Fasting builds a favorable environment for effective gut microbiota modulation by microbiota-accessible carbohydrates

Abstract

Dietary nutrients are an important determinant of gut microbial composition (Asnicar et al, Nat Med 27:321–332, 2021; Arifuzzaman. et al, Nature 611:578–584, 2022; Bolte. et al, Gut 70:1287–1298, 2021). Commensal bacteria compete and cross-feed on host-derived nutrients to maintain stable gut microbial communities (Kolodziejczyk. et al, Nat Rev Microbiol. 17:742–753, 2019; Ma. et al, Gut Microbes 12:1785252, 2020). However, the changes to the gut bacteria induced by fasting are not well-defined. Here, we propose a powerful method to selectively and effectively increase specific gut bacteria by combining fasting and administration of microbiota-accessible carbohydrates (MACs). Fasting alters the gut microbial community structure, and the fasting + MAC intervention has profound effects on the gut microbiome with increased specific bacteria and fecal IgA levels than MAC administration alone. The changes in gut microbiota composition are specific to the type of MAC administered. We identified the most effective protocol to combine with fasting + MAC to increase the levels of specific bacteria such as Bifidobacterium. Overall, the integrating fasting with MACs effectively alters the gut microbiome, suggesting that fasting can prepare the environment for gut microbial modulation by MACs.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-025-04140-y.

Introduction

The gut microbiome can be conceptualized as a superorganism comprising trillions of diverse microorganisms [1, 2]. The molecules and metabolites associated with the gut microbiome impact the human inflammatory state [3, 4] metabolism [5] and cognition [6]. As the diet shapes the gut microbial structure [3, 5, 7] dietary interventions are conducted to obtain health benefits.

Dietary fiber can alter the gut microbiome composition and influence the types and activities of metabolites produced [8, 9]. Dietary fibers are complex carbohydrates from plants that are resistant to digestion by humans, and are functionally classified as microbiota-accessible carbohydrates (MACs) because these are consumed by gut bacteria [10]. Gut bacteria ferment MACs into metabolites, which serve as a primary energy source and contribute to host health by supporting intestinal homeostasis and suppressing disease development [9, 11]. The capacity to utilize various types of MACs varies among gut bacteria, leading to differing impacts on the gut microbiome and host physiology [11, 12]. Thus, MACs can serve as “gut microbiota modulators” and enhance host physiological functions based on individual conditions.

The gut microbiota are resilient, and a greater diversity of gut bacteria results in microbiome stability and resistance to dietary changes [13]. Additionally, the gut microbiota contributes to the complexity and stability of the gut ecosystem by competing and sharing nutrients in the limited intestinal environment [14]. As the robustness of gut bacteria is not easily disrupted [13] dietary interventions including MACs have struggled to alter the gut microbiome significantly [13, 15–17].

Substantial studies have shown that fasting can alter the gut microbiome composition and influence the physiological state of the host [18–23]. Fasting can disrupt the stability of gut bacteria, resulting in a distinct microbiome that can last for several months after the end of the fasting period [24, 25]. Therefore, we hypothesized that fasting can make the gut microbiome less resilient, enabling effective changes in the gut microbiome by MACs. However, the effects of fasting on gut bacteria depend on the specific type of fasting employed—such as intermittent or long-term fasting—and the existence of underlying health conditions, as shown by studies in mouse models [18–21, 26]. Moreover, the changes to the gut bacteria induced by fasting are not well-defined.

In this study, we demonstrated that fasting induces significant changes in gut microbiota structure and that administration of MACs during fasting induces selective bacterial growth and host IgA production within a short time frame.

Results

Fasting affects the gut microbial structure

We first examined how a 36-hour fasting period influences the gut microbial composition in C57BL/6J mice. The mice that underwent fasting showed a significantly altered gut microbial community based on β-diversity analysis with no notable differences in α-diversity (Fig. 1a and b). Fasting increased the relative abundance of Bacteroidota and Pseudomonadota, whereas the abundance of Bacillota decreased (Fig. 1c and d). In particular, the relative abundance of Lactobacillus was dramatically decreased in the fasting group (Fig. 1d). In addition, the relative abundance of Ruminococceae UCG-014, Ruminiclostridium 5, and the uncultured bacterium Canditatus Saccharibacteria was significantly decreased, and the abundance of the [Eubacterium] coprostanoligenes group and Alistipes was significantly increased after fasting (Fig. 1d). A comparison of the fold change for the number of each bacterial species before and after fasting revealed the dynamics of bacterial modulation. Blautia and Ruminococcaceae UCG-010, from the phylum Bacillota, showed a significant decrease. Conversely, the abundance of several bacteria, including Alloprevotella, Alistipes, and Escherichia-Shigella, showed an increase (Fig. 1e). Fasting drastically remodeled gut microbial structure through decreased numbers of Bacillota and an increase in abundance of Bacteroidota and Pseudomonadota. Thus, fasting induces rapid remodeling of the gut microbial structure.

