Pepper power: short-term impact of pepper consumption on the gut bacteriome composition in healthy volunteers

Introduction

The human gut microbiota, composed of diverse microorganisms inhabiting the gastrointestinal tract, plays a pivotal role in maintaining health and preventing disease (Ayeni et al., 2018). This complex ecosystem, influenced by a myriad of factors, including genetics, age, and environmental exposures, has been the subject of extensive research due to its profound implications for human well-being (Cénit et al., 2014; Cahana & Iraqi, 2020). Among these factors, diet stands out as a significant modulator of gut microbial composition and function (Zmora, Suez & Elinav, 2018; Kurilshikov et al., 2021). Dietary choices not only shape the diversity of microbial species residing in the gut but also influence their metabolic activities, impacting various aspects of host physiology, including immune function, metabolism, and even mental health (David et al., 2014; Berding et al., 2021). Thus, understanding the intricate interplay between diet and the gut microbiota has become a paramount area of investigation in contemporary biomedical research.

One food item that has garnered attention for both its culinary appeal and potential health implications is pepper, derived from various species of the Capsicum genus. The Capsicum genus comprises more than 200 varieties; the five major species are C. annuum, C. baccatum, C. chinense, C. frutescens and C. pubescens., with C. chinense reported by some authors as the hottest pepper in the world (Sosa-Moguel et al., 2017). The active compound in pepper is capsaicin and it is involved in its health properties (Rosca et al., 2020). Peppers belonging to the genus Capsicum have characteristic pungency and flavor with a long history of human use. They are recognized as one of the earliest spices employed in cooking (Perry et al., 2007; Hill et al., 2013). Pepper is a ubiquitous spice that is used to enhance the flavors of numerous regional and global dishes. Beyond its culinary significance, pepper has been recognized for its potential health benefits, echoing ancient beliefs in spices as remedies for humoral imbalances, a concept rooted in the teachings of Hippocrates and Galen (Nam & Nam, 2014).

Modern scientific investigations, both in vitro and in vivo, have substantiated the therapeutic potential of Capsicum sp. its bioactive constituents, and phytochemicals (Ao, Huang & Liu, 2022). The phytochemical activity of pepper compounds, such as capsaicin and piperine, has been associated with various health-promoting properties, including anti-inflammatory, antioxidant, and antimicrobial effects (Dludla et al., 2023). Its antimicrobial properties have been demonstrated against pathogens such as Helicobacter pylori, other bacteria, and fungi (Lai & Roy, 2012; Tharmalingam et al., 2014; Füchtbauer et al., 2021; Manjunath et al., 2022). Additionally, emerging research suggests that Capsicum sp. pepper may exert profound effects on the intestinal microbiota and influence the pathogenesis of metabolic disorders (Qin et al., 2012; Tang et al., 2013; Rosca et al., 2020).

However, despite its popularity, the consumption of Capsicum sp. pepper has been occasionally associated with gastrointestinal discomfort, alterations in stool patterns, and could ultimately result in gastrointestinal disease conditions (Esmaillzadeh et al., 2013; Chaiyasit & Wiwanitkit, 2016). This momentary diarrhea has been attributed to the irritation of the digestive system (stomach and intestinal linings) by capsaicin, a potent ingredient in chili peppers which triggers certain receptors in the digestive system, resulting in burning sensation followed by rapid and hence inadequate processing of the food (Chaiyasit & Wiwanitkit, 2016; Xiang et al., 2022). Conversely, other studies have reported the alleviating effect of Capsicum sp. pepper in calves, pigs, and piglets (Cairo et al., 2018; Moraes et al., 2022; Su et al., 2023; Satitsri et al., 2023). However, there is lack of information about the direct effect of pepper consumption in human. This lack of information has prompted scientific inquiry into the effects of Capsicum sp. pepper consumption on gut health and its influence on the composition of the human intestinal microbiota.

This study seeks to address the potential effects of short-term dietary intervention with pepper on the gut bacteriome composition in humans. We aimed to elucidate whether the consumption of pepper leads to discernible alterations in the microbial landscape of the human gut. Additionally, we sought to identify any associations between pepper intake and the abundance of specific bacterial l taxa.

