No changes in the abundance of selected fecal bacteria during increased carbohydrates consumption period associated with the racing season in amateur road cyclists

Introduction

A long-distance cyclists’ training schedule runs year round, but the competitions usually start at the end of March and, due to the required climatic conditions, are continued until October. Exhausting exercise, long-lasting racing season, and difficult weather conditions are the main reasons why cycling can be considered one of the most demanding sports (Lombardi et al., 2013).

Exercise or competition of long duration require proper nutrition strategy. Planning water and nutrients intake is an important part of the competition preparation, as prolonged exercise may lead to splanchnic hypoperfusion and gut dysfunction (Van Wijck et al., 2011). Long-distance cycling puts the human body under extreme stress, as it causes a shift in the distribution of the blood in the body. It increases abdominal organs ischemia, which is potentially harmful to the intestinal epithelium (through mucosal erosion and ischemic colitis) (Van Wijck et al., 2012). Local hypoxia depletes adenosine triphosphate (ATP), resulting in cell death and inflammation (Colgan et al., 2015). This, combined with hyperthermia, may lead to tight junction (TJ) proteins breakdown, which in turn increases intestinal permeability and endotoxemia. The exercise-induced endotoxemia intensifies the production and release of pro-inflammatory cytokines (Lambert, 2008). Endotoxemia may occur due to the “leakage” of undigested food particles, bacteria and pathogens from the intestinal lumen into the blood circulation (Camilleri, 2019). Besides the biochemical consequences of highly demanding training and exhausting competition there is an additional factor negatively affecting the intestinal barrier in cyclists. It is the specific body position, in which the cyclists spend most of the time. They lean forward to get as much aerodynamics as possible and it increases the abdominal pressure causing stomach distress (De Oliveira, Burini & Jeukendrup, 2014).

Exercise is a potential tool for the gut microbiota modulation, as it may increase the beneficial bacteria abundance and biodiversity. Evidence suggests that effects of exercise on individual taxa are variable, but usually they lead to an increase in commensal taxa such as Bifidobacterium, Lactobacilli, and Akkermansia (Hughes & Holscher, 2021). However, exercise-induced intestinal barrier impairment is an important risk factor for gut microbiota alterations (Karl et al., 2018; Kulecka et al., 2020). Gut distress during professional endurance sports participation promotes inflammation and affects recovery through the number of metabolic pathways (Snipe et al., 2018). It has been previously shown that dietary modifications required for endurance training and competition may alter the gut microbiota profile, intestinal barrier function, and, in turn, affect the athletes’ immune system (Jang et al., 2019). Strenuous physical exertion may also alter the circulation of bile acids, a group of mediators in the gut microbiota communication (Meissner et al., 2011).

Besides the physical activity levels or antibiotic therapies, dietary factors are generally considered as the main determinants of gut microbiota composition (David et al., 2014). Carbohydrate, fibre and protein intake gain the most attention in case of macronutrients impact on microbiota, but proper hydration is another important factor often difficult to control in athletes (Son et al., 2020; Campbell & Wisniewski, 2017). It is recommended for athletes to consume high amounts of monosaccharides to optimize glycogen storage and sustain blood glucose during exercise. Long-distance runners or cyclists are often instructed to avoid excessive dietary fibre intake, as it may slow down the digestion and cause gastrointestinal disturbances during physical activity (Rodriguez, DiMarco & Langley, 2009). On the other hand, low dietary fibre intake may lead to a bowel movements deterioration, thus potentially reduce the abundance of Akkermansia muciniphila and Faecalibacterium prausnitzii, responsible for short chain fatty acids (SCFAs), such as propionate and butyrate, production (Simpson & Campbell, 2015). It is suggested that these microbiota by-products regulate several biological processes, e.g., the inflammation through monocyte/macrophage and neutrophil suppression (Bermon et al., 2015). Gut microbiota modulation might become an important direction of research in the field of athletes’ recovery (Petersen et al., 2017).

