been shown to improve longevity and reduce the onset of disease in experimental animals. Conversely, some human studies in which ketogenic diets were examined, suggest negative impacts on microbial ecology and gut health. These studies, however, were carried out in small cohorts with specific metabolic conditions, limiting the generalization to larger populations .
Vegan/vegetarian diets are both plant-rich diets associated with positive health outcomes and reduced risk of some diseases . The beneficial effects of these diets on human health could also be linked to intestinal microbiota modulation. Plant-based foods are the primary source of dietary MACs, and it has been found that individuals who consume vegetarian or predominantly plant-based diets have a microbiota metabolically optimized for MAC fermentation. However, some intervention and cross-sectional studies have found only modest differences in microbiota composition between omnivores and vegetarians, and suggest that the effects of dietary patterns on the microbiota are greatest at the level of genus and species, but relatively minimal on broader compositional features such as diversity . Despite the absence of a wide microbiota compositional shift, the species-level changes appear to be sufficient to alter metabolic outputs as SCFA production, which in vegetarians is typically increased. It is still unclear to what extent these microbiota-dependent metabolic outputs can mediate the beneficial effects of vegetarian diets.
Plant-based foods, in addition to supplying MACs, provide a diverse source of vitamins, polyphenols and other biologically active phytochemicals. Many phytochemicals may often reach the lower intestinal tract and have direct antimicrobial and anti-inflammatory effects in the intestine. Furthermore, microbial enzymes can modify phytochemicals into metabolites with increased bioactivity [118, 119]. So, microbiome-mediated changes in phytochemical bioavailability can be an additional mechanism underlying the beneficial effects of plant-based diets.
Several studies classify the Mediterranean diet as the most healthy and balanced human diet. It is characterized by a beneficial fatty acid profile, rich in both monounsaturated and polyunsaturated fatty acids, high polyphenols and other antioxidants and high fiber intake. Fruits, vegetables, cereals, legumes and nuts are at the basis of this diet, as well as consumption of fish and red wine . The potential benefits of Mediterranean diet on the gut microbiota are linked to the increased levels of fecal SCFAs together with an increase of
Even if there are different types of Mediterranean diet, as well as several ketogenic diets (e.g. normo- or iper-proteic) and even vegetarian diets (with or without eggs, with or without fish), what can be concluded in general about the effects of dietary patterns on the intestinal microbiota is that all those patterns which, for various reasons, tend to restrict the amount of vegetables, seem to be inadvisable. Thus the Western diet, which is poor in fruit and vegetables, and the ketogenic diets, which necessarily eliminate fruit for its carbohydrate content, appear to be diets with a probable negative impact on the intestinal microbial ecology. Despite this, comparative controlled clinical trials are needed to fully evaluate the possible short-term and long-term effects of these dietary patterns on the gut microbiome.
5. Use of fungus and officinal plants for the modulation of the intestinal microbiota and immune system
Microbiota and its multiple connections, already described in the previous paragraphs, remind us that every human being is an unrepeatable and unique Psycho-Neuro-Endocrine-Immuno-Somatic-Environmental unit that is constantly dynamic and interactive in its parts . From this perspective, the gastrointestinal system should be evaluated and treated as a neuro-immuno-endocrine-visceral-microbial interface of the human body. The modulation of the gut microbiota and, consequently, of the immune system is a key function of this complex network. Any disorder of the gastrointestinal tract, be it functional or with organic inflammatory basis, involves cells belonging to multiple tissues, including the sphere of the microbiota, and is therefore continuously reflected at the systemic level.
Consequently, even medicinal plants can, indeed should, act at multiple levels of the organism through direct and indirect actions that certainly, with various types of mechanisms, involve the Intestinal Immune System (IIS) and the intestinal microbiota. The action of fungi and medicinal plants is exerted on the gastrointestinal system through the immunomodulating, antioxidant and protective properties of the microbiota. Furthermore, the protection of the biofilm and the intestinal barrier, in the structuring of which the microbiota directly and actively participates, also fall within these therapeutic actions. These effects on the intestinal barrier and on the gastrointestinal system can obviously also have systemic consequences.