Materials and Methods

Sample collection

Individuals recruited for the study consumed 200 ml of 0.14 g/ml of fresh Habanero Pepper (Capsicum chinense) once each day for the first 4 days after which they abstained from all form of pepper consumption for the subsequent 4 days. The last stage involved the subjects resuming their normal pepper consumption in their diet, 4 and 10 days into which samples were likewise collected (Fig. 1). Fecal samples were obtained from all 10 volunteers at five time points (initial, after 4 days of pepper consumption, after 4 days without pepper, after 4 days of a normal diet, and after 10 days of a normal diet). The fecal samples were preserved according to the method reported by Ayeni et al. (2018) and subsequently transported to Vassar College, Poughkeepsie, NY, USA for further analysis.

Results

Ten healthy adults (five males and five females, mean age = 27.3 years) provided 52 fecal samples in total. The subjects underwent four pepper consumption phases: baseline, higher pepper consumption, no pepper consumption, and normal pepper consumption. The V3–V4 of the microbial 16S rRNA gene was sequenced using the Illumina MiSeq platform, generating a total of 651,463 high-quality paired-end reads, with an average of 12,528 reads per subject. These reads were further clustered into 1,838 OTUs/ASVs.

We confirmed that the data significantly deviated from the normal distribution (p = 1.089432 × 10⁻⁹¹), as is expected for microbiome data. Alpha diversity indices, including Chao1, Pielou’s evenness, and Shannon entropy index, displayed no significant variation across all time points (p > 0.05, all groups, and pairwise). Additionally, UniFrac (Weighted and Unweighted), Bray Curtis, and Jaccard emperor did not reveal distinct clusters between different intervention time points.

The most abundant genera present in all groups were Corynebacterium (17.7–41.2%, 25.6 ± 3.5), Bacteroides (7.7–36.45%, 24.1 ± 3.9), and Mycoplasmas (10.71–28.67%, 16.4 ± 2.7) (Fig. 2). During the 10-day normal pepper consumption there is heightened Bacteroides populations (36.45%). Shigella and Staphylococcus, initially present (12.43% and 6.6%), decreased in subsequent interventions, with the lowest recorded abundance in the 10-day normal pepper consumption time point (3.79% and 1.6% respectively) (Fig. 2). Streptococcus, Weissella, and Acinetobacter presented in minimal levels solely in the gut of volunteers during the 4-day pepper consumption phase, 4-day normal pepper consumption phase, and 10-day normal pepper consumption phase, respectively (Table S1).

Members of Bifidobacteriaceae showed a potential differential abundance during the period of abstaining from pepper for 4 days compared to the time of returning to the normal pepper consumption for 4 days (p = 0.005) and 10 days (p = 0.006). Similarly, members of Bacteroidaceae (p = 0.038) displayed a differential potential during the period of consuming pepper for 4 days compared to those reverting to the normal pepper consumption for 4 days (p = 0.015), albeit not significant upon adjusting the p-values (Table S2).

At the phylum level, the microbiota composition across all time points showed notable abundance of Firmicutes (19.6 ± 19.6), Bacteroidetes (27.2 ± 26), Proteobacteria (10.5 ± 6.5), Actinobacteria (18.3 ± 10.5), and Tenericutes (21.2 ± 13). Notably, participantshad increased levels of Firmicutes and Bacteroidetes on returning a 10-day normal pepper consumption. During the pepper intervention stage, Verrucomicrobia dominated, constituting 5.6% of the microbial composition (Fig. 3, Table S3).

Significant variations were observed in the abundance of Tenericutes (p = 0.024), which appeared notably lower during the baseline period (Initial) compared to the period when pepper was consumption was increased for 4 days. Additionally, Firmicutes showed a lower abundance (p = 0.000788) during the baseline period compared to the samples at the 4-day normal diet, while Proteobacteria exhibited lower abundance (p = 0.013) during the baseline period compared to the samples at the 10-day normal pepper consumption. However, upon correction for multiple testing, no significant differences in the abundance of these phyla were found between the study time points (Table S3).

