Detection of typical indigenous gut bacteria related to kanpyo Lagenaria siceraria var. hispida powder in murine caecum and human faecal cultures

Abstract

Kanpyo (KP) is an edible dried product produced by peeling the fruit of the gourd Lagenaria siceraria var. hispida; it is used in the traditional Japanese cuisine. The health functionality of KP due to its rich dietary fibre is expected to include a possible combined effect of KP-responsive indigenous gut bacteria (KP-RIB). However, its effect on the gut microbiota is unclear. To determine the effects of the KP on the gut microbiota and their host, Institute of Cancer Research mice were fed a high-sucrose diet containing no fibre (NF) or 5% (w/w) KP for 14 days, and their caecal microbiota was analysed by 16S rRNA (V4) amplicon sequencing. Higher faecal frequency and weight and lower spleen weight and spleen tumour necrosis factor-α levels were observed in KP-fed mice than in NF-fed mice (p < 0.05). KP increased and decreased the abundance of short-chain fatty acid producer Lachnospiraceae and obesity-inflammation related Allobaculum species, respectively. In the case of human faecal cultures, stool samples from five healthy volunteers were inoculated and incubated at 37 °C for 24 h anaerobically; 3.2% (w/v) KP suppressed putrefactive compounds (indole, phenol, and ammonia). KP increased butyrate-producer Faecalibacterium, acetate/lactate-producer Bifidobacterium, and Lachnospira. Furthermore, KP cultures showed high antioxidant and RAW264.7 macrophage cell activation capacities. These results suggest that KP-RIB and KP intake may synergistically affect host health. However, further studies are required to clarify the synergistic effects of KP and KP-RIB.

Introduction

Kanpyo (KP) is an edible dried product produced by peeling the fruit of the gourd Lagenaria siceraria var. hispida into long, thin strips; it is used in traditional Japanese cuisine, such as sushi (Lowry 2005). According to the "Food Composition Database" of the Ministry of Education, Culture, Sports, Science and Technology in Japan (https://fooddb.mext.go.jp/index.pl), KP contains 30% (w/w) dietary fibre. Semi-purified KP dietary fibre comprises 50% cellulose, 23% insoluble hemicellulose, 9.7% soluble hemicellulose, and 1.6% lignin (Sannoumaru et al. 1996). The health benefits of KP have been anticipated because of its rich dietary fibre content. For example, several reports suggest that the liver protects against the effects of some toxic compounds, such as amaranth (Ropan et al. 2016). There are also reports by non-Japanese researchers on the functional properties of gourd fruit, such as antioxidant, anti-inflammation, anti-hyperlipidaemic, anti-hypertensive, and cardioprotective activities (Turroni et al. 2022).

The gut environment is a critical part of the human microbiota ecosystem, with hundreds of bacterial species colonising the gut and contributing to the metabolic health of the human host (David et al. 2014). The gut microbiota is a changing ecosystem continuously affected by factors, such as dietary habits, seasonality, lifestyle, and disease, with a pronounced and rapid effect exerted by the diet (Ecklu-Mensah et al. 2022; Xu et al. 2022). Specific metabolites produced by the gut microbiota, such as short-chain fatty acids (SCFAs) and branched-chain amino acids, are considered beneficial to host health (Hu et al. 2022; Shen et al. 2023). SCFAs, such as acetate, propionate, and butyrate, can improve gut barrier integrity, glucose levels, and lipid metabolism, regulate the immune system, inflammatory response, and blood pressure, and function as crucial metabolites in maintaining intestinal homeostasis (Nguyen et al. 2015; Seethaler et al. 2022). To elucidate the dynamics of host–gut microbiome interactions in host nutrition and disease development, mice and rats are widely used as models (Čoklo et al. 2020; Nagpal et al. 2018). However, limited data are available regarding the host species-specific signatures of the gut microbiome and its metabolites in these models and their similarities/differences relative to those in humans (Ye et al. 2022).