The main aim of the study was to determine the potential effects of repetitive and intense cycling and carbohydrates-enriched diet on the abundance of the targeted gut microbiota in road cyclists at the amateur level. Additionally, we have compared targeted bacteria species abundance of similarly aged people with moderate physical activity level and a balanced diet (control group), to the participants from the study group. The primary outcome measure was the comparison of abundance of selected fecal bacteria between cyclists and healthy controls. Body mass and physical capacity indices, as well as fecal pH were considered as secondary outcomes.

Materials & Methods

Results

Nutrients intake in cyclists and control group at baseline (1st term of the study)

Figure 1 shows the proportions of nutrients consumed by the study participants. The dietary survey analysis presented significant differences in protein, carbohydrates, sugar and fat consumption between the examined groups (p < 0.01; Table 2). Road cyclists ate more calories from carbohydrates, especially from sugar, and less calories from protein and fat than controls. There was no significant difference in daily energy consumption between control group and cyclists at an amateur level before the competition season. The mean fiber intake in both groups reached the lower range of reference values for daily consumption.

Table 2:

Comparison of cyclists’ and control group daily energy [kcal] and carbohydrates fractions intake.

	Cyclists (n = 14)		Control group (n = 11)		T-test/ Mann–Whitney (p-value)
	Mean± SD	MedianIQR	Mean± SD	MedianIQR
Energy [kcal]	2612.1 ± 873.9	2396.3 1 491.9	2500.5 ± 1208.0	2459.2 1 610.1	0.7907
Sugar [%]	11.1 ± 4.0	9.7 4.0	5.7 ± 8.0	3.7 2.6	0.0009*
Sugar [g]	69.3 ± 22.7	65.2 33.5	53.3 ± 110.9	25.2 23.8	0.0016*
Fiber [g]	26.8 ± 13.9	24.5 14.9	25.6 ± 12.6	22.9 23.1	0.8481
Lactose [g]	8.4 ± 8.0	6.8 9.1	5.6 ± 4.4	5.4 7.6	0.3961
Starch [g]	160.6 ± 48.6	158.1 57.6	133.5 ± 54.6	128.3 90.9	0.2026

DOI: 10.7717/peerj.14594/table-2

Notes:

SD

standard deviation

IQR

interquartile range

*p < 0.01.

Cyclists’ nutrients intake analysis at 1st and 2nd term (pre- and post-competition season)

The dietary surveys collected from cyclists at the 1st and the 2nd stage of the study revealed significant differences in energy, protein and carbohydrate consumption (Table 5). The further analysis showed that cyclists have covered their increased carbohydrate demands mainly through starchy products intake increase. Small, but insignificant decrease of sugar and fiber intake was observed (Fig. 2).

Table 5:

Comparison of daily energy [kcal], protein [gkg b.w.], carbohydrates [gkg b.w.], and fats [gkg b.w.] intake of cyclists at baseline (1st term) and after the competition season (2nd term).

	Cyclists (1st term)		Cyclists (2nd term)		T-test/ Wilcoxon (p-value)
	Mean± SD	Median IQR	Mean ± SD	Median IQR
Energy [kcal]	2612.1 ± 873.9	2396.3 1491.9	2898.9 ± 900.6	2431.4 1258.6	0.0231*
Protein [g/kg b.w.]	1.32 ± 0.62	1.11 0.31	1.58 ± 0.50	1.43 0.61	0.0157*
Carbohydrates [/kg b.w.]	4.48 ± 1.56	4.34 0.6	5.18 ± 1.99	4.91 1.70	0.0355*
Fat [g/kg b.w.]	1.24 ± 0.51	1.14 0.45	1.39 ± 0.69	1.38 0.69	0.6934

DOI: 10.7717/peerj.14594/table-5

Notes:

SD

standard deviation

IQR

interquartile range

*p < 0.05.

b.w.

