1. Introduction
Obesity has become an epidemic of the 21st century, which is confirmed by the alarming data from the WHO on the NCD-RisC indicating a rapid increase in the prevalence of metabolic disorders in well-developed countries [
1,
2]. Records show that in 2016 more than 1.9 billion adults were overweight, of whom 13% were obese, meaning they had a BMI higher than 30 [
3]. Similar findings concern children and adolescents. It was estimated by the WHO that over 340 million children aged 5–19 years were overweight in 2016, and over 30% of these were obese [
4]. This is alarming given the fact that obese children have a significantly higher chance of becoming obese adults than non-obese children, which will directly impact the number of people suffering from obesity-related health implications [
5]. Since then, there has been no record of improvement in the situation. According to several predictions, by the year 2030 the number of obese children may double and further increase with time [
2,
6,
7].
There are various factors that promote the development of metabolic disorders, such as lifestyle changes that favor sitting or less active occupations, high stress levels, rush, and ill-considered dietary decisions [
8]. It is well-established knowledge that diet influences the gastrointestinal microbiota, which is reflected in several studies, i.e., comparing the microbiota of normal-weight and obese people or examining how certain types of food impact the gut microorganisms [
9,
10,
11,
12,
13]. Apart from contributing to energy metabolism, gut microorganisms produce a variety of bioactive compounds, such as vitamins, short-chain and branched-chain fatty acids (SCFAs, BCFAs), and fecal enzymes, which participate in several metabolic pathways where they exhibit anti-inflammatory, anticarcinogenic, and antioxidative effects (genera Lactobacillus, Bifidobacterium) [
14,
15,
16]. In contrast, a dysbiosis of gut microorganisms, or their low diversity favoring the domination of certain genera (Bacteroides, Clostridium, Escherichia, Enterococcus), may cause reduced synthesis of bioactive compounds and promote synthesis with the possible accumulation of potentially harmful ones [
17,
18].
SCFAs have a variety of beneficial effects on the host organism, i.e., regulation of energy metabolism, immunoregulation, and stabilization of the integrity of the intestinal barrier [
8,
19]. BCFAs, however, are often associated with the process of protein fermentation, which may lead to the accumulation of potentially harmful bioactive compounds and can increase the risk of developing colonic cancer [
20]. Microbial diversity in the gastrointestinal tract is similarly a factor that influences enzymatic activity in the colon. Bacteria produce a variety of enzymes that help to digest food ingredients while producing several bioactive compounds as byproducts. Reductases and hydrolases, which are the main classes of enzymes secreted by the intestinal bacteria, can help to produce beneficial SCFAs, but may likewise be responsible for the formation of toxic or carcinogenic compounds [
21,
22]. Due to the high impact of the above-mentioned bioactive compounds on the human organism, they can be considered health markers, which help to assess the physical status of a person.
Based on these facts, the aim of this study was to further confirm differences in the activities of fecal enzymes and the profiles of fatty acids in fecal samples between a group of healthy overweight or obese children and a group of children of normal weight. Accordingly, concentrations of SCFAs, namely formic, acetic, propionic, butyric, and valeric acids, together with concentrations of lactic acid were determined using the HPLC method. Furthermore, concentrations of BCFAs such as isobutyric and isovaleric acids were measured. The next part of the study involved the determination of activity of fecal enzymes, namely α-glucosidase, α-galactosidase, β-glucosidase, β-galactosidase, and β-glucuronidase, in the same fecal samples. The outcome of the experimental work allowed for the evaluation whether the group of obese individuals without dietary supervision has significantly different health markers (fecal enzyme activity and concentration of fatty acids). The obtained results were subject to statistical analysis to verify possible correlations among the mentioned health markers and elucidate more comprehensive conclusions.