Fig. 1.

Fasting affects the gut microbial structure. a Principal Coordinate Analysis (PCoA) plot based on UniFrac distance shows differences in the microbial community structure in different treatment groups. Each symbol and color indicate the timepoint and group, respectively. b Violin plot showing α-diversity after 36 h of fasting. Left; Shannon Index, center; Chao1, right; Faith’s phylogenetic diversity. c Stacked bar plot showing the microbiota composition before and after fasting. Each gradient color indicates the representative bacterial phylum and genera. d Representative bacterial genera that exhibited significant differences in enrichment. e Heatmap showing the comparison of bacterial enrichment comparing an ad libitum diet vs. fasting. Plots represent the mean ± S.E.M. Pairwise Palmanova, ** P < 0.01 (a). Welch’s t-test (b, c, e). *** P < 0.001, ** P < 0.01, * P < 0.05

Combination of fasting and MACs drastically promoted the growth of specific gut bacteria

Lactobacillus were responsive to the fasting and the nutritional state in the gastrointestinal tract (Fig. 1d and e) [27–29]. Furthermore, fructo-oligosaccharides (FOS) can be utilized by Lactobacillus and promote their growth [30–33]. Therefore, we investigated how fasting impacts the levels of Lactobacillus with FOS administration. To assess the influence of host genetic background on the gut microbiota response to fasting, BALB/cAJcl mice were treated with 10% FOS and fasted for 36 h (Fig. 2a). Fasting or FOS conditions changed the gut microbiota composition and reduced α-diversity. However, the combination of fasting + FOS drastically altered gut microbial community composition, with a drastic reduction in α-diversity of the Fasting + FOS group compared with the control group (Fig. 2b and c). Consistent with the effects of fasting on C57BL/6J mice (Fig. 1c and d), abundances of bacteria from the phyla Bacteroidota and Pseudomonadota were increased, with a concomitant decrease in Bacillota (Fig. 2d). Notably, the relative abundance of Lactobacillus showed no increase in the Ad libitum + FOS group but was drastically increased in the Fasting + FOS group (Fig. 2d–f). In contrast, the relative abundance of Bacteroides was increased in the Ad libitum + FOS group but not in the Fasting + FOS group. Moreover, the combination of fasting + FOS diminished the increase in the [Eubacterium] coprostanoligenes group that was observed after fasting and abolished the relative abundance of Ruminococcaecae UCG-014 observed in other groups (Fig. 2e and f). FOS intake and the presence of Lactobacillus is associated with enhanced IgA production in gastrointestinal tracts [34–36] and IgA binding to bacteria plays a crucial role in regulating bacterial colonization and metabolic functions [37–40]. Therefore, we evaluated fecal IgA levels in mice. In the Fasting + FOS group, total IgA levels in feces were upregulated 18-fold (Fig. 2g), indicating that fasting enhances the responsiveness of the gut microbiota to FOS, increasing specific bacteria and gut IgA levels.

Fig. 2.

Fasting + MAC intervention drastically alters the gut microbiome structure. a Diagram illustrating the protocol for prebiotic administration during fasting. b Principal Coordinate Analysis (PCoA) plot based on UniFrac distance showing differences in the microbial community structure. Each symbol and color indicate the timepoint and group, respectively. c Violin plot showing α-diversity after 36 h of fasting. Upper left; Shannon Index, lower left; Chao1, lower right; Faith’s phylogenetic diversity. d Stacked bar plot showing the microbiota composition before and after fasting. Each gradient color indicates the representative bacterial phylum and genera. e Representative bacterial genera that exhibited significant differences. f Heatmap showing the comparison of bacterial behavior by fasting and prebiotics. g Bar plot showing the total IgA antibody levels in feces. Plots represent the mean ± S.E.M. Pairwise Palmanova, * P < 0.05 (b). One-way ANOVA followed by Dunnett’s test. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 (c, e-g)

