J Physiol Biochem
DOI 10.1007/s13105-017-0564-2

REVIEW ARTICLE

Food additives, contaminants and other minor components:
effects on human gut microbiota—a review
Paula Roca-Saavedra 1 & Veronica Mendez-Vilabrille 1 & Jose Manuel Miranda 1 &Carolina Nebot 1 & Alejandra Cardelle-Cobas 1 & Carlos M. Franco 1 & Alberto Cepeda 1

Received: 25 January 2017 / Accepted: 10 April 2017
# University of Navarra 2017

Abstract Gut bacteria play an important role in several
metabolic processes and human diseases, such as obesityand accompanying co-morbidities, such as fatty liver disease, insulin resistance/diabetes, and cardiovascular
events. Among other factors, dietary patterns, probiotics,
prebiotics, synbiotics, antibiotics, and non-dietary factors,
such as stress, age, exercise, and climatic conditions, candramatically impact the human gut microbiota equilibriumand diversity. However, the effect of minor food constituents,
including food additives and trace contaminants, on humangut microbiota has received less attention. Consequently, thepresent review aimed to provide an objective perspective of
the current knowledge regarding the impacts of minor foodconstituents on human gut microbiota and consequently, onhuman health.
Keywords Antibiotics . Bacteroidetes . Dietary emulsifier .
Firmicutes . Food additive . Gut microbiota . Non-nutritive
sweetener . Proteobacteria

Introduction
Humans have approximately 10 times as many microorganisms (approximately 100 trillion) within their gastrointestinal* Jose Manuel Miranda
josemanuel.miranda@usc.es

1

Laboratorio de Higiene Inspección y Control de Alimentos. Dpto. deQuímica Analítica, Nutrición y Bromatología, Universidade deSantiago de Compostela, 27002 Lugo, Spain

tract (GI) than the number of somatic cells (10 trillion cells)
within their body [16, 28, 42]. Indeed, the gut microbiota(GM) contributes to health and disease in humans, being
sometimes referred to as the Bforgotten organ^ [27].
The GM play an important role in a number of humandiseases, such as obesity [3, 51], diabetes [15, 94], [56, 74,
89, 114], cardiovascular diseases [38, 116], metabolic syndrome [27, 39, 68], non-alcoholic fatty liver disease [2, 38,70], and in several psychiatric disorders [10, 45], which gut
microorganisms produce a large number of bioactive compounds that can influence human health [6]. Some (such asvitamins) are beneficial, but other products can be harmful[28]. Additionally, the GM interacts with the immune system,
providing signals to promote the maturation of immune cells
and the normal development of immune functions [11, 35]. In
this context, GM microbes contribute to maintaining the integrity of the intestinal epithelium, preserving cell-to-celljunctions, promoting epithelial repair following injury, andin the regulation of enterocytes turnover [103]. Thus, imbalance in GM can result in a pro-inflammatory luminal environment that could contribute to the progression of low chronic
inflammation and metabolic disorders [38].
The association between the GM and non-transmissible
chronic diseases have been widely investigated [33]. Amongthem, the link between the human GM and obesity has received great attention [51]. Thus, the modulation of the GM
can have beneficial effects to controlling obesity, and several
mechanisms that may contribute to microbiota-induced susceptibility to obesity and metabolic diseases have been proposed [80]. Changes in dietary patterns, and specific functional foods, prebiotics or probiotics intake, have the potential tofavorably influence host metabolism by targeting the GM andmay be a useful approach for the management of obesity andother adverse metabolic conditions [80]. Various nonnutritional factors, such as stress, age, exercise or climaticRoca-Saavedra et al.

conditions, can also dramatically affect the human GM diversity and equilibrium [28, 63, 68]. Additionally, the ability ofminor food components, or additives and chemical contaminants, to modulate specific components of the GM has been
acknowledged. However, the attention paid to the effect ofthese minor food constituents, food additives and trace contaminants on the GM has received less attention.
Consequently, the present descriptive aimed to provide an
objective perspective of the current knowledge surrounding
the effects of these minor foods constituents on the humanGM, and, consequently, on human health.

Composition and evolution of human gut microbiota
There is a continuum increase in the number of bacterial cells
occurring in the human gut that ranges from 101 to 103 bacteria per gram of contents in the stomach and duodenum, from
104 to 107 in the jejunum and ileum, culminating in 1011–1013
in the colon, particularly in the distal part [2], were around
300–500 different species live [42]. The GM also varies incomposition depending on the location along the GI and axial
depth (mucosal versus luminal) [49]. Globally, the microbial
mass in the intestine represents about 1 kg or more bodyweight and is essential to human metabolic functions [90].
Out of 53 known bacteria phyla on earth, only five to sevenphyla (predominantly Firmicutes and Bacteroidetes, comprising 90% of the total) usually colonize the human gut [107].
Firmicutes (the most predominant phyla in people living indeveloped countries) encompasses mostly Gram-positive bacteria with a DNA that has a low G + C content but also include
Gram-negative bacteria. The Gram-negative bacteria aremainly represented by the Bacteroides genus in the humangut [87]. The relative proportions of these two dominant phyla
vary and can be influenced by a range of factors, but mostpeople have similar proportions of each [28]. Lesser (but also
important) contributions from members of the Cyanobacteria,
Proteobacteria, Actinobacteria, Fusobacteria andVerrumicrobia phyla comprise the rest of the microbiota [90].
Bacteroidetes, Faecalibacterium, Bifidobacterium andEubacterium are numerically the most important genera
among GM and may account for more than 60% of the bacteria present in human stool, but their relative abundance ishighly variable across individuals [28, 101]. Clostridium,Enterobacteriaceae and Streptococcus are also important genera but less numerous [28].
One metagenomic analysis suggested that the GM of eachhuman is typified by one of three enterotypes, with eachenterotype featuring distinct dominant groups of microbes[6], namely Bacteroides, Prevotella and Ruminococcus.
However, subsequent studies, including those of The Human
Microbiome Project, have been unable to provide conclusive
evidence that supports this concept [55, 57].