2. Materials and Methods
2.1. Biological Material
The fecal samples were collected from 97 overweight and obese children (participants of the PreSTFibre4kids research program) and 26 children of normal weight (both males and females) aged 6–10 years (
Table 1). Patients from the PreSTFibre4kids program were children with one of the following health implications: simple obesity and overweight without organ complications, fatty liver disease, and high blood pressure. Volunteers were recruited in Poland. Every participant had voluntarily signed a consent form that confirmed his/her involvement in the project.
Exclusion criteria from the study included organ failure, metformin treatment, and allergy to any component of the prebiotic preparation that would be used in further phases of the research. The exclusion criteria did not include a medical history of taking antibiotics or the usage of dietary supplements. The overweight status of children was assigned according to the WHO’s BMI classification charts [
23].
Participants were given general dietary advice by a trained professional.
Fecal samples were transferred into sterile containers and frozen immediately (−20 °C) prior to transport to the laboratory.
The project was accepted by the Research Ethics Committee of the Children’s Memorial Health Institute in Warsaw, Poland (18/KBE/2021).
2.2. Activity of Fecal Enzymes
Prior to the enzymatic assays, the samples were prepared according to the following protocol. Firstly, 0.7 g of sample was suspended in 3.5 mL of 0.2 M phosphate buffer and vortexed thoroughly (Vortex RS-VA 10, Phoenix Instrument, Garbsen, Germany). Secondly, the samples underwent sonification (Time = 2 min, Amplitude = 60, Pulse = 6 s, Cole–Parmer Instrument Co., Vernon Hills, IL, USA). Afterwards, the samples were centrifuged (12,000 rpm, Time = 20 min, Centrifuge MPW-251, MPW, Warszawa, Poland) and the supernatant was transferred to the sterile Eppendorf tubes.
With the use of spectrophotometric methods, the activity of the fecal enzymes α-glucosidase, α-galactosidase, β-glucosidase, β-galactosidase, and β-glucuronidase was determined. The protocols used in the study were based on the reaction of α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase, and β-glucuronidase with 4-nitrophenyl α-D-glucopyranoside (TCI, Tokyo, Japan), 4-nitrophenyl β-D-glucopyranoside (TCI, Tokyo, Japan), 4-nitrophenyl α-D-galactopyranoside (TCI, Tokyo, Japan), 4-nitrophenyl β-D-galactopyranoside (TCI, Tokyo, Japan), and 4-nitrophenyl β-D-glucuronide (TCI, Tokyo, Japan), respectively.
The used substrates were specific to the respective enzymes present in the fecal sample. The reaction mixture contained 0.5 mL of phosphate buffer (pH = 7, 0.02 M), 0.05 mL of substrate solution (20 mM), and 0.25 mL of sample. Incubation was performed at 37 °C for 15 min (α-glucosidase, α-galactosidase and β-glucuronidase) or 60 min (β-glucosidase and β-galactosidase).
An observed hue shift in the sample to yellow proved the reaction had taken place. The intensity of the color was directly proportional to the amount of p-nitrophenol released. The reactions were inhibited using 0.25 M sodium carbonate after the given reaction time. The absorbance of the samples was measured using the spectrophotometer Rayleigh UV-2601 (BFRL, Beijing, China) at a wavelength of λ = 400 nm. The unit of enzyme activity refers to the amount of p-nitrophenol (expressed in µM) which was released during 1 h of reaction for 1 mg of protein in 1 mL of sample [µMh·mg−1].
2.3. Concentration of Lactic Acid, SCFAs and BCFAs
Prior to the HPLC analysis the samples were prepared according to the following protocol. Firstly, 0.5 g of sample was suspended in 3 mL of demineralized water and vortexed thoroughly (Vortex RS-VA 10, Phoenix Instrument, Garbsen, Germany). Afterwards, samples were centrifuged (12,000 rpm, Time = 20 min, Centrifuge MPW-251, MPW, Poland) and the supernatant was filtered (0.22 µm filters, ALWSCI Technologies, Shaoxing, China) and transferred to the sterile autosampler vials.