Several medicinal plants and fungi are described in the scientific literature as being able to act positively on various acute and chronic inflammatory disorders of the gastrointestinal system, most of these are also part of the medical tradition of one or more regions of the world.
Medicinal mushrooms that have been used in most preclinical and clinical studies are
In this brief discussion, we will limit ourselves to analyzing the scientific literature supporting possible therapeutic use of some of these fungi and these plants, in the modulation of intestinal inflammation and dysbiosis, the two components that are always associated in almost all pathologies of the gastrointestinal tract.
5.1 Microbiota-modulating fungi
The drugs used are the fruiting body and/or the mycelium in aqueous or hydroalcoholic or alcoholic extracts titrated and standardized in one or more of the following components: polysaccharides and beta-glucans (with anti-inflammatory and antibacterial action), alpha-glucans, diterpenes and triterpenes and polyphenols [125, 126].
The most studied activities of this fungus relate to its immunomodulatory effects on the gut, its anti-inflammatory systemic activity, but also its prebiotic activities on the intestinal microbiota .
A single protein, called HEP3, isolated from
Similar results were obtained in a model of dextran sulfate sodium (DSS)-induced colitis in mice. DSS treatment resulted in increased relative abundances of Verrucomicrobia and Actinobacteria and decreased amounts of Bacteroidetes in fecal samples, compared to the control group. Treatment of colitic mice with dry power of fermented
The most studied activities of this mushroom are the immunostimulatory effect exerted on the gut but also at systemic level. However, there is also evidence of prebiotic activity on the microbiota, although this could be secondary to a direct effect on immune system components. Its powerful immunomodulatory effects led to extend its field of use also to the therapy of tumors, a topic which, however, goes beyond the themes of this chapter . In DSS-induced colitis in rats,
In a mouse model of pancreatitis, induced by diethyldithiocarbamate (DDC), polysaccharides from
Finally, it should be emphasized that even if all these mushroom preparations can be easily found for free sale, and even if they do not seem to have side effects, it is a good practice to never use them in self-prescriptions as their direct interactions with drugs, or their effects on detoxifying enzymes such as CYP, have not yet been studied or poorly known. For example, Chaga extract inhibited platelet aggregation in mice. It may also have synergistic effects when used with anticoagulant/antiplatelet drugs, but the clinical relevance in humans is not known . Chaga may also interact with hypoglycemic agents drugs, since it has demonstrated to possess hypoglycemic activity in animals [144, 145]. A single case-report described oxalate nephropathy as a side effect associated with the ingestion of Chaga mushroom powder (4–5 teaspoons daily for 6 months), in a 72-year-old Japanese woman with liver cancer .
Chaga effects on detoxifying enzymes such as CYP have not yet been studied. Reishi may increase the risk of bleeding, interfering with anticoagulants/antiplatelets drugs . Reishi can also enhance immune response and this effect should be taken into account in patients on immunosuppressive therapy. Finally, at least
5.2 Microbiota-modulating plants
6. Aromatic plants end essential oils (EOs) as bowel “Eubiotics”
6.1 Intestinal microbiota modulation exerted by essential oils and aromatic plants
Aromatic plants are a wide group of herbs with characteristics aroma due to the presence of high amounts of volatile compounds known as EOs. Consequently, aromatic plants have always constituted a characteristic aspect of the gastronomic traditions. In recent years, the use of these aromatic plants has been replaced, especially in countries with high per capita income, with artificial flavors that allow the elaboration of more sophisticated aromas that in many cases are kept secret by the food industry, to avoid plagiarism. This replacement is certainly part of the transition from the traditional cuisines to the so-called western diet, the process called westernization of the diet that has taken place in many countries, parallel to the increase in the incidence of many intestinal diseases related to alterations of the gut microbioma, such as Inflammatory Bowel Diseases (IBD) . EOs have multitarget effects on the intestine due to their antioxidant, anti-inflammatory but also antimicrobial properties directed on the bacterial, yeasts, fungi and viruses components of the human microbioma . The antibacterial activity of EOs depends on the concentration that they reach into the gut, but also on the species of bacteria that they encounter. In fact, some OEs have more marked effects (i.e. lower Minimum Inhibitory Concentrations or MICs) for bacterial species considered pathogenic, while showing less activity (i.e. higher MICs) towards components of the microbiota such as bifidobacteria and lactobacilli . This multitarget positive effects of EOs on the intestinal microbiota, different from those obtained with the use of probiotics and prebiotics, has not found a definition in the literature yet. Hence, we propose here for the first time the term “eubiotic” activity since EOs restore the intestinal microbiota back to a physiological state of eubiosis, when a dysbiosis has been established into the gut.