Subsequently, we assessed whether fasting could amplify the microbiota-modulating effects of various MACs. We first tested the effects of galacto-oligosaccharides (GOS) and α-cyclodextrin (α-Cyd), which are known to promote the growth of specific gut bacteria [31, 41–44]. Administration of GOS and α-Cyd during fasting differentially altered the murine gut microbiota composition, including increased relative abundance of Lactobacillus, Erysipelatoclostridium, and Bacteroides (Supplementary Fig. 1). We also investigated the effects of combining fasting and human milk oligosaccharides (HMOs) administration. HMOs such as 2’-fucosyllactose (2FL), 3’-sialyllactose (3SL), and 6’-sialyllactose (6SL) promote the colonization of specific gut bacterial populations and shape the infant gut microbiota [45–51]. Compared with the effects of other MACs, the intake of HMOs—including 2’-FL, 3’-SL, 6’-SL, and their cocktail—altered the abundance of multiple bacterial taxa. This was characterized by increases in the relative abundance of Atopobiaceae, Lactobacillales, Parabacteroides, Bacteroides, and Bifidobacterium, along with suppression of the increase in the that of Verrucomicrobiaceae and Eggerthellaceae DNF00809 (Supplementary Fig. 2c). Thus, the combination of fasting + MACs induced marked changes in the gut microbiota, and these effects appear to be dependent on the structural characteristics of the administered oligosaccharides, such as FOS, GOS, α-CYD, 2’-FL, 3’-SL, and 6’-SL.

Integrated regimen of fasting + MACs robustly enhanced gut microbial modulation effects

We then sought to determine the optimal strategy for combining fasting with MAC administration to promote the enrichment of specific gut bacterial taxa. There are several reports showing that Bifidobacterium has various benefits in maintaining human health and is often used to induce prebiotic effects on the gut microbiota [52, 53]. We evaluated the change in relative abundance of Bifidobacterium using the combination treatment of fasting and FOS administration. The human gut microbiome can efficiently degrade inulin/FOS within 24 h [54] and FOS supplementation increases Bifidobacterium growth within 24–48 h [55, 56]. In addition, a high dose of FOS significantly increases Bifidobacterium levels in mouse feces [57, 58].

To determine the most effective FOS administration pattern and volume, we tested several dose patterns and volumes over a 36-h interval (Fig. 3a). Administration of FOS every 12 h within 24 h of the onset of a 36-hour fasting was the most effective treatment pattern that increased the relative abundance of Bifidobacterium (Fig. 3b). Analysis of the most effective fasting duration (Fig. 3c) showed that fasting for 36 h resulted in a higher increase in the relative abundance of Bifidobacterium (Fig. 3d). These results indicate that FOS administration promotes Bifidobacterium growth when taken three times within 24 h, with each dose taken every 12 h. The increase is particularly effective when FOS was administered during a 36-h fast.

Fig. 3.

Fasting + MAC approach effectively increases Bifidobacterium levels in the gut of mice. a Diagram illustrating prebiotic administration strategies employed in the study. b Relative abundance of Bifidobacterium after fasting. c Illustration of prebiotic administration protocols of various durations with an ad libitum diet or fasting. d Relative abundance of Bifidobacterium after fasting. Plots represent the mean ± S.E.M. One-way ANOVA followed by Dunnett’s test. **** P < 0.0001 *** P < 0.001, ** P < 0.01, * P < 0.05 (b, d)

To evaluate whether the treatment regimen described above enhanced the growth of Bifidobacterium after administration of other oligosaccharides, C57BL/6J mice were fed 6SL and fasted for 36 h (Fig. 4a). The administration of 6SL during fasting strongly modulated the gut microbial community composition and significantly decreased the α-diversity (Fig. 4b and c). Administration of 6SL increased the relative abundance of Actinobacteria and Pseudomonadota (Fig. 4d). In particular, the relative abundance of Bifidobacterium, Parabacteroides, Atopobiaceae, and Burkholderiaceae were significantly increased in the fasting + lactose group and in the fasting + 6SL group (Fig. 4e and f). Bifidobacterium stimulate the secretion of mucosal IgA in the gastrointestinal tract [59–62]. Administration of 6SL during fasting resulted in a significant increase in total fecal IgA levels (Fig. 4e and f). Overall, our results indicate that the regimen of fasting and MAC administration has a marked effect on gut microbiota structure and promotes selective growth of gut bacteria.