HPLC with the Surveyor liquid chromatography system (Termo Scientifc, Waltham, MA, USA) was employed in the experiment. The following parameters of the process were used: Aminex HPX-87H column (300 × 7.8 mm), UV detector, 0.005 mL−1 sulphuric acid as eluent, flow rate 0.6 μLmin−1, single sample analysis time 40 min.
2.4. Statistical Analysis
The normality of the distribution of variables was examined with the Shapiro–Wilk test, whereas the homogeneity of variances was assessed with Bartlett’s test. After the confirmation of normality and equal variance, the results were analyzed using the one-way ANOVA test and Tukey’s post hoc test. Python was used for statistical testing, where the p-value of 0.05 was considered significant. The data is presented in a mean ± standard deviation (SD) format.
4. Discussion
In the presented study, the main objective was to evaluate whether obesity has a significant effect on the metabolites present in the large intestine of children, namely lactic acid, SCFAs (formic, acetic, propionic, butyric, and valeric acid), BCFAs (isobutyric and isovaleric acid), and selected fecal enzymes (α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase, β-glucuronidase). Additionally, these parameters were also compared by gender.
The obtained results confirmed the stated hypothesis, that obese children have noticeably different activity of fecal enzymes and profiles of fatty acids. The observed disparity is mostly negative, as obese children when compared to children of normal weight had increased concentrations of BCFAs and increased activity of potentially harmful enzymes such as β-glucosidase and β-glucuronidase. Contrastingly, children of normal weight exhibited significantly increased concentrations of lactic acid and SCFAs (especially formic and butyric acids). Nonetheless, there was no linear correlation found between the increase in obesity status (value of BMI) and shifts in the activity of fecal enzymes and concentrations of investigated metabolites. The changes were observed only among two compared groups and among certain individuals, where participants with the highest BMI also exhibited extreme values for tested metabolites. This finding might suggest that other factors are very likely also affecting the state of metabolic activity in the large intestine.
Furthermore, the activity of beneficial fecal enzymes (α-glucosidase and α-galactosidase) was drastically higher when compared to the group of obese children. Such an observation should be considered positive given that these enzymes are beneficial for the host. α-glucosidase allows individuals to digest more fiber, thus producing more SCFAs, while α-galactosidase contributes to the digestion of dairy products which are of high importance for developing children. In comparison, significantly elevated activity of β-glucuronidase may promote the formation of colon cancer [
24,
25,
26]. Moreover, β-glucuronidase (similarly to β-glucosidase) has the ability to convert heterocyclic aromatic amines, polycyclic aromatic hydrocarbons, and some bile acids into harmful compounds (the products of such conversion are carcinogenic aglycons) [
27,
28,
29]. Furthermore, it was found by Li et al. [
30] that by inhibition of β-glucosidase it is possible to increase the effectiveness of chemotherapy against colorectal cancer and even to suppress the growth of cancer.
Another important aspect to consider is that, although α-glucosidase is a beneficial fecal enzyme, the α-glucosidase inhibitors are the major antidiabetic agents. A-glucosidase inhibitors are a class of drugs that are used to manage hyperglycemia, or high blood sugar levels, in people with type 2 diabetes. These drugs work by blocking the action of the α-glucosidase enzyme in the gut, which is responsible for breaking down carbohydrates into simple sugars. This results in a slower digestion of carbohydrates and a slower release of glucose into the bloodstream. Studies have shown that α-glucosidase inhibitors can have an effect on the gut microbiota and fecal enzyme activity [
31,
32,
33]. In general, these drugs have been shown to cause alterations in the gut microbial community, including changes in the abundance of certain bacterial species and the overall diversity of the gut microbiome. In terms of fecal enzyme activity, α-glucosidase inhibitors can decrease the production of certain enzymes, including α-glucosidase, which is the target of these drugs. This decrease in enzyme activity can affect the ability of the gut microbiome to digest and utilize carbohydrates, potentially leading to changes in the composition of the gut microbiota. It is important to note that the effects of α-glucosidase inhibitors on the gut microbiota and fecal enzyme activity may vary between individuals, and more research is needed to fully understand the implications of these changes. Additionally, the effects of these drugs on the gut microbiome may depend on the dose and duration of treatment, as well as other factors such as diet and lifestyle. Overall, the effects of α-glucosidase inhibitors on the gut microbiota and fecal enzyme activity are an area of active research, and it is important to monitor and understand these effects in order to optimize the use of these drugs in managing hyperglycemia in type 2 diabetes. Further studies are needed to determine the long-term implications of these changes on human health, and to develop strategies for mitigating any negative effects.
