Trillions of microorganisms coexist within the human body, comprising a diverse ecosystem of beneficial and harmful bacteria. These microorganisms play a pivotal role in human health and disease by actively participating in metabolic processes, pathogen defense, immune system regulation, and influencing various physiological functions (1). The advent of next-generation sequencing (NGS) methods has provided valuable insights into the abundance, diversity, and functional significance of the human microbiome (2). Since the late 2000s, extensive research has highlighted the microbiome’s involvement in crucial physiological processes, including nutrient absorption, drug metabolism regulation, immune system modulation, brain and behavioral development, and prevention of infectious diseases (3–5).

In recent years, the significance of intestinal health in boosting immunity and promoting skin health has sparked interest, leading to the emergence of various biotic products such as probiotics, prebiotics, synbiotics, and postbiotics. Prebiotics are non-digestible carbohydrates that serve as a substrate for the gut microbiota, demonstrating resistance to gastric acid and digestive enzymes, thus arriving intact in the intestine. Here, they undergo bacterial fermentation, encouraging the proliferation of beneficial microflora, and providing substantial health advantages (6–8). They are prevalent in an array of dietary sources including diverse fruits, vegetables, cereals, and legumes such as tomatoes, bananas, various berries, sweet potatoes, asparagus, allium vegetables, chicory, leafy green vegetables, pulses, oats, flaxseed, barley, and wheat (8).

Among these, bananas are one of the most extensively cultivated tropical fruits worldwide, with over 1,000 varieties produced. However, a significant portion of the banana harvest goes to waste due to consumers’ prevalent preference for ripe bananas. Fresh green bananas contribute substantially to the overall loss and are generally unpalatable due to the presence of soluble phenolic compounds, such as tannins, imparting an astringent taste (9). Consequently, researchers have explored alternative derived products from green bananas, such as green banana flour and green banana biomass, to mitigate waste. Green banana flour, in particular, has garnered attention within the food industry due to its high resistant starch (RS) content, ranging up to 68% w/w (10). Upon reaching the colon, RS undergoes fermentation by the intestinal microbiota, yielding short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate (11). RS exhibits diverse therapeutic and preventive effects, including colon cancer prevention, blood glucose reduction, cholesterol reduction, fat accumulation inhibition, gallstone formation reduction, and enhanced mineral absorption (12).

Despite previous studies discussing the prebiotic effects of green banana flour, which is rich in RS, limited research has been conducted to substantiate significant changes in the gut microbiome and perform comprehensive metabolic pathway analyses through in vivo studies (10). To validate the effectiveness of green banana flour as a prebiotic, further investigations are warranted to elucidate its precise impact on the gut microbiome. This study aims to investigate the effects of green banana flour consumption on mice, with a specific focus on validating alterations in the composition of the intestinal microbiota and identifying associated metabolic pathways that may hold relevance to specific diseases. Our findings aim to provide compelling evidence of the prebiotic potential of green banana flour. Moreover, our research demonstrates that green banana flour can be applied as a prebiotic or functional food ingredient through the improvement of gut microbiota, and it provides a solution to the environmental issues caused by the disposal of unused green bananas, thereby emphasizing its novel contribution to the field of environmental science.

The prevalence of individuals routinely consuming biotic products like probiotics and prebiotics has escalated recently, kindling an upsurge in biotics-related research. Many past studies have demonstrated improvements in index markers and symptom alleviation through prebiotics administration, as evidenced by animal trials, cell experiments, and human application tests. Next-generation sequencing (NGS) has allowed us to analyze microbial communities, thereby enabling us to confirm changes in the intestinal flora, identify valuable microbial target strains for specific diseases, and validate their potential as prebiotics.

In our study, we administered varying doses of green banana flour to normal mice to ascertain the resultant changes in gut microbiota and infer the functional profile of the microbiome. In all groups, the body weight of mice tended to increase over time and there was no significant difference between groups (Supplementary Figure S2). Although control animals were given distilled water instead of standard diet used for feeding via oral gavage, considering that the absolute intake quantity of green banana flour used in this study ranged from only 10 to 40 mg (approximately 0.01% of daily food intake) per animal, the stress associated with force-feeding appeared to be minimal. Following the ingestion of green banana flour, a significant increase in Actinobacteriota was observed (Figures 1B,D). Actinobacteriota, one of the largest bacterial phyla, is a gram-positive bacterium with high G+C DNA, ubiquitously found in aquatic and terrestrial ecosystems. The phylum is known for its ability to produce short chain fatty acids (SCFAs) from resistant starch, which stimulate salt and water absorption, provide energy, and induce trophic effects on the mucous membranes of the colon and small intestine (20). Especially, Bifidobacteriaceae and Atopobiaceae, members of the Actinobacteriota phylum increased by green banana flour consumption (Figures 1C,E). They are known to promote health through dietary fiber digestion, infection prevention, and the production of critical chemicals. They also produce beneficial lactates and SCFAs, including acetate and butyrate (21, 22). Although there was an increase in proportions of SCFAs producing bacteria, as SCFAs were not determined, it remains unclear whether this bacterial changes directly influenced SCFA production. Therefore, additional experiments, such as measuring SCFA production levels, are required to substantiate this.