body weight

Discussion

There is a growing body of evidence indicating that endurance exercise may be an important, external factor that increases gut bacteria species richness (alpha diversity) (Batacan et al., 2017). Human studies have reported that subjects with higher cardiorespiratory fitness present higher gut microbiota diversity, especially in case of butyrate-producing bacteria (Bycura et al., 2021; Estaki et al., 2016; Barton et al., 2018; Allen et al., 2018). To date, the bacterial production of metabolic mediators, including short-chain fatty acids or secondary bile acids is the most explored mechanism involved in gut microbiota modulation of muscle function (Frampton et al., 2020; Hooper & Gordon, 2001; Wahlström et al., 2016). SCFAs can modulate the glucose metabolism via stimulation gluconeogenesis, or through the increase of the glucose, glycogen, and fatty acids bioavailability during exercise (Ticinesi et al., 2017). The effects of SCFAs on skeletal muscles metabolism is mediated for example by activating AMP kinase (Hu et al., 2010; Den Besten et al., 2013).

Although cyclists in our study presented better cardiorespiratory fitness than controls (Table 1), there were no significant differences in terms of targeted gut bacteria abundance (Table 3). A significant decrease in exercise capacity and VO₂max was shown in the cyclists after the competition season (Table 4), but it did not result in significant changes of the abundance of bacteria such as Bifidobacterium spp., Bacteroidetes spp., Faecalibacterium prausnitzii, Akkermansia muciniphila (Table 3).

It is proposed, that shift in the gut pH may be an important factor in creating the right environment for microbial diversity increase and metabolic functions maintenance. As exercise-induced increased fecal SCFAs concentrations can decrease luminal pH (Walker et al., 2005), fecal pH in our study participants was expected to drop. However, there were no significant differences in fecal pH between the group of cyclists and controls, as well as between cyclists in the 1st and the 2nd term of the study (Table 3). Values of fecal pH in study participants remained unchanged and within the neutral range.

It has been previously suggested, that the increase in microbial diversity occurs mostly when a maximal oxygen uptake (VO₂ max) is twice as high as VO₂ max in sedentary subjects (Joyner & Coyle, 2008). In our study the cyclists who competed at an amateur level had an average pre-competition VO₂max of 56.1 ml/kg/min, whereas top-level cyclists’ maximum aerobic capacity can reach values as high as 70–85 ml/kg/min (Faria, Parker & Faria, 2005). Although the baseline fecal abundances of Bifidobacterium spp., Faecalibacterium prausnitzii, and Akkermansia muciniphila in cyclists were below reference values for these bacteria, the competition season did not affect it significantly (Table 3). In the post-competition tests, we have noticed the effects of accumulated fatigue in athletes, which were proved by a decrease in physical capacity indices in the GXT test in the 2nd term of the study (Table 4).

Besides the endurance training and frequent racing, athletes’ nutrition could be an additional factor influencing individual taxa abundances in the gut (Mohr et al., 2020). It has been previously found, that diet composition modifies the A. muciniphila and F. prausnitzii, but there is still more room for improvement in this field of knowledge. Taxa such as A. muciniphila and F. prausnitzii are proposed to be a potential target in preventing inflammatory and metabolic conditions. However, it’s difficult to track the effects of different dietary approaches and particular foods or nutrients on the gut microbiota of athletes, as there are many other factors that could modify the effects (baseline gut microbiota, sex, sport characteristics, energy intake, history of medication and many others) (Verhoog et al., 2019).

In our study, the habitual dietary nutrients intake was different in cyclists and control group, and also in cyclists pre- and post-competition season (1st and 2nd term). The cyclists’ group (1st term) and control group ate similar amounts of calories, but the cyclists’ diet contained significantly more carbohydrates, especially sugar, and less protein and fat (Fig. 1; Table 2).

Energy, protein and carbohydrates intake have significantly increased during the cyclists’ racing season (Table 5). Carbohydrate loading is a common strategy used by endurance athletes to maximize glycogen concentrations before a competition (Burke & Hawley, 2018). Amateur cyclists’ carbohydrates intake fitted the recommended in endurance athletes range, which is 5–12 g/kg b.w. per day (mean 4.48 g ± 1.56 g/kg b.w. and 5.18 ± 1.99 g/kg b.w. on the 1st and 2nd term of the study, respectively) (Jeukendrup, 2011). It should be noted, that cyclists’ in the 1st term of the study almost doubled the control group in terms of saccharose consumption (Table 2). However, it has changed in the competition season, as the cyclists’ starch products consumption significantly increased alongside the small and insignificant sugar intake decrease (Fig. 2).