It is also important to note that elevated glucose levels in the bloodstream can contribute to the development of insulin resistance, which is a hallmark of type 2 diabetes [
34]. High α-glucosidase and β-glucosidase activity can increase glucose levels by breaking down complex carbohydrates into simple sugars that are rapidly absorbed into the bloodstream. In response to elevated glucose levels, the pancreas secretes insulin to bring glucose levels back to normal. However, if this process occurs repeatedly over time, the cells in the body may become resistant to insulin, leading to higher glucose levels that are harder to control [
35]. Another important aspect to consider is the role of incretins in regulating glucose levels. Incretins are hormones that are released in response to food intake and stimulate insulin secretion to help regulate glucose homeostasis by increasing insulin secretion and decreasing glucagon secretion in response to a meal. High α-glucosidase activity can increase incretin secretion and enhance insulin secretion, leading to improved glucose control [
36]. This enzyme is also important for the metabolism of dietary fibers, which are indigestible carbohydrates that are fermented in the large intestine by the gut microbiota. Increased activity of these enzymes can lead to an increase in the production of short-chain fatty acids (SCFAs), which can stimulate the release of incretins such as glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) [
37,
38].
This highlights the interplay between fecal enzyme activity, glucose levels, insulin secretion, and incretin levels, and underscores the importance of considering all these factors when trying to understand and manage glucose metabolism. It is also worth noting that fecal enzyme activity can vary between individuals and can be influenced by a variety of factors, including diet, genetics, and gut microbiome composition. For example, diets high in complex carbohydrates can increase the demand for α-glucosidase and β-glucosidase, while diets high in simple sugars can lead to decreased demand for these enzymes. Understanding the factors that influence fecal enzyme activity can help us better understand the factors that contribute to glucose metabolism and inform the development of new therapeutic approaches for metabolic disorders. In summary, the effects of fecal enzyme activity on glucose, insulin, and incretin levels are complex and multifaceted, and a better understanding of these effects can induce the development of new approaches to manage glucose metabolism and metabolic disorders.
An interesting finding was discovered after a comparison of profiles of metabolites by gender. These results clearly indicated that fatty acids were affected by gender, in several cases by a significant amount in both groups of children (obese and with healthy weight). Nonetheless, this phenomenon was observed to a lesser extent in the case of the activity of fecal enzymes, where differences between genders were found only within group of children of normal weight.
Furthermore, the in-depth analysis of the results suggested that there may be some differences in the ratios of fecal enzymes between male and female children, with male children generally having higher mean activities of α-glucosidase and β-glucosidase, while female children generally having higher mean activities of α-galactosidase and β-galactosidase. However, these differences may not be significant, and further research is needed to fully understand the impact of gender on fecal enzyme activities.
Nonetheless, when comparing the ratios of the enzyme activities within each group, it is important to keep in mind that the ratios will vary depending on the specific activity of each enzyme. The ratios can provide insight into the relative proportions of the different enzymes present in each group, but they should not be used to make direct comparisons between the groups. When comparing the enzyme activity ratios between the two groups, it is important to note that the control group had higher levels of all five enzymes studied, which suggests that the gut microbiota in the control group may be more diverse and healthier compared to the gut microbiota in the overweight group. In conclusion, the results of this study suggest that there are differences in the gut microbiota and fecal enzyme activity levels between overweight children and children of normal weight, but further research is needed to better understand the underlying mechanisms and the potential implications of these differences for health and disease.