The development of the human GM is a large and complex
process that begins during the fetal age [46]. Recent studies
have reported that microbial contact is initiated throughout
the course of fetal development and continues thereafter in an
accelerated manner [31, 36, 46]. The diversity of the GM in theinfant gut is initially very low, and the GM are generally
aerotolerant, as the gut initially contains oxygen, however, after
birth, they are replaced by anaerobes that are typically found in
the adult GM [27]. The GM alters considerably from birth to
6 months, when the GM appears to be relatively similar to thechildhood-type population [36]. At this age, one of the mostimportant factors contributing to the formation of the GM is thetype of lactation [31, 63]. The bacterial composition begins toconverge toward an adult-like GM by the end of the first year oflife and fully resembles the adult GM by 2.5–3 years of age [31,65, 125]. In terms of ecological succession, theBifidobacterium-dominated GM of the infant changes overtime into the Bacteroidetes- and Firmicutes-dominated GM of
the adult, which can be affected by several factors [6, 49, 55,112]. Among dietary factors, it was observed that subjects consuming a diet particularly rich in animal protein and fat (such as
the typical Western diet) were associated with the Bacteroides
enterotype, whereas the GM of subjects ingesting more carbohydrates were dominated by the Prevotella enterotype [125].
An increase in the phylum Firmicutes and a decrease in the
Bacteroidetes (mainly expressed as the Firmicutes/
Bacteroidetes ratio; FBR) were associated with obesity in someoccasions, but this feature is still not consistent [6].
Additionally, an increase of Actinobacteria in obese individuals
was also reported [6]. These changes are probably not a mere
consequence of obesity but could be involved or a cause fasterobesity pathogenesis [28, 68].
Once the GM has reached maturity, it remains mostly stable until old age, although some differences can be found in
the GM of the elderly from that of young adults [27].
Particularly, Bacteroidetes phyla and Clostridium genus predominate in the GI of elderly people compared to higher proportions of Firmicutes in young adults [63]. Elderly people arealso noted to have significant decreases in Bifidobacterium[63]. Young adults have variability in community composition
than for old age and vary greatly among individuals, ranging
from 3 to 92% for Bacteroidetes and 7–94% for Firmicutes[23, 27]. These findings could be related to the greater numberof morbidities associated with the elderly and the complex
repertoire of drugs used to treat them that are likely to affectthe microbiota [23].

Impact of the human gut microbiota on humanhealthThe GM equilibrium is essential for several physiological
functions associated with impact on human health, affectingFood additives, contaminants and other minor components

almost all organ systems that contribute to metabolic control[41]. Thus, the GM modulates appetite and food intake [42,125], absorption of nutrients from the gut, hepatic steatosis,
inflammation, triglyceride accumulation in adipose tissue[14], and fatty acid oxidation in skeletal muscle and the liver[125] and synthesis of vitamins. However, there is still limited
knowledge on the exact mechanisms by which the GM affects
human metabolism.
The GM express the enzymatic machinery to process otherwise non-digestible carbohydrates, such as fructooligosaccharides, galactooligosaccharides and inulin, and thus, releasemonosaccharides that can be used by the host for metabolic
purposes [63]. In addition to the conversion of complex carbohydrates into absorbable substrates, the GM also benefits
the human host by producing SCFAs, with great impact in thecolonic epithelial cells maintenance, and vitamins, like vitamin K, as well as some water-soluble B vitamins, such as
biotin, cobalamin, folic acid, nicotinic acid, pantothenic acid,pyridoxine, riboflavin and thiamine [2].
The GM also influences the host health status through the
enzymatic transformation of bile acids, natural detergents withnovel signaling functions including regulation of cholesterolsynthesis and absorption, modulation of inflammatory responses, and energy homeostasis [19]. Moreover, the GM
synthesizes amino acids, influences iron absorption, and it isinvolved in the conversion of dietary polyphenolic compounds and in the bile acid biotransformation process [63].
The intestinal microbiota is able to transform potentially carcinogenic compounds, such as N-nitroso compounds and heterocyclic amines, and to activate bioactive compounds including phytoestrogens [104].
Globally, although the healthier GM is not yet fully
established, it is well known that the richness and diversityof bacterial species in the human gut may be an indicator of
wellbeing, and consequently, alterations in GM can affectmultiple health issues [49]. In this context, compositionaland functional alterations in the GM have been linked to malnutrition [109], obesity and adiposity-related diseases [68,95], cardiovascular events [12, 76], type 2 diabetes [64], inflammatory bowel disease [85], colorectal cancer [127],
neurodevelopmental disorders [52] and aging-related disturbances [59, 76]. Considering the increasing global incidenceof many of these conditions, changes in the lifestyle and dietin the post-industrialization/westernization era have been argued to contribute to their emergence by shifting the GM
ecology [125].
Knowledge of the effects of specific microbial phyla is still
limited. However, the presence of Firmicutes, from diverse
families, namely Clostridiales, Erysipelotrichaceae,
Ruminococcaceae, Eubacteriaceae, and Lachnospiraceae
have been shown to be associated with healthy populations[63]. Additionally, certain bacterial genus such as Bacteroides,
Bifidobacterium, Clostridium clusters XIVa/IV, Eubacterium,

Faecalibacterium, Roseburia or Lactobacillus and even specific species, such as Akkermansia muciniphila ,Faecalibacterium prausnitzii or Roseburia intestinalis, havebeen shown to prevent health disorders such as obesity or
diabetes, or to improve immunity and inflammatory status[49, 51, 55, 63].