Fig. 4.

Fasting HMO intervention changes the structure of the gut microbiome. a Diagram illustrating the protocol for prebiotic administration during fasting. b PCoA plot based on UniFrac distances showing differences in the microbial community structure. Each symbol and color indicate the timepoint and group, respectively. 6’-sialyl lactose; 6SL (c) Violin plot showing α-diversity after 36 h of fasting. Upper left; Shannon Index, lower left; Chao1, lower right; Faith’s phylogenetic diversity. d Stacked bar plot showing the microbiota composition before and after fasting. Each gradient color indicates representative bacterial phylum and genera. e Representative bacterial genera that exhibited significant differences in enrichment. f Heatmap showing the comparison of bacterial behavior between the fasting and prebiotic administration groups. g Bar plot showing the total IgA antibody levels in feces. Plots represent the mean ± S.E.M. Pairwise Palmanova, ** P < 0.01, * P < 0.05 (b). One-way ANOVA followed by Dunnett’s test. **** P < 0.0001 *** P < 0.001, ** P < 0.01, * P < 0.05 (c, e–g)

Fasting with paramylon administration increases specific gut bacterial levels and fecal IgA production

To explore whether fasting can potentiate the microbiota effects of saccharides structurally distinct from conventional oligosaccharides, we focused on β-glucans and their impact on the gut microbiota. β-Glucans are non-digestible polysaccharides found in sources such as yeast, grains, and fungi, and have been reported to modulate the gut microbiome, stimulate immune responses, and improve insulin sensitivity [63–66]. Among them, paramylon—a β−1,3-glucan derived from the microalga Euglena gracilis, accounting for 30–40% of its cellular content—is a commercially available bioactive compound with potential to influence both gut microbial composition and host physiology [66, 67].

We tested the effects of Paramylon and Euglena administration on the gut microbiota in C57BL/6J mice during fasting (Fig. 5a). Mice were administered a dose equivalent of 10 mg/kg body weight which was calculated based on the recommended human dosage for glucan dietary supplements [66]. In contrast to the optimized protocol used in Fig. 3, we applied a single administration of paramylon or Euglena followed by 36 h of fasting. This design was intended to test whether fasting could enhance the microbiota-modulating effects of these polysaccharides within a short timeframe, as compared to previous studies requiring daily administration over 45 days [66]. Fasting dramatically altered the gut microbial community composition (Fig. 5b). Administration of Paramylon during fasting had a modest effect on the gut microbial community composition compared to fasting alone (Fig. 5b). Notably, the administration of Euglena without fasting resulted in increased α-diversity, whereas the combination of Paramylon and fasting decreased α-diversity (Fig. 5c). The administration of Paramylon during fasting increased the relative abundance of the Bacteroides and the [Eubacterium] coprostanoligenes group, whereas Euglena and Paramylon intake decreased the relative abundance of Lactobacillus (Figs. 5e-f). Euglena administration resulted in increased gut bacteria such as Oscillibacter, Ruminiclostridium 5, Ruminiclostridium 9, uncultured Ruminococcaeceae, and Ruminococcaeceae UCG-014. However, this increase was abolished due to fasting (Fig. 5f). Additionally, Bacteroides induce IgA production [68, 69] and fasting with Paramylon intake significantly upregulated total fecal IgA levels (Fig. 5g). These findings suggest that, in addition to oligosaccharides, fasting can potentiate the microbiota-modulating effects of polysaccharides such as paramylon, even within a short intervention period, as reflected by alterations in bacterial composition and diversity.

Fig. 5.

Fasting with β-Glucan (Pramylon) administration effectively increased specific gut bacteria and fecal IgA levels. a Diagram illustrating the protocol for prebiotic administration during 36 h of fasting. b PCoA plot based on UniFrac distances showing differences in the microbial community structure. Each symbol and color indicate the timepoint and group, respectively. c Violin plot showing α-diversity after 36 h of fasting. Upper left; Shannon Index, lower left; Chao1, lower right; Faith’s phylogenetic diversity. d Stacked bar plot showing the microbiota composition before and after fasting. Each gradation color indicates the representative bacterial phylum and genera, respectively. e Representative bacterial genera that exhibited significant differences. f Heatmap showing the comparison of bacterial behavior by fasting and prebiotics. g Bar plot showing the total IgA antibody levels in feces. Plots represent the mean ± S.E.M. Pairwise Palmanova, ** P < 0.01, * P < 0.05 (a). One-way ANOVA followed by Dunnett’s test. **** P < 0.0001 *** P < 0.001, ** P < 0.01, * P < 0.05 (c, e-g)