All these differences can be explained by shifts of balance in the gut microbiota or changes in its diversity caused by unhealthy (or gender-influenced) diet or associated with the occurrence of obesity, which can have a major impact on the secretion of active compounds such as lactic acid, SCFAs, and BCFAs and the activity of fecal enzymes [
14,
39,
40]. However, further studies on the subject are necessary to assess the differences in dietary habits between children of different genders, and to link these habits with the occurrence of specific strains or makeups of microbiota present in the large intestine. It may be suspected that specific genders, even at young age, tend to follow a different dietary trend either imposed by parents, society, or their own preferences, which finally lead to differences in microbiota composition. Another factor might be connected with hormonal development or differences in children, which may also influence the composition of fecal microbiota, which was demonstrated in several studies [
41,
42,
43].
Fatty acids are produced in the large intestine as byproducts of the fermentation of dietary fibers and resistant starches by several species of bacteria residing in this environment. As mentioned before, SCFAs have many confirmed positive effects on the human body, while BCFAs are instead associated with negative impacts. Nonetheless, many studies have shown their beneficial effects, i.e., anti-inflammatory, anticarcinogenic, and anti-obesity, which suggests that their concentration might play a crucial role in the properties they exhibit [
44,
45].
Even though the topic of links between the anthropometric status of patients and the metabolites of their microbiota has been raised in the scientific community, to the author’s current knowledge there are not many studies that directly compare these differences. An interesting study by Covian et al. [
46] revealed quite similar findings to our study. It was shown that concentrations of BCFAs (isobutyrate and isovalerate) were directly correlated with the BMI. The higher the BMI, the higher the BCFA concentration. Nonetheless, contrary to our findings, the overall effect on fatty acids in the above-mentioned study was considered non-significant.
Moreover, several studies have indicated the association of specific genera of gut bacteria with either health benefits or the development of diseases. Bacteria from genera Clostridium, Bacteroides, Enterococcus, and Escherichia are usually presented as non-desired microbiota and are related to increased activity of β-glucuronidase (EC 3.2.1.31) and β-glucosidase (EC 3.2.1.21), which both have a significant correlation with the development of colorectal cancer [
27,
47,
48]. In contrast, genera such as Bifidobacterium and Lactobacillus present a positive impact on the enzymatic activity in the gastrointestinal tract. Various studies have demonstrated their anticarcinogenic properties and ability to decrease the activity of carcinogen-metabolizing enzymes [
49,
50]. First and foremost, these species are also directly connected with the prevalence of obesity. Bacteria from the genera Clostridium, Bacteroides, Enterococcus, and Escherichia are more prevalent in obese patients, while species from the genera Bifidobacterium and Lactobacillus are often considered probiotics and are associated with a healthy and balanced diet rich in fibers and vitamins [
8,
51,
52].
Furthermore, the gut microbiota and its metabolites might be affected by various other factors such as the amount and bioavailability of amino acids in the intestines, different metabolic pathways not necessarily directly linked to the gut, or several diseases such as depression and anorexia [
46,
53,
54,
55]. Hence, the studied metabolites can be treated as health markers, but with serious consideration and caution.
Further limitations of this study include that the determination of metabolites, both fatty acids and fecal enzymes, was performed in fecal samples, which does not necessarily reflect the actual concentrations and activity of these metabolites in different parts of the colon, but are mainly derived from the end of the gastrointestinal tract. Additionally, the tested metabolites are in constant fluctuation due to their absorption in the large intestine. Therefore, the true concentrations or activities of these metabolites might vary slightly. A further important factor that was not considered in this study was the water content of feces, which could also influence the results. Nonetheless, this problem was partially prevented by the selection process in which children with gastric problems were excluded from the study.
In light of these findings and the limitations, further studies on the comparison of profiles of metabolites produced by the gut microbiota in patients with different anthropometric statuses should be conducted to acquire further insight in the field of relationships in the gastrointestinal environment of humans.