6.2 Eubiotic proprieties of EOs on gut microbiota of animals and humans
There is no doubt that EOs are able to modulate the intestinal microbioma for their antimicrobial activities, which is one of the reasons why nature has selected these complex mixtures of active molecules with evolution. EOs may have “eubiotic” effects thanks to their capability to control and modulate bacterial growth, acting both as bacteriostatic or bactericidal agents . In fact, due to their lipophilic properties, EOs can penetrate membranes, and damage bacterial cells structure making their membranes more unstable and permeable. Membrane disruption may also lead to bacterial death caused by the significant leak of ions and other essential cytosolic components. These EO effects are generally more pronounced on Gram positive bacteria respect to Gram negative ones . However, it has been demonstrated that EOs can also affect bacterial cell wall in Gram-negative bacterial strains . Despite this, there are very few clinical studies of their eubiotic activity on humans, while the scientific data obtained on animals bred for human consumption or on experimental animal models are numerous and really convincing.
In broiler chickens, EOs have been widely adopted to improve intestinal microbiota and, as a consequence, to boost the growth performances of farmed animals. For example, the effects of liquidambar essential oils (LEO) isolated from Turkish sweet gum leaves (
Broiler chicken is not the only farmed animal treated with EOs for the purpose of modifying microbiota and reduce the susceptibility to infection by pathogenic bacteria. In farmed rainbow trout, the treatment with a mixed EO (containing eucalyptus, oregano, thyme and sweet orange EOs) caused significant microbiota changes in alpha and beta diversity, increasing also their growth performance and the final product quality. . In farmed pigs, oral administration of a EO mixture (containing cinnamon and oregano EOs) caused a significant decrease of infections caused by two porcine diarrhetic enterotoxigenic
Two different essential oils were tested on farmed ducks, again in order to improve their growth performance and also to replace the use of antibiotics in animal farming. One consisted of oregano oil, the second of thyme and cinnamon oil. Both of these EO preparations were able to decrease the cecal populations of coliforms and lactose-negative enterobacteria, demonstrating also in these animals an eubiotic effect of these OE on the gut microbiota .
Even on farmed crustaceans, a blend of organic acids and essential oils was tested for the improvement gut microbiota and disease resistance of Pacific white shrimps. Results demonstrated that this mixture was capable to enhance microbiota diversity and richness, increasing the abundance of Firmicutes and reducing the abundance of Proteobacteria. Also, a significant increase in the abundance of
All these studies as a whole demonstrate without doubt the eubiotic potential of orally administered EOs. Furthermore, they clearly demonstrate that dosages effective for modulating the microbiota are free of toxic effects on animals. Nevertheless, it remains rather difficult to understand which components of EOs are most active for modulating the microbiota, because of their natural complexity and their use in mixtures. For these reasons, several studies have explored the eubiotic properties of EO single molecules. The most studied was certainly geraniol, for its interesting antimicrobial potential. Geraniol antibacterial activity seems to be linked to his property to destabilize bacterial cell wall and damage transmembrane efflux pumps . Despite being absorbed very quickly and in an active manner by the small intestine mucosa, geraniol is reported to positively modulate the colitis-associated dysbiosis when administered by oral route by using a controlled delivery system based on microencapsulation . In mice but also in humans, geraniol has demonstrated to act as an excellent modulator of intestinal microbiota, capable to boost populations of butyrate-producer bacteria such as
Another interesting EO molecule with antibacterial activities is eugenol (2-Methoxy-4-(prop-2-en-1-yl) phenol), the major compound present in clove oil, but also found in many other EOs. Eugenol has demonstrated antimicrobial activities based on a non-specific permeabilization of the bacterial membrane with depletion of adenosin triphosphate (ATP), an energy moiety necessary for bacterial metabolism and survival . This effect has been observed against gut pathobionts such as
Cinnamaldehyde (2E-3-Phenylprop-2-enal) is a phenylpropanoid naturally present in the plant of the genus
Other molecules, such as thymol do not seem to show eubiotic effects in the gut, being non-selective and affecting all the intestinal bacteria and therefore behaving like a broad-spectrum antibiotics, depleting the microbiota even when administered at low dosages with a negative impact also on commensal bacteria .