Effect of minor food compounds on the human gut
microbiota
Although dietary patterns have an important effect on the human GM, the individual effects of minor food compounds
have received less attention than conventional diets, with different proportions of macronutrients. Micronutrients are pivotal for several health-related functions, like energy metabolism, cellular growth and differentiation, and organ and immune function [9]. A diet low in micronutrients, but not necessarily low in energy, is frequent in populations of lowincome countries, but may also be present in povertyaffected settings in middle- and high-income countries [9]. It
is estimated that more than three billion people worldwide
suffer from various types of micronutrient deficiencies (predominantly vitamin A, iron and zinc), with the majority being
women and children [9]. Vitamin A can modulate the immune
response of the intestine by interactions with immune cells of
modulation of the microbiota [11]. Iron deficiency or anemia
is related to a depletion of Lactobacillus in women [7].
Some reports have shown that prophylactic doses of Zn invarious animal models increased the presence of Gramnegative facultative anaerobic bacterial groups, the colonicconcentration of short chain fatty acids (SCFAs), as well as
overall species richness and diversity [96]. Likewise, others
have found a gut microbiota enriched in members of the phylum Firmicutes, specifically Lactobacillus, following ZnO administration [101]. Moreover, even mild zinc deficiencies canprofoundly impact growth and development, as well as block
immune differentiation and maturation [96]. Supplementation
with high levels of zinc has been shown to result in an increaseof Lactobacillus in the GM of weaned pigs [108]. Using
chicks as a model, one study recently demonstrated that zinc
deficiency results in a remarkable change in the microbiota,
with metabolic changes, such as decreased SCFAs output[96].
Various other dietary constituents, including various compounds belonging to polyphenols, also nourish colonic microbes [28]. Polyphenols are secondary metabolites foundabundantly in a wide variety of foods, such as fruits, vegetables, herbs, seeds and cereals, and in beverages, such as coffee, tea, cocoa and wine [84]. The beneficial activities of polyphenols on the prevention of cancer and cardiovascular disease and, specifically, on the GM have been widely investigated in recent years [28, 39, 84]. Most polyphenols passRoca-Saavedra et al.

through the SI without being absorbed, thus encountering theGM, which colonizes the colon [84]. Once reached the colon
the interaction polyphenols-GM results in a two-way mutual
reaction. First, polyphenols are biotransformed in vivo bysome GM bacteria, increasing their bioavailability, and thusincreasing their effects on human wellbeing [39]. Second,
polyphenols modulate the composition of the GM mostly
through the inhibition of pathogenic bacteria and the stimulation of beneficial bacteria [39, 40, 42, 84]. Several phenolic
compounds have been recognized as potential antimicrobial
agents with bacteriostatic or bactericidal effects, and have various effects on bacterial species or genus [42, 76, 84]. About
90% of the dietary polyphenols escape digestion and absorption in the SI [116, 118] and can have a significant influenceon the microbial populations and their activities [69, 77, 119],but our understanding of the microbial bioconversion processes is still limited [69].
Flavonols [69], quercetin [37, 54], catechin and puerarin[54], anthocyanins [50, 52], ellagitannins [74], resveratrol[94], trans-resveratrol [37] and pterostilbene [41] are all reported to impact the GM. Quercetin supplementation resulted
in an altered composition of the GM at different taxonomic
levels, including the FBR and inhibiting the growth of bacterial species associated with diet-induced obesity, such as
Erysipelotrichaceae, Bacillus, and Eubacterium cylindroides[37]. In other recent work, it was demonstrated that different
types of flavonoids can modulate the growth of different phyla
and genus from GM [63].
Li et al. [73] demonstrated that ellagitannins can stimulate
the growth of several bacterial genera with beneficial properties for human health, such as Akkermansia muciniphila,
Butyrivibrio, Escherichia, Lactobacillus or Prevotella.
Proanthocyanidins from grape seed can increase
Lachnospiraceae, Clostridiales, Lactobacillus andRuminococcacceae in female pigs [22]. In another research,
Qiao et al. [94] found that resveratrol ameliorated thedysbiosis in the GM of mice induced by a high-fat diet.
Specific effects included an increase in the FBR, significant
inhibition of the growth of Enterococcus faecalis, and increased growth of Lactobacillus and Bifidobacterium.
Flavonols can also increase the relative abundance ofBifidobacterium and Lactobacillus at the expense of potentially pathogenic bacteria, notably Clostridium histolyticum [77].
In a recent work, it was demonstrated that pterostilbene (adimethoxy resveratrol derivative) supplementation in rats
exerted protective antiobesity effects, improved insulin sensitivity and modified GM by decreasing Firmicutes and increasing Verrucomicrobia. Regarding specific genus, pterostilbene
supplementation increased mucin-degrading bacterial members, such as A. muciniphila and Odoribacter spp. [41].
Besides polyphenols, other minor compounds such as conjugated linoleic acid [17], L-carnitine [61], choline [2],sphingomyelin [76] or ellagitannins [69] have been reported

to modulate the GM and consequently, impact human health.
Thus, Chaplin et al. [17] found that conjugated linoleic acidincreased A. muciniphila levels, that was associated with several beneficial associations with metabolism [16]. It was also
reported that L-carnitine, present in red meats, can be metabolized to trimethylamine and trimethylamine oxide, and increase atherosclerosis risk [66]. Colonic bacteria can hydrolyze choline to form dimethylamine and trimethylamine,
which are precursors of dimethylnitrosamine [2], a potent
hepatotoxin, and carcinogen.
Mice fed diet with 0.25% sphingomyelin showed a higher
relative phylogenetic abundance of the predominately Grampositive Firmicutes phylum and significantly lower numbersof the Gram-negative Bacteroidetes phylum and some intestinal pathogens [83]. Milk sphingomyelin supplemented mice
had a significantly relative abundance of the beneficial bacteria Bifidobacterium, and higher relative abundance of
Bacteroides, one of the few microbes that synthesize and utilize sphingolipids [83]. A summary of some previously published works regarding effects of food minor compounds inhuman GM is displayed in Table 1.