Discussion

We explored the influence of short-term dietary interventions on the diversity and abundance of the gut bacteriome in a diverse group of healthy individuals. The gut microbiome’s dynamic nature and its role in human health make it a compelling target for investigations into the impact of diet. This aligns with a previous study (David et al., 2014) highlighting that dietary changes can promptly alter gut microbiota composition, evident within 1‒3 days of intervention. The study further established that even short-term dietary interventions consisting of plant and animal products can reshape microbial communities and override inter-individual differences in microbial gene expression. Although many dietary intervention studies extend over 15–30 days, our study, in concordance with prior research, underscores that diet modifications can swiftly alter gut microbiota composition and population within a few days. Our analysis of alpha and beta diversity metrics provided valuable insights into the response of gut microbial communities to different dietary regimens. We did not observe significant variations in alpha diversity metrics, including Chao1, Pielou’s evenness, and Shannon entropy index, across all dietary times. These results suggest that the overall richness and evenness of microbial communities remained relatively stable, regardless of short-term dietary changes. This finding aligns with previous studies that highlight the robustness of the gut microbiota in response to acute dietary alterations (Lozupone et al., 2012; Sommer et al., 2017; Fragiadakis et al., 2020; Fassarella et al., 2021; Jian et al., 2021).

However, an intriguing observation emerged concerning the influence of pepper consumption on the gut microbiota. The gradual depletion of Shigella and Staphylococcus from baseline is an interesting observation. A possible reason could be the antimicrobial effects of the pepper, which has a lingering effect till 10 days of resumption of normal diet, testifying to its antimicrobial effects on some specific pathogenic microbes (Füchtbauer et al., 2021; Manjunath et al., 2022). However, this warrants further investigation. Consumption of higher amount of pepper over a 4-day period exhibited a higher abundance of Verrucomicrobia, a phylum rarely found in significant proportions in other dietary time. Phylum Verrucomicrobiota are Gram-negative bacteria that have been isolated from fresh water, soil environments and human faeces. Lower proportions of phylum Verrucomicrobia has been associated with poorer sleep quality in the older people and in prediabetes (Barlow, Yu & Mathur, 2015; Anderson et al., 2017). Interestingly, the beneficial Akkermansia muciniphila which has been noted for its beneficial activities like anti-inflammatory and immunostimulatory properties, improvement of gut barrier function and insulin sensitivity is a member of phylum Verrucomicrobia (Cani et al., 2022). The dominance of this beneficial phylum in this study during pepper consumption requires further investigation into potential prebiotic and beneficial properties of pepper.

At the genus level, Bacteroides, Corynebacterium, and Mycoplasmas were consistently abundant across all time points. These genera appeared to be resilient to short-term dietary changes, this suggests their potential roles as core members of the gut ecosystem. In contrast, certain genera, such as Streptococcus, Weissella, and Acinetobacter, exhibited time point-specific associations, indicating that short-term dietary interventions could influence the relative abundance of certain microbial taxa. Notably, members of the Enterobacteriaceae family were not prevalent and were not significantly abundant across the dietary groups, a phenomenon that could be linked to the dominance of the other genera. The persistent abundance of these genera might create a competitive environment, potentially restraining the proliferation of Enterobacteriaceae. It’s important to note that the dynamics of gut microbial communities are intricate, and there is still a lot to be understood about interplay between different microbial species, as well as their responses to external factors like dietary components.

Importantly, our study has some limitations that warrant consideration. The relatively small sample size and the short duration of dietary interventions may have limited our ability to capture more subtle changes in microbial diversity and composition. Long-term dietary interventions and larger cohorts are needed to further investigate the complex and dynamic interactions between diet and the gut microbiome. Despite these limitations, our pilot study contributes valuable information to the growing body of research on diet-microbiome interactions. Understanding the impact of dietary interventions on the gut microbiome has implications for personalized dietary recommendations and the development of targeted interventions to promote gut microbial health. Future studies with more extended intervention periods and diverse dietary profiles will be essential for unraveling the intricate relationships between diet and the gut microbiota and their potential impact on human health.

Conclusions

Our study contributes valuable insights to the field of diet-microbiota interactions, with implications for personalized dietary recommendations and the development of targeted interventions aimed at supporting gut microbial health. We provided baseline data for dietary intervention in humans with Capsicum sp. pepper consumption, which has been lacking. The findings presented herein provide a foundation for further research in this burgeoning field, offering the potential for novel strategies to promote human well-being through dietary modulation of the gut microbiota.

Supplemental Information