Discussion

In this study, we showed that fasting induces significant changes in gut microbial structures within a short period. Furthermore, the combination of fasting with MAC administration influenced the trajectory of the microbial community changes by promoting the selective growth of specific gut microbes. In addition, the direction of these microbial shifts was dependent on the type of MAC administered. We also observed a significant increase in fecal IgA production in the fasting + MAC group of mice. These results suggest that our approach is promising for altering gut microbial structure and selectively enhancing beneficial bacteria.

Nutritional exposure is a significant factor influencing gut microbial composition, and the gut microbiota also exhibits stability in response to external perturbations [13]. The response of gut microbiota or their metabolic functions to MACs, such as prebiotics and dietary fiber, can manifest within a few days to several months. However, the response time varies considerably among individuals, depending on their baseline and previous dietary habits [54, 60]. The gastrointestinal nutritional status, such as that in oligotrophic environments, has a profound impact on the colonization of gut bacteria and their metabolic functions, influencing the responses to MACs [28, 70]. Fasting has been shown to remodel the gut microbial structure [18–23]. Indeed, the gut microbiome changes that result due to fasting can be maintained for several months compared to pre-intervention levels [24, 25]. Consistent with previous findings [21, 25, 71, 72] our results demonstrated that fasting alters gut microbial communities, decreasing the relative abundance of Bacillota while increasing that of Pseudomonadota, independent of the mouse strains. Additionally, in line with previous studies [27–29] Lactobacillus species exhibit sensitivity to the host’s nutritional status. Under malnourished conditions, the expression of surface glycans on Lactobacillus is reduced [28] which in turn diminishes their interaction with secretory IgA and impairs intestinal colonization [28]. These mechanisms may partly explain the observed decline in Lactobacillus populations during fasting. However, it remains unclear whether these fasting-induced changes confer beneficial effects on human health. Furthermore, gut microbiota exhibits differential responses to MACs in both oligotrophic and eutrophic conditions [70]. Based on our analyses, the administration of prebiotics during fasting significantly enhances the selective growth of beneficial bacteria within a short time frame. Further investigation of the minimal dose required to achieve the observed effects will strengthen a major advantage of our strategy. Different oligosaccharides exerted distinct effects on bacterial growth, particularly for beneficial species such as Lactobacillus, Bifidobacterium, and Bacteroides. The increase in the levels of these beneficial bacteria, in conjunction with fasting + MACs, were comparable or more pronounced than those documented in previous studies where MACs were administered over two weeks to one month [32, 43, 44, 49, 50, 57, 73, 74].

Several studies have reported that the gut microbiota tends to gradually revert to its baseline composition after the cessation of prebiotic interventions [74–76]. Recent evidence also suggests that prior dietary exposure can condition the microbiota to more efficiently utilize specific prebiotics, such as inulin and FOS, during subsequent interventions, thereby enhancing SCFA production [54]. Although the long-term stability of microbial shifts induced by our approach remains to be determined, intermittent or lower-dose supplementation may help maintain the desired microbial profiles.

Bifidobacterium and Bacteroides utilize human milk oligosaccharides (HMOs) [49, 77–80] which play a critical role in the maturation and stabilization of the human gut microbiome [81] and facilitate recovery after antibiotic treatment [80]. The colonization of Bifidobacterium or Bacteroides by fasting + HMOs may promote the stabilization and maintenance of the remodeled gut microbiota. In our results, the Fasting + Lactose intervention did not lead to a Bifidobacterium increase comparable to that seen in the Fasting + 6’-SL group, despite the lactose-utilizing capacity of many Bifidobacterium strains. This may be due to the broader utilization of lactose by various gut microbes [51, 82, 83] whereas only specific Bifidobacterium strains can metabolize HMOs such as 6’-SL. In addition, 6’-SL has been shown to promote Bifidobacterium growth through epithelial adhesion and microbial cross-feeding [84–86]. These mechanisms may explain the differential responses observed. Further, we identified genera such as Parabacteroides, and bacterial families such as Atopobiaceae and Burkholderiaceae, that increased abundance after fasting and HMO administration. However, it remains unclear whether these bacteria possess the necessary enzymes for HMO degradation.