During both, the training and competition seasons, endurance athletes usually limit their intake of indigestible carbohydrates, such as fiber and resistant starch. It is recommended to avoid dietary fiber excess to minimize the risk of potential gastrointestinal problems, such as gas and bloating (Tiller et al., 2019; Rogerson, 2017). In our study, cyclists’ 1st term fiber consumption didn’t differ from that of the control group, and from the data taken at the 2nd term of the study (Table 2; Fig. 2). Both groups’ mean fiber intake didn’t reach the doses recommended in healthy population, which is 30–35 g per day for men (26.8 ± 13.9 vs 25.6 ± 12.6; cyclists and control group respectively) (Stephen et al., 2017). Fiber-consuming, SCFA-producing bacteria abundances, such as Akkermansia muciniphila and Faecalibacterium prausnitzii, also were lower than reference values in both groups and both terms of the study (Table 3).

The analysis performed in our study showed that cyclists’ daily protein consumption in pre-competition season was 1.3 ± 0.6 g/kg b.w., and it has significantly increased to 1.6 ± 0.5 g/kg b.w. throughout the competition season (p < 0.05; Table 5). This intake levels covered (1st term), and then exceeded (2nd term) the protein consumption recommendations for endurance athletes’, which are set within the ranges of 1.2–1.4 (Vitale & Getzin, 2019; Kato et al., 2016). It should be noted that the protein intake of men in the control group was significantly higher than that of cyclists’ and exceeded their RDA (Table 2). The concentration of dietary protein, it’s source and amino acid ratio may affect the colonic fermentation processes and furtherly influence the microbial metabolites levels (Ma et al., 2017). Despite the significant difference between the groups in terms of protein consumption, there were no statistically significant differences in the examined gut bacteria abundances (Table 3). It stands in line with our previous observations on amateur bodybuilders consuming high-protein diet (Szurkowska et al., 2021). Participants of this research have consumed on average 2,1 g of protein per kilogram of body mass (1,58 g in cyclists in the 2nd term), significantly more than sedentary control group, yet had only fecal pH increased.

In presented study, there were significant differences in fat consumption between cyclists and control groups (p < 0.01; Table 2). Cyclists’ fat intake was 32.4 ± 5.1% of total energy intake in the pre-competition tests, while during the racing season it was 31.1 ± 6.2% of total energy, which stands in line with current fat intake recommendations for athletes (approximately 30%) (Kerksick et al., 2018). The fat intake of the control group was 37.9 ± 4.7% of the total energy intake, which significantly exceeded cyclists’ fat consumption (Table 2) and the Dietary Reference Intakes (DRIs). High-fat diet may affect the amounts of dietary fat that actually reaches the distal gut, thus affect the gut microbiota (Rinninella et al., 2019). An increase in the pool of bile acids that eludes epithelial absorption in the GI tract is one of the mechanisms behind the potential reduction of the abundance of Faecalibacterium spp., as they are known as bile sensitive (Lopez-Siles et al., 2017; Mach et al., 2021). The LCHF (low-carbohydrate high-fat) diet is associated with a significant reduction in the relative abundance of Faecalibacterium spp. and an increase in the relative abundance of Bacteroides (Murtaza et al., 2019). In our study, both groups showed the relative abundances of Faecalibacterium spp. below the reference values (p < 0.01; Table 3).

Conclusions

It can be assumed that differences between the groups of amateur cyclists and more sedentary controls in terms of diet composition and physical activity levels were not large enough to distinguish the groups in regard to the gut bacteria composition determined in the study (Bacteroides spp. Bifidobacterium spp., Faecalibacterium prausnitzii, and Akkermansia muciniphila). Further research with bigger groups of cyclists with higher or low carbohydrates intake and including follow-up study is needed to clarify the effects of sport-related dietary interventions on gut microbiota composition.

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