Effects of food additives on human gut microbiota
An important change in human diets since the mild-twentiethcentury is the increasing consumption of food additives thatare incorporated into almost all processed foods, often to aid
stability, shelf-life, taste, and texture improvement, particularly in processed foods [19]. The primary basis for approving
the use of these agents is the notion that they do not cause
acute toxicity at concentrations reasonably greater than theirapproved concentrations. However, only few prospective interventional human studies address the possible impact of additives on the human GM, presumably due to difficulties inallocation of cohorts of healthy individuals who have not beenpreviously exposed to food additives, and the need for robust
stratification of potentially confounding factors, such as genetics, lifestyle and dietary patterns [19]. Consequently, researchers have turned to animal models to study the effect offood additives on the GM. Recent studies have demonstrated
that the consumption of non-nutritive sweeteners (NNS) anddietary emulsifiers (DEs) can alter the GM, resulting in intestinal disturbance and inflammation, favoring the developmentof the metabolic syndrome [19, 26] (Table 2).
Nowadays, most processed foods contain one or more DEsin order to seek for specific textures. Some authors have suggested that DEs may be a factor resulting from industrializationthat has resulted in a reduction of GM diversity, altered hostmicrobiota interactions and, consequently, have contributed tothe increased incidence of metabolic syndrome and other inflammatory diseases in industrialized societies [19, 27]. Two
DEs, namely carboxymethylcellulose and polysorbate 80, haveModels

Observational study (8 anemic
and 26 normohemic females)
Pigs

Pigs

Rats

Rats

In vitro model of human gut

In vitro model of the human gut

Mice

Uncontrolled study including
22 healthy human volunteers

Case-controlled study including
22 healthy human volunteers

Mice

Mice

Chicken

[7]

[17]

[22]

[37]

[41]

[50]

[54]

[66]

[72]

[77]

[83]

[94]

[96]

Zinc

Resveratrol

Sphingomyelin

Flavonols

Ellagitannins

Flavonoids (quercetin, catechin,puerarin)
L-carnitine

Malvidin-3-glucose, gallic acidand a mixture of anthocyanins

Pterostilbene

Polyphenols

Proanthocyanidins

Conjugated linoleic acids (CLA)

Iron

Micronutrients

Zinc oxide supplementation at 42 μg/g or
2.5 μg/g

200 mg/kg per day

High-fat diet with 0.25% of milk sphingomyelin
added 45% Kcal as fat

Dark chocolate at 50 g/day for 1 week

Pomegranate extract at 1000 mg/day for 4 weeks

250 mgGallic acid (150 mg/L and 1000 mg/L),
malvidin-3-glucoside (20 mg/L and
200 mg/L), and enocianin (4850 mg/L
and 48,500 mg/L)
Each flavonoid at 0.15 g/L

15 mg/kg body weight and day for 6 weeks

Diet containing 1% (w/w) of grape seed extract
daily for 6 days
Trans-resveratrol (15 mg/kg body weight/day],
quercetin (30 mg/kg/day) or a combinationof both polyphenols at those doses

Cocoa flavonols increased the relative abundance ofBifidobacterium and Lactobacillus at the expense of
potentially pathogenic bacteria, notably the
C. histolyticum group
Decrease in Gram-negative bacteria, such as Bacteroidetes
or Tenericutes phyla and increase in Gram-positivebacteria, such as Firmicutes and Actinobacteria phyla
Resveratrol increased GM dysbiosis induced by a high-fat
diet, with an increase in the FBR, Lactobacillus andBifidobacterium growth and a significant decrease inEnterococcus faecalis
The zinc-deficient group had a significantly lower
cecal microbial diversity

Ellagitannins from pomegranate stimulated A. muciniphila,
Butyrivibrio, Enterobacter, Escherichia, Lactobacillus
and Prevotella and inhibited Collinsella

Catechin and puerarin presented different activities
on regulating the GM, but all increased GM diversity
L-carnitine supplementation significantly alteredcecal microbial composition, markedly
enhanced synthesis of trimethylamine/trimethylamineoxide, and increased atherosclerosis

All the anthocyanins tested significantly enhanced
the growth of Bifidobacterium spp. and
Lactobacillus−Enterococcus spp.

Quercetin attenuated the Firmicutes/Bacteroidetes
ratio and inhibited the growth of Erysipelotrichaceae,
Bacillus and Eubacterium cylindroides.
Trans-resveratrol supplementation alone or in
combination with quercetin scarcely modified the GM
Pterostilbene decreased Firmicutes levels and increased
Verrucomicrobia, A. muciniphila and Odoribacter spp.

Dramatic increase in fecal Lachnospiraceae,
Clostridiales, Lactobacillus and Ruminococcacceae

Fecal Lactobacillus were significantly lower
in anemic women
CLA supplementation exerted a prebiotic action on
Bacteroidetes/Prevotella and Akkermansia muciniphila.
However, it was not able to override the negative
effects of a high-fat diet on Bifidobacterium spp.

–
6 mg of CLA/day was given to mice consuming
both a normal-fat diet and a high-fat diet

Main conclusion

Supplementation dosage

Recent works regarding the effects of micronutrients on gut microbiota (GM)Reference

Table 1

Food additives, contaminants and other minor components

Case-control study including
22 healthy human volunteers[119]

Cocoa flavonols

High-cocoa flavonol (HCF) group received
494 mg cocoa flavonols/day), while low-cocoa
flavanol and low-cocoa flavanol group received
23 mg cocoa flavonols/day, for 4 weeks

Pronounced reductions were observed forEnterobacteriaceae and the Escherichia groupas well as for Lactobacillus spp. and for threeof five studied Lactobacillus spp.
Consuming the HCF drink for 4 weeks significantly
increased the Bifidobacterium and Lactobacillus
populations but significantly decreased theClostridia counts in fecal samples
57 (low) or 2425 (high) mg/kg zinc oxidefor 5 weeks
Pigs
[108]

Zinc

ModelsReference

Table 1 (continued)

Micronutrients

Supplementation dosage

Main conclusion

Roca-Saavedra et al.