The increase in Bacteroides by α-Cyd treatment is associated with higher levels of short-chain fatty acids (SCFAs) and upregulation of gluconeogenesis-associated genes in the liver, contributing to anti-obesity effects and improved endurance [43, 44]. Due to its complex structure, paramylon may require more time to be metabolized compared to simpler oligosaccharides [64] which may explain its relatively delayed impact on gut bacterial composition. Nonetheless, the increase in Bacteroides observed following fasting combined with paramylon intake appeared qualitatively similar to that reported after prolonged daily administration of paramylon [66].

Follow-up studies are needed to determine how specific bacterial taxa, such as Bifidobacterium, are selectively promoted under fasting conditions, and how the remodeled microbiota are maintained after refeeding and contribute to host physiology. In particular, the rapid enrichment of primary degraders such as Lactobacillus, Bifidobacterium, and Bacteroides may help stabilize and enhance the outcomes of subsequent interventions by amplifying beneficial effects and mitigating undesirable ones. This is because the acquired dietary memory of the gut microbiota improves the metabolism of the entire gut microbiome [54]. Furthermore, despite the presence and abundance of these primary degraders, their effects on the host may vary due to the influence of different substrates on various metabolites, short-chain fatty acid composition and production, and cross-feeding bacteria [42, 50]. Therefore, the combination of fasting + MACs may provide a more flexible intervention that considers the individual baseline or pathology.

Fasting + MACs increased the levels of fecal IgA, which regulate the composition and function of the gut microbiota [28, 39, 40, 87]. IgA binding to bacteria helps protect against infection and inflammation [88–90]. Gut bacteria stimulate IgA production through several mechanisms. For example, Lactobacillus and Bifidobacterium enhance IgA production through Toll-like receptor 2 signaling via the membrane or membrane vesicles [28, 61, 91]. Additionally, Bacteroides stimulate immune cells and promote IgA production by metabolizing short-chain fatty acids such as acetate and butyrate [36, 69, 92–95]. The type of bacteria and sites recognized and bound by IgA remain unclear. However, the upregulation of IgA using the method described in this study may facilitate the colonization of the gastrointestinal tract by gut bacteria.

To summarize, our results show that fasting induces significant changes in gut microbiota structure, and the administration of MACs during fasting induces selective bacterial growth and host IgA production within a short time frame. For the evaluation of the effects on some diseases and the application of clinical, follow-up studies are required to evaluate the to elucidate how these changes are sustained after refeeding and their influence on the host and the underlying mechanisms. On the other hand, these results indicate that our methods have the potential to be combined with more diverse substrates, such as bacterial membrane vesicles [96, 97] peptides [98]and unsaturated fatty acids [99] as well as to enhance the function of oligosaccharides as gut microbial modulators. These combinations may allow the treatment of various non-communicable diseases without the administration of antibiotics or excessive doses. These findings indicate that fasting and MACs administration can provide an effective approach for modulating the gut microbiota composition.

Methods

Mice

Eight-week-old female C57BL/6JJcl and BALB/cAJcl mice (CLEA Japan Inc.) were housed under standard conditions, under controlled conditions for light (12 h light/dark cycle), temperature (24 ± 0.5 °C), and humidity (40 ± 5%), at the animal facility of the Faculty of Pharmacy, Keio University (Tokyo, Japan). Mice had free access to food, CE-2 (CLEA Japan, Inc.), and tap water. Following a 5–7-day acclimatization period, the mice were randomly assigned to three to five mice per group. The mice in the fasting group were fasted for 12–36 h and given a solution containing 10% (w/v) of various prebiotics (fructo-oligosaccharides; FOS, galacto-oligosaccharide; GOS, or α-cyclodextrin; α-Cyd. All products were manufactured by FUJIFILM Wako Pure Chemical Corporation, Japan). Human milk oligosaccharides (2’-fucosyllactose; 2FL, 3’-sialyllactose; 3SL and 6’-sialyllactose; 6SL) were obtained from KYOWA HAKKO BIO CO. LTD., Japan. The HMO cocktail was prepared with each HMO included to achieve a final concentration of 10% (w/v). MACs were administered either via drinking water (Fig. 2a and Supplementary Figs. 1 and 2) or by oral gavage (Figs. 3, 4 and 5). Oral administration was used in fasting experiments to ensure consistent intake, as voluntary water consumption can decrease under fasting conditions [100]. Euglena and paramylon were obtained from Euglena Co., Ltd. (Tokyo, Japan). Paramylon was isolated as previously described [101]. Euglena and Paramylon were orally administered [66] at a final concentration of 375 µg in 200 µL PBS after the start of the fasting period. All mice were unconscious under inhalant isoflurane anesthesia and sacrificed by exsanguination with cardiocentesis at the end of the experiment. To avoid coprophagia, the mice were kept in cages without bedding chips on a stainless-steel mesh floor. The animal experiments conducted in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Kitasato University (Approval No. 24 − 10) and Keio University (Approval No. A2022-078).