Carvacrol, a major component of oregano EO, showed to inhibit bacterial adhesion, invasion and biofilm development in cultured intestinal cells [144, 145]. In farmed broiler, treatment with carvacrol-rich EO was tested to control the pathogenic bacteria spreading inside the farms. Results of these studies demonstrated that carvacrol reduced the microbial counts of
Limonene (1-Methyl-4-(prop-1-en-2-yl) cyclohex-1-ene) is a cyclic monoterpene present in high amount in EO of citrus fruit peels that has widely demonstrated antimicrobial and eubiotic effects
Eucalyptol (1,3,3-Trimethyl-2-oxabicyclo[2.2.2]octane) is a cyclic ether and a monoterpenoid. It is the major compound in
Menthol (5-Methyl-2-(propan-2-yl)cyclohexan-1-ol) is a chiral alcohol and the main molecule present in cornmint and peppermint EOs. It has been well known for its use in foods as a cooling and minty-smell aroma. Many
6.3 EOs in the modulation of gut mycobiome
Fungi were reported to represent about 0,1% of all the microorganisms present in the gastrointestinal tracts. Maybe also for this reason, despite the presence of fungi in the intestine has been known for many years, in depth studies of the human mycobiome were only recently performed . Together with bacteria, fungi contribute to the modulation of the intestinal immune system . Many of them have a clear pathogenic potential even if, physiologically, they are commensals in our bodies. Only in some specific conditions their overgrowth can lead to well-known mycosis. The best known fungal pathogen of humans is certainly
EOs antimycotic activities are characterized by a broad spectrum of actions .
Limonene has shown to possess strong antifungal properties  and in particular an excellent anti-
Mentha EOs have demonstrated good antimycotic activities against different fungi genus, including
Clove EO has been traditionally used in dentistry for its anesthetic and antimicrobial activities . Its anti-fungal action has been attributed to eugenol, the major clove oil molecule. A recent study indicated that Clove EO, at concentrations that ranged between 0,03% and 0.25% (v/v), inhibited the biofilm formation in many
EOs have also been shown to have strong antiviral activities, which could affect the gut virome, which is an integral part of the human microbiota . To date, no study has been performed to understand the impact of EOs on the intestinal virome. The main physiological viral component of the gastrointestinal tract is represented by prophages or phages . The bacteriophage component is mainly composed by temperate virus of the Caudovirales order, but most of the detected viral sequences in human gut virome could not be attributed to known viruses  and to date it is estimated that the number of virus in human stools is up to 109 per gram . Despite it is clear that EOs may impact on the intestinal virome composition by modulating all the microbiota components, it could be really difficult to understand the direct impact of EOs on the intestinal viruses and the consequences of this modulation on the intestinal ecology.
The scientific data present in the literature undoubtedly demonstrate that some EOs and some of their components are able to positively modulate the human intestinal microbiota, acting in a differentiated way on pathobiontic microorganisms, without altering or even improving the component of microorganisms defined as healthier commensals. This selective antimicrobial activity is certain for the bacterial component of the intestinal microbiota, conceivable for fungi, but at the moment completely unknown for viruses. It is therefore possible to define with certainty an eubiotic activity for some EOs and some of their components, such as for example geraniol, eugenol, cinnamic aldehyde and limonene, which can properly be considered as eubiotics. Finally, it is interesting to note that the antibacterial activities of these compounds are always multitarget and that for this reason the bacteria are unable to develop resistance. These data associated with the low toxicity of these compounds (by oral administration), suggests that these EOs may be part of a long-term therapy aimed at restoring an eubiotic and resilient microbial ecosystem.
The authors thank Dr. Alberto Sardo for illuminating us on the infinite potential of essential oils.
Conflicts of interest
The authors declare no conflict of interest.
This research received no external funding.