demonstrated to promote bacterial overgrowth in the murine SI
and facilitate translocation of bacteria across a model gut epithelia [19]. Additionally, they reduced the mucus layer thickness and were involved in the onset of intestinal inflammation,
obesity, and diabetes. These effects were also associated withan increased food intake, from still unknown origin [27]. A
previous study, also carried out in mice, showed as polysorbate
80, enhances the translocation of E. coli across M-cells [35].
An increase in numbers of E coli have been found in association with Crohn’s mucosa [99]. There are studies showing that
the E. coli translocation can increase in 59 folds. Thus, thisemulsifier may contribute to the impact of dietary factors on
Crohn’s disease pathogenesis [99].
As a result of the many negative health conditions associated with the intake of excessive sugar, there has been an
upsurge in the consumption of NNS as an alternative [105,111]. NNS are synthetic compounds that are several hundredfold sweeter than sucrose. Thus, they can be used in small
amounts with negligible added caloric value. Some NNS are
excreted unchanged from the mammalian body, and are, therefore, considered metabolically Binert^ [105, 111].
Theoretically, NNS would only aid in weight loss if compensatory sugar intake did not occur. However, the common perception that NNS may promote weight loss by reducing calories is misguided because it was reported that consumption ofsaccharin-sweetened liquids might increase overall food intake [1, 105]. Furthermore, positive correlations between
NNS consumption and increased body mass index in children
and adolescents have been described in several observational
studies [43, 105].
The effects of NNS on the GM could be due to the bacteriostatic effects of the NNS, such as saccharin, sucralose, aspartame and stevia [1, 86, 110, 111]. Data from studies inanimals [1, 86] and from a small study in human subjects[110] suggests that the bacteriostatic effects of NNS are notlimited to the microbial inhabitants of the mouth, but extend to
those in the gut, thereby affecting the host metabolic phenotype and disease risk [89]. Pioneer work showed that 12 weeks
of exposure to Splenda significantly altered the GM composition by decreasing beneficial bacteria and was associatedwith weight gain in rats [1]. In another work, it was confirmed
and extended these findings by identifying a microbemediated mechanism by which NNS might influence metabolism [110], inducing higher glucose intolerance, mediated byalterations in the GM. Consistent with previous findings, it
was showed that 8 weeks of aspartame exposure in a dose
equivalent to human subjects consuming 2–3 diet soft drinks
per day, perturbed the GM and resulted in elevated fasting
glucose levels and impaired insulin tolerance in rats [1, 86].
Other additives reported to significantly alter the GM areessential oils (EOs), which were used to prevent the growth ofpathogenic bacterial species that are generally more sensitive
to EOs than most commensal bacteria [113]. It wasFood additives, contaminants and other minor components
Table 2

Recent works regarding the effects of food additives on gut microbiota (GM)Reference Models

Additive

Supplementation dosage

[1]

Rats

Splenda100, 300, 500, or 1000 mg/kg
for 12 weeks

[19]

Mice

Carboxymethylcellulose
and polysorbate-80

[29]

Rats

Aspartame

[32]

Pigs

Saccharin

[86]

Rats

Aspartame

5–7 mg/kg/day for 8 weeks[97]

In vitro trial

Sucralose

1.1–11 mg/kg

[110]

Mice

Saccharin

0.1 mg/ml in water for 11 weeks[113]

In vitro model of Thymol, nerolidol, eugenol, 100–500 mg/kg
the human gut
methyl isoeugenol
and geraniol

Main conclusion

Total anaerobes, Bifidobacterium, Lactobacillus,
Bacteroides, clostridia, and total aerobic bacteria
were significantly decreased. No significant
changes were found in the Enterobacteriaceae
1% of each emulsifier
Reduction in the microbial diversity, Bacteroidales,for 12 weeks
Verrucomicrobia phyla (particularly Akkermansia
muciniphila) and enriched mucosa-associatedinflammation-promoting Proteobacteria
Chow and high-fat feed added with Increase in total bacteria associated with aspartame
addition, and reductions in Lactobacillus
0.4 g/100 mL of aspartame inand Bacteroides
water for 8 weeks
Diet supplemented with 0.015%
Saccharin + neoesperidin dihydrochalcone
saccharin + neoesperidin
dramatically increased the cecal population
dihydrochalcone
abundance of Lactobacillus

demonstrated that EOs (mainly thymol), selected for their effectiveness against gut pathogens (C. difficile) did not havesignificant effects on the abundance of F. prausnitzii, whichplays an important anti-inflammatory role in the gut [113]. In
particular, EOs may have potential use as an adjunct to chemotherapeutic agents used to treat colorectal cancers [89].
Patients receiving chemotherapy for cancer treatments sufferfrom gastrointestinal disturbances due to damage to the mucosal cells of the GI and disrupt the gut ecological balance.
Consequently, chemotherapy increases the risk of bacterial
infections, such as the overgrowth of C. difficile [113] anddecreases beneficial microbial populations such as
B i f i d o b a c t e r i u m , L a c t o b a c i l l u s , Ve i ll o n e l l a a n d
F. prausnitzii. Consequently, EOs might be exploited as prophylactic agents and as adjuncts in chemotherapy to protectcommensal bacteria, including Bifidobacterium spp. andF. prausnitzii [126].

Toxic compounds produced by gut microbiota
metabolism
In addition to their action on certain populations of the GM,
some compounds can be metabolized by gut microorganisms

Aspartame increased total bacteria,Enterobacteriaceae and Clostridium leptum inthe feces, and attenuated the increase in
Firmicutes/Bacteroidetes ratio
Sucralose had little effect on E. faecalis and C.
sordellii, while there was a concentrationdependent inhibition of the growth of Bacteroides,
B. fragilis and B. uniformis
Saccharin induced an increase of the Bacteroidetes
and reduction in Firmicutes
Thymol and geraniol suppressed pathogens,such as C. difficile, with no concern for
beneficial colonic bacteria in the distal gut

and exert potentially toxic effects to their consumers. A summary of previously published work, describing toxic compounds produced by its metabolization by GM, is reported
in Table 3.
In particular, alcohol can be metabolized by bacteria toaggravate their intrinsic negative effects. Thus, oral bacteria,such as Streptococcus, have the capacity to convert ethanol inwine to acetaldehyde, which is an in vitro and in vivo
genotoxin and a recognized human carcinogen [15].
Furthermore, the GM is suggested to play an important rolein alcohol-induced liver injury, apparently through dysbiosis
of the intestinal ecosystem caused by alcohol intake [15].
Other example was reported that the occurrence of renal
injury in infants and children exposed to melamine-tainted
milk in China could also be attributed to the metabolism of
the GM [60]. Certain gut bacterial species, like Klebsiellaterrigena, can convert melamine to cyanuric acid, which thenforms complex precipitates that lead to kidney stone formationand causes renal toxicity [60].
Another group of compounds that can be metabolized by
the GM and cause harmful effects are contaminants, such as
drugs, heavy metals or environmental chemicals [25]. An interesting study showed how the GM has the ability to inactivate drugs delivered into the intestine, with the potential toRoca-Saavedra et al.
Table 3