16S ribosomal RNA sequencing and analysis

Bacterial DNA was extracted from mouse feces using the E.Z.N.A. Stool DNA Kit Pathogen Detection Protocol (OMEGA) and purified using an magLEAD 12 gc Nucleic Acid Extraction Instrument (Precision System Science Co., Ltd.). DNA was amplified by PCR using primers specific to the V3-V4 regions of the 16 S ribosomal RNA gene (Key Resources Table). Sequencing was performed by Cancer Precision Medicine (Japan) using an MiSeq System (Illumina, Inc., USA). Raw FASTQ files were processed using QIIME2 (Version 2022.2.0) [102] with denoising using DADA2 [103]. The taxonomy was assigned with the DADA2 implementation of the Ribosomal Database Project classifier using the SILVA database [104, 105]. Sequences were deposited into the SRA database of NCBI: PRJNA1272289.

Total IgA ELISA

Fecal samples were lyophilized overnight. Freeze-dried feces (10 mg) were disrupted using 3.0 mm Zirconia Beads (Biomedical Science, Japan) and a Complete Mini Protease Inhibitor Cocktail (Roche, Basel, Switzerland) by vigorous shaking (1,500 rpm for 15 min) using a Shake Master (Biomedical Science, Japan). The lysate was centrifuged at 18,000 ×g at 4 °C for 15 min and the supernatant was collected as a fecal extract. The samples were stored at − 80 °C until use. Total IgA levels were measured using a Mouse IgA ELISA Quantitation Set (Bethyl Laboratories, USA) and the IgA concentration in 1 mg feces was estimated.

Statistical analysis

Dunnett’s test was used for statistical analyses of the two groups. Analyses were performed using GraphPad Prism software (version 10.0.3, GraphPad Software Inc.). Statistical significance is indicated with * denoting P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Statistical analysis was performed with GraphPad Prism 10 software (Version 10.2.3) and Qiime2 using Pairwise Palmanova, Welch’s t-test, and One-way ANOVA followed by Dunnett’s test. Data were visualized using Python (version 3.8.3), the matplotlib (version 3.5.0), seaborn (version 0.11.2) packages, and GraphPad Prism 10 software (Version 10.2.3).

Abbreviations

α-Cyd

α-cyclodextrin

FOS

Fructo-oligosaccharides

GOS

Galacto-oligosaccharides

HMOs

Human milk oligosaccharides

MACs

Microbiota-accessible carbohydrates

SCFAs

Short-chain fatty acids

2FL

2’-fucosyllactose

3SL

3’-sialyllactose

6SL

6’-sialyllactose

Authors’ contributions

K.S., J.I., and Y.-G.K. conceived the study and designed the experiments; K.S. performed the experiments and analyzed the data; K.S., A.N., J.I., S.F, and Y.-G.K. provided resources; and K.S. and Y.-G.K. wrote the manuscript with contributions from all the authors. Y.-G.K. supervised the study.

Funding

This study was supported in part by JSPS KAKENHI (JP23H02718 and JP 23K18223 to Y.-G.K.), KYOWA HAKKO BIO CO. LTD (to Y.-G.K.), Euglena Co., Ltd. (to Y.-G.K.), JST SPRING (JPMJSP2123 to K.S.), the Young Leaders Fellowship Fund (to K. S.), Sylff Research Grant (2346 to K.S.), and research funds from the Yamagata Prefectural Government and the City of Tsuruoka (to K.S.).

Data availability

The sequencing data were deposited to the Sequence Read Archive National Center for Biotechnology Information (NCBI) with accession number: PRJNA1272289.

Declarations

Ethics approval and consent to participate

The animal experiments conducted in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Kitasato University (Approval No. 24 − 10) and Keio University (Approval No. A2022-078).

Consent for publication

Not applicable.

Competing interests

Ayaka Nakashima is a researcher at Euglena Co., Ltd. The other authors declare no competing interests.