The consumption of green banana flour had no effect on the diversity or richness of the gut microbiome. Although, changes in the composition of the microbial community were significant in all groups on day 21 after ingestion (Figure 2). Based on LEfSe analysis, specific bacteria that showed consistent increase in the low and high-dose groups compared to the control group were Coriobacteriaceae_UCG-002, Turicibacter, Gastranaerophilales_ge, RF39_ge, and Parasutterella. Conversely, bacteria that consistently decreased were Clostridia_UCG-014_ge, Candidatus_Arthromitus, Roseburia, Ruminococcus, Lachnospiraceae_NK4A136_group, and Faecalibaculum. Altered microbes in our study show associations with various disease models. Turicibacter, typically reduced in obese mice due to a high-fat diet, increased in our study; Candidatus_Arthromitus, usually elevated, decreased (23, 24). Coriobacteriaceae_UCG-002, elevated in our study, correlates positively with synthesizing polyunsaturated fatty acids (PUFAs), lowering low-density lipoprotein (LDL) cholesterol. Parasutterella improves LDL levels; decreased Roseburia is prominent in mild strok group (25–27). Diminished Lachnospiraceae_NK4A136_group is linked to intestinal diseases. Clostridia_UCG-014_ge, Ruminococcus, and Faecalibaculum relate to impaired intestinal barrier in Crohn’s patients (28–31). Increased RF39_ge in our study correlates negatively with diabetes and cognitive impairment (32) (Figure 3).

Differences in metabolic pathways related to altered microbiomes were observed between groups. Green banana flour supplements increased pathways commonly increased pathways like aspartate superpathway, superpathway of L-alanine biosynthesis, taxadiene biosynthesis (engineered) and the superpathway of L-lysine, L-threonine, and L-methionine biosynthesis I. In contrast, chondroitin sulfate degradation I (bacterial), myo-, chiro-and scillo-inositol degradation, pyridoxal 5′-phosphate biosynthesis I, and the superpathway of pyridoxal 5′-phosphate biosynthesis and salvage were reduced. It was predicted that consuming green banana flour may have various beneficial effects on metabolic syndrome, diabetes, and cardiovascular health, as indicated by a taxonomic-based analysis of metabolic pathways. After consuming green banana flour, myo-, chiro-and scillo-inositol degradation pathways decreased. These pathways break down inositols, affecting insulin action and playing roles in signal transduction. Inositol deficiency relates to conditions like metabolic syndrome, neural tube defects, and diabetes (33–35). Similarly, reduced pyridoxal 5′-phosphate (PLP) biosynthesis I, the superpathway of pyridoxal 5′-phosphate biosynthesis and salvage pathway synthesize metabolically active vitamin B6 coenzyme PLP. PLP sources include meat, dairy, beans, nuts, and produce; it associates with cardiovascular health (36, 37). Increased pathways post-green banana flour ingestion were aspartate superpathway, superpathway of L-alanine biosynthesis, taxadiene biosynthesis (engineered), superpathway of L-lysine, L-threonine, and L-methionine biosynthesis I. Aspartate superpathway increased in weight-loss group due to lifestyle changes (38). Synthesis of essential amino acids like L-alanine, L-lysine, L-threonine, and L-methionine also rose. L-alanine is an essential component of cellular proteins and peptidoglycan, synthesized for cell biosynthesis and involved in transamination (39). The biosynthesis pathways of L-lysine, L-threonine, and L-methionine stem from TCA cycle’s oxaloacetate. The taxadiene biosynthesis (engineered) is essential for membrane synthesis protein assembly (40) (Figure 4).

In summation, our investigation did not ascertain definitive therapeutic or prophylactic effects of green banana flour consumption within a specific disease model. Nevertheless, it did substantiate conspicuous alterations in the intestinal microbiota and associated biochemical pathways resulting from the administration of varied quantities of green bananas to murine subjects. Our findings revealed an augmented prevalence of beneficial bacterial taxa such as Coriobacteriaceae_UCG-002 and Parasutterella, coupled with heightened engagement of pathways implicated in amino acid biosynthesis and secondary metabolite production, subsequent to green banana flour ingestion. Beyond its conventional merits encompassing gastrointestinal, renal, hepatic, and metabolic dimensions, our data underscores the latent prebiotic attributes of green banana flour. While acknowledging the intrinsic limitations of our study, the results here reported suggest a causal nexus between changes in gut microbial ecology and concomitant metabolic reprogramming, thereby shedding light on the potential salutary effects of green banana flour.

The original contributions presented in the study are publicly available. This data can be found at: https://www.ncbi.nlm.nih.gov/bioproject/991718.

The animal study was approved by Institute Animal Care and Use Committee of Korea Research Institute of Bioscience and Biotechnology. The study was conducted in accordance with the local legislation and institutional requirements.

J-SK: designed the study. GB, Y-JK, S-CJ, and HS: performed the experiments. YL: provided critical materials and scientific insight. GB and J-SK: analyzed and visualized the data. GB, HS, and J-SK: wrote the paper. All authors contributed to the article and approved the submitted version.