Recent works regarding foods that can become toxic by the metabolism of gut microbiota (GM)Reference Models

Food/substanceDosageMain conclusion

[15]

Mice

Alcohol

The GM plays an important role in alcohol-induced
liver injury, apparently through dysbiosis of theintestinal microbial ecosystem caused byalcohol intake

[20]

Mice

Mixture of polychlorinatedbiphenyls (PCBs) congeners

10% v/v in drinking water for
7 days, plus an additional
oral gavage of 5 mg/kg
on day 7
150 μmol/kg for 2 days

[62]

Rats

Chlorpyrifos

1 mg for 30 days

[92]

Mice

Arsenic[106]

In vitro trial

Glyphosate

Cecal content of mice was added Thioarsenicals were found in soluble andwith 0, 200, 1000 andparticulate fractions of the reaction mixtures,2000 μg/kg arsenic
suggesting interactions with anaerobic
microbiota
Reduction of beneficial bacteria, such as some
0.05, 0.15, 0.075, 0.3, 0.6,
Bifidobacterium spp. or Lactobacillus spp. that
1.2 and 2.4 mg/ml
could disturb the normal gut bacterial
for 5 days
community, whereas limited effect was shownon the intestinal pathogens[120]

In vitro model Polycyclic aromaticof human gut
hydrocarbons (PAHs)

Hypothetical soil ingestionof 5 g/day

[122]

Mice

Nitric oxide (NO)

[124]

Mice

Nitrogen compounds

Daily intrarectal bolus treatment
with an NO donor in two
doses + 4% dextran sodium
sulfateDiet supplemented with 1.0%
betaine, 1.0% choline, 0.12%
trimethylamine N-oxide or
1.0% dimethylbutanol for
3 weeks

[129]

Rats

2,3,7,8-tetrachlorodibenzofuran 24 μg/kg for 5 days

[60]

Rats

Melamine

0.2 mg/kg

generate toxic compounds, like hydrogen sulfide [104]. The
gut normally converts luminal hydrogen sulfide to thiosulfate,
which can be further oxidized to tetrathionate. High concentrations of hydrogen sulfide severely inhibit cytochrome C
oxidase, blocking mitochondrial activity [104, 122].
Regarding effects of heavy metals by the GM, Pinyayev
et al. [92] reported that anaerobic microbiotas of the mouse
cecum convert arsenate into oxyarsenicals and thioarsenicals.
Additionally, it was reported that exposure to mercury altered

PCBs decreased the levels of Proteobacteria
and induced substantial changes in the gut
microbiome, which may then influence
their systemic toxicity
Chronic, low-dose exposure to chlorpyrifos wasfound to induce dysbiosis in the microbialcommunity with the proliferation of Bacteroides
sp. and decreased levels of Lactobacillus andBifidobacterium spp.

PAHs biotransformation potency of colon
microbiota suggests that the current risk
assessment may underestimate the risk fromingested PAHs
NO-producing microorganisms in the gut lumen
should be considered a modulating processduring colitis
Mice fed diets supplemented with trimethylamine
species (choline or trimethylamine oxide)
showed increased peritoneal macrophagecholesterol content and raised plasma levels oftrimethylamine oxide
Dietary 2,3,7,8-tetrachlorodibenzofuran altered the
GM by shifting the Firmicutes/Bacteroidetes
ratio. The cecal content was enriched with
Butyrivibrio spp. but depleted in Oscillibacter
spp. These changes in the GM were associatedwith altered hepatic lipogenesis,
gluconeogenesis, and glycogenolysis
Melamine is converted to cyanuric acid in vitro byKlebsiella terrigena cultured from normal rat
feces. Rats colonized by K. terrigena showed
exacerbated melamine-induced nephrotoxicity

the bacterial community in the gut of a terrestrial isopod(Porcellio scaber) [20].
Contaminants may be poorly absorbed after ingestion, andsubsequently can reach the distal SI and caecum by peristalsis.
Additionally, environmental chemicals (or their metabolites)
may also be excreted in the bile [25]. There is increasing
evidence that chronic exposure to environmental chemicals
through the diet, particularly persistent organic pollutants,
may promote the development of obesity and type 2 diabetes

Food additives, contaminants and other minor components

in humans, even without inducing dysbiosis [25]. Of particular interest is the role of the aryl hydrocarbon receptor, which
is bound and activated by a variety of persistent organic pollutants including coplanar polychlorinated biphenyls and halogenated aromatic hydrocarbons [129]. For instance, it wasrecently reported that a persistent organic pollutant, 2,3,7,8tetrachlorodibenzofuran, can dramatically alter the GM by
shifting the FBR, increasing Butyrivibrio spp. and decreasing
Oscillibacter spp. These changes in the GM were associatedwith altered BA metabolism and subsequent host metabolic
disorders as a result of an altered hepatic lipogenesis, gluconeogenesis, and glycogenolysis [25].
Conversely, the GM can regulate the expression of cytochrome P450 enzymes, which are involved in the metabolism
of a variety of environmental chemicals [24]. Polycyclic aromatic hydrocarbons are among the most widespread organic
pollutants and can be transformed by the GM to estrogenic
metabolites [120]. Furthermore, it has been shown that the rat
and human GM could regenerate benzo(a)pyrene from its hepatic conjugate, reversing the endogenous detoxification process, which is of potential toxicological relevance [24]. Choiet al. [20] reported that after exposure to polychlorinated biphenyls in mice, the most striking change in the intestinal
microbial profiles was a decrease in bacterial species.
Other environmental chemicals, for example, pesticides or
herbicides, can also exert increased harmful effects on humanhealth via the action of the GM [57, 98]. Indeed, chronic
exposure to chlorpyrifos, an organophosphate insecticidecommonly used to treat fruit and vegetable crops andvineyards has been shown to induce dysbiosis of the GM in
both human and rats and was associated with the proliferationof Bacteroides sp. and decreased levels of Lactobacillus sp.
and Bifidobacterium sp. [62]. Glyphosate, the most widelyused herbicide worldwide, has been shown to have important
effects in poultry GM [109]. The sensitivity to glyphosate isdependent on the bacterial strain. Some typical pathogens,such as Salmonella or Clostridium, are highly resistant,whereas beneficial bacteria, like Lactobacillus spp. or
Bifidobacterium spp. are moderately or high susceptible. No
trials were performed using human models, but if it were
demonstrated that glyphosate acts similarly in human GM,
this would be of a toxicological relevance [106].

Specific effects of antibiotics on the human gut
microbiota
Several drugs can modulate the GM. Although it was reported
that other pharmacological treatment can alter the GM [44,71], the drugs that primarily play the most significant action
on the GM are antibiotics [26, 78, 100, 102]. Antibiotics are
one of the most prescribed drugs in human medicine, particularly in pediatrics and neonatal nursing in developed countries[47, 67]. The effect of these drugs on the human GM, bothduring and after the treatment has been widely investigated inrecent years, although it is not yet fully understood [87].
Interestingly, although the effects of therapeutic doses ofantibiotics employed in human medicine have been widely
investigated in recent years, the effects on GM of antibiotics
residues present in foods at trace concentrations, derived fromveterinary medicine, have received little attention [18, 21, 82,98]. Only a few investigations focused on the effects of low
concentrations of antibiotics on the GM [30, 34, 121]. This is
surprising because antibiotics are the most widely used drugs
in the livestock industry in the world [8] and their residues canreach humans through animal feeds, vegetables and surface
waters [98]. Paradoxically, while humans are interested inmodulating their microbiota to aid in weight loss, producersof animal feed have used antibiotics for decades to increase
the weight gain of the animals. Antibiotics in livestock production are incorporated in animal feed either as growth promoters in countries where such use is allowed [30, 73, 98] oras prophylactic or therapeutic agents in the European Unionand other countries where antibiotic use as growth promotersis banned. Importantly, these antibiotic effects are not limited
to oral administration, but may also be present and, therefore,
have effects on microbiota when administered parenterally[31] (Table 4).
As a general rule, it was reported that antibiotic intake inmice increased adiposity [3, 18, 30, 67, 81, 117], and thus
favored the development of obesity and type II diabetes[79], besides affecting normal metabolic activity, hormonaland immune development. However, antibiotic treatment does
not always display adverse effects on the GM of experimental
animals. Indeed, in some instances, antibiotic treatment improved the insulin response in Bio-Breeding diabetes-prone
rats [13].
Antibiotics exert very different actions on the individual
groups that constitute the GM. Overall, for a variable period
after antibiotic treatment ceases, the microbiota usuallyregains its original composition. However, some bacterial species have been reported to irreversibly disappear in certain
individuals [27]. This can influence the health of the host,particularly if the bacterial group that is suppressed affects aphysiological health-related function [27].
Cho et al. [21] found a significant increase in the FBR
as a result of the administration of beta-lactams and vancomycin. An increase in this ratio, as explained previously in this review, is associated in diverse studies withobesity and other metabolic disorders. Other authors [30]found significant decreases in the taxa associated with
beneficial health properties, such as Lactobacillus spp.
and Bifidobacterium spp. and significant increases ofEnterobacteriaceae family that includes many genres considered potentially pathogenic. Other authors [102] treatedmice with antibiotics, such as amoxicillin, metronidazole,Prospective trial in 28,354 mother-childdays for 7 years

Prospective trial in 27 preterm infantsand 13 full-time babies

Mice

Mice

Prospective trial in 3 people before
and after antibiotic treatment
Observational study in 74 infants

Prospective trial on 6 patients

Prospective study in 12 males

Retrospective cohort study of 74,946
children with asthma and /or allergies

Prospective study in patients withno digestive diseases
In vitro model of the human gut

Mice
Mice

Observational study in 96 males

CalvesRandomized controlled trial
in 20 obese males

Mice

[3]

[5]

[21]

[30]

[34]

[48]

[58]

[79]

[81]

[87]

[100]
[102]

[115]

[121]

[123]

[128]

[91]

Models

Tetracycline and ampicillin

Vancomycin

Ampicillin, ceftiofur, penicillin andoxytetracycline

Vancomycin plus other antibiotics

Vancomycin
Vancomycin or streptomycin

Ampicillin + sulbactam and cefazolin

Different antimicrobials

Different antimicrobials

Vancomycin, gentamicin andmeropenem

Clarithromycin + metronidazole

Ampicillin and gentamicin

Ciprofloxacin

Penicillin

Penicillin, vancomycin, tetracycline orvancomycin + penicillin

Different antibiotics

Different antibiotics

Antimicrobial

Effects of antibiotics and concentrations on the gut microbiota (GM)Reference

Table 450 mg/kg or 2 mg/kg (for tetracycline)
and 30 mg/kg (for ampicillin)

500 mg for 7 days

0.005, 0.01 and 0.3 mg/ml and 0.1 g/ml,
respectively from birth to weaning

Different doses depending on the typeof disease and patient characteristics

Several antibiotics and doses dependingon the type of disease and patientcharacteristics
Several antibiotics and doses depending on thetype of disease and patient characteristics
Ampicillin + sulbactam on first days andintravenous cefazolin during 14 days
100 mg/L in drinking water
200 mg/L in drinking water

500 mg vancomycin, 40 mg gentamicin and
500 mg meropenem

250 mg + clarithromycin and 400 mg
metronidazole

Various dosages and treatment and durations

1 g/day for 5 days

Subtherapeutic dosages

Subtherapeutic dosages at 1 μg/g bodyweight per day

Several antibiotics and doses depending on thetype of disease and patient characteristics

Several antibiotics and doses depending on
the type of disease and patient characteristics

DosageEarly exposure to antibiotics increased the risk of being
overweight in later childhood by decreasing thediversity of the GM
Prematurity and perinatal antibiotic administration caused
lower percentages of Lactobacillaceae or Bacteroidaceae
and increased Enterobacteriaceae on GM
Antibiotic treatment induced significant changes in GM,
increased adiposity and modified lipid metabolism andcholesterol
Modified the GM and induced long-term changes in themetabolism of the host, inducing obesity
Ciprofloxacin treatment reduced the GM diversity, withsignificant effects on 1/3 of the bacterial taxa
Infants who received 5–7 days of antimicrobials in the firstweek had an increased relative abundance of Enterobacterand lower bacterial diversity in the second and third weeks
of life
Antibiotic treatment affected the GM by decreasing
Actinobacteria and this disturbance on the GM persisted
after 4 years
Antibiotic treatment caused significant shifts in the GM.
Nevertheless, the changes observed did not haveimportant effects on glucose metabolism
Exposure to antibiotics during the first year of life wasassociated with an increase in the body mass index of
5–8-year-old children
Both fluoroquinolones and beta-lactam reduced the GM
diversity in more than 25% of the patients
Antibiotic treatment caused a marked decrease in
Bacteroidetes and increase in Firmicutes
Different antibiotics had specific effects on the GM
Vancomycin caused a loss in Bacteroidetes, which were largely
replaced by Firmicutes, Paenibacillaceae, Verrucomicrobia(specifically Akkermansia), and Enterobacteriaceae. In
contrast, streptomycin increased the Bacteroidetes,
particularly Porphyromonadaceae and Bacteroidaceae
Vancomycin plus gentamicin treatment increased the risk ofobesity in men. High levels of Lactobacillus were found,possibly related to the use of vancomycin as a growthpromoter
Antibiotic residues resulted in discriminate GM
communities, although they did not result in
disruption of the taxonomic levels above the genus
Vancomycin reduced fecal microbial diversity with adecrease in Gram-positive bacteria (mainly
Firmicutes) and a compensatory increase inGram-negative bacteria (mainly Proteobacteria)
Antibiotic oral administration had important effects on
the selection and extent of antibiotic resistance genes

Main conclusion

Roca-Saavedra et al.

Food additives, contaminants and other minor components

cefoperazone, and a combination of all three. As a result,the Proteobacteria and, in particular, the Enterobacteriaceae,
become dominant in the intestinal tract of the treated mice,accounting for 73% of the total microbiota. Two weeks after
ceasing the antibiotic treatment, the microbiota of these animals recovered a relatively low proportion of Proteobacteria(5.77%), although it remained considerably more abundant
than the percentage of the total microbiota representing this
phylum in untreated mice (1.2%).
Indeed, although Proteobacteria usually represent about
15% of the intestinal microbiota, they accumulate morethan 35% of the antibiotic resistance genes contained in
the microbiome. In contrast, despite representing 31% of
the total microbiota, Bacteroidetes accumulate only 6% of
the antibiotic resistance genes [53]. Hence, it is highly
feasible that an antibiotic treatment can cause fewer declines in the population of Proteobacteria (or even increase, occupying the space left by other bacterial groups
more sensitive to the action of the antimicrobial) thanBacteroidetes, for instance. Similarly, it is also reasonable
that once the Proteobacteria reach a high proportion within the microbiota, before gradually declining, its population will be maintained at high levels compared to prior to
the administration of the antimicrobial.
Another study developed in experimental animals showedthat after treatment with cefoperazone (a broad-spectrum antibiotic), there was a significant loss of microbial diversity,
without recovery, even at 6 weeks post therapy [4, 47]. In
another research work [102], in which mice were given vancomycin or streptomycin in their drinking water, no significant changes regarding the action of streptomycin were found,while vancomycin was associated with significant variations
in both the bacterial load and diversity. An almost total removal of Bacteroidales and a marked enrichment of Lactobacillus
were observed.
However, humans have a greater variation in diet and lifestyle than experimental mice, which introduces factors affecting the recovery of metabolic disturbances or susceptibility toweight gain [30]. Hence, the influence of antibiotics on the
GM of humans, particularly children, has been studied.
Children are often the most exposed to antibiotic treatments
within the human population and typically experience thegreatest effects [47]. Indeed, some reports suggest that exposure to antibiotics within the first 6 months of life predisposes
the individuals to a significant increase in body mass in later
life [3, 80, 117]. However, other authors found conflicting
results, suggesting important differences according to the antibiotic regimens, the routes of administration, the choice of
methods of statistical analysis, or other poorly controlled factors [47].
Antibiotic treatments can also significantly alter the microbiota composition of the adult GI, causing a decrease in the
microbial diversity to between one-quarter to a third of the

pre-antibiotic state [47]. However, in this stage of life, theGM is relatively strong and, in most instances, recovers after
several weeks of ceasing the antibiotic treatment [87].
However, other studies have shown that after cessation oftreatment, the microbiota requires several months to fully recover [34, 58, 75, 87]. However, in some cases, it has evendemonstrated that some bacterial groups eliminated by an antibiotic treatment not reappear again in several years after
discontinuation of treatment [27, 31, 123]. These effects canbe more severe in elderly people, in whose GM is less diverse
compared to younger adults and a more unstable balance thatcan easily lead to the emergence of various pathologies [23,93].
It has also been shown that upon contact with antibiotics,
the GM is perhaps the most accessible reservoir of genes
encoding antibiotic resistance due to their high density within
the gut ecosystem, which can have important consequences
for human wellbeing [31]. The GI is also an open system,
which incorporates everyday bacteria from the environment[88]. These incoming bacteria often possess antibiotic resistance genes, and besides being a potential risk to the host,because these resistance encoding genes can be transferredto the host.

Conclusions
The vast majority of experimental evidence supporting theeffects of food minor components and contaminants on GM
has been generated in animal (especially mice) models.
However, mice and humans differ in their microbiota composition, immune function, diets, and metabolism and the results
obtained in mice are not totally extrapolable and valid tohumans. Thus, interventional studies in humans are also needed, although are seriously limited by ethical concerns. In thissense, the use of in vitro models of the human gut enables
investigating the effects of minor compounds (even those dangerous for humans) without health risks and ethical concerns.
In view of the results explained in the present work, there are alarge variety of food minor components, additives and chemical contaminants that can dramatically affect GM. Thus, there
is a profound need for more in-depth investigations into theeffects on the human GM of the cited compounds.

Acknowledgments The authors want to thank the European Regional
Development Funds (FEDER), grant GRC 2014/004 for covering thecosts.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict ofinterest.

Roca-Saavedra et al.

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