Dietary fibres that the host's digestive enzymes cannot break down reach the large intestine, where they become nutrients for the bacteria; their metabolites are generally considered to have the desired effect on the host (Xu and Marques 2022). For example, SCFAs (acetate, propionate, butyrate) derived from fibre fermentation by the gut microbiota have various health functions against the lifestyle diseases mentioned above (Takei et al. 2022; Shen et al. 2023). Although there are many reports on soluble and fermentable dietary fibres, the effects of insoluble fibres, such as cellulose, hemicellulose, and lignin, on the gut microbiota have also been reported. For instance, wheat bran insoluble fibre suppresses gut Allobaculum in mice fed a high-sucrose and low-fibre diet (Takei et al. 2022). In addition, Allobaculum is reportedly positively associated with obesity and liver injury phenotypes in mice (Rienzi and Britton 2022; Wang et al. 2022). However, modern dietary habits are altered by reducing dietary fibre consumption and increasing fat, sugar, and animal protein intake, adversely affecting the gut microbiota (Xia et al. 2022b).

KP is considered functional because of its rich dietary fibre, as mentioned above; however, its effect on the gut microbiota is unclear. In this study, to clarify the potential use of KP as a new source of dietary fibre, KP-responsive indigenous gut bacteria (RIB) in the caecal microbiota of Institute of Cancer Research (ICR) mice fed a high-sucrose and a low-fibre diet containing 5% (w/w) KP were detected using 16S rRNA (V4) amplicon sequencing. We also determined the effects of KP on organic acids, putrefactive compounds, and the microbiota in human faecal cultures.

Materials and methods

Animal experiments

Animal experiments were performed in compliance with the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology. The study was approved by the Animal Experiment Committee of the Tokyo University of Marine Science and Technology (Approval No. R3-1).

KP products harvested, shaped, and semi-dried in Heilongjiang, People's Republic of China, were obtained from local contract farmers. The KP product was powdered after additional hot air drying in our laboratory. Twelve 5-week-old male ICR mice were purchased from Tokyo Laboratory Animal Science Co. (Tokyo, Japan) and housed in metal-wire cages (3 mice/cage) at 22 ± 2 °C. Mice were provided with food and distilled water ad libitum and housed under a strict 12-h light–dark cycle. The mice were acclimatised to a high-sucrose and low-dietary fibre diet (NF; Table 1) for 7 days. Subsequently, the mice were divided into two groups (n = 6/group) and fed a diet lacking or with 5% (w/w) KP powder for 14 days. During the experiments, faecal weight and frequency were measured on days 11–13. After completion of the feeding regimen, the mice were anaesthetised using isoflurane (Fujifilm Wako Pure Chemical, Osaka, Japan) and sacrificed by exsanguination via the abdominal aorta, after which the liver and epididymal fat pads were removed and weighed. After ligation with yarn, the caecum was excised and placed on ice before microbial analysis.

Table 1.

Composition of test diets

	Feeding groups
	NF	KP
Kanpyo (dried gourd)		5.0
Corn starch	51.4	46.4
Alpha-corn starch	15.5	15.5
Sucrose	10	10
Milk casein	14.0	14.0
L-Cystine	0.18	0.18
Soy oil	4.0	4.0
Vitamine mix (AIN-93)*	1.0	1.0
Mineral mix (AIN-93)*	3.5	3.5
Choline bitartrate	0.25	0.25
tert-Butylhydroquinone	0.0008	0.0008

*AIN American Institute of Nutrition

Plasma lipid and glucose levels and spleen pro-inflammatory cytokines

Plasma triacylglycerol, total cholesterol, and glucose levels were determined using commercial kits (Triglyceride E Test, Total Cholesterol E Test, and Glucose CII Test, respectively; Fujifilm Wako Pure Chemicals). The spleen was homogenised in a tenfold volume of phosphate-buffered saline and centrifuged at 10,000 × g at 4 °C for 30 min. Following centrifugation, tumour necrosis factor (TNF-α) and interleukin-6 (IL-6) levels were measured using an enzyme-linked immunosorbent assay kit (Chondrex, Woodinville, WA, USA) according to the manufacturer's instructions and read using a grating microplate reader at 450 nm with 630 nm as the correction wavelength.

Human faecal cultures

Experiments using human stool samples were performed as per the "Ethical Guidelines for Medical and Health Research Involving Human Subjects" under the authority of the Ministry of Education, Culture, Sports, Science, and Technology and the Ministry of Health, Labour and Welfare, Japan. The Committee for Research Involving Human Subjects of Tokyo University of Marine Science and Technology approved the study protocol (Approval No. R03-001). The volunteers were recruited by posting announcements at the school; all volunteers were gratuitous.

Gifu Anaerobic Medium (GAM) 1/4 broth was prepared as previously described (Nakata et al. 2017). Briefly, 250 mL of GAM semi-solid without dextrose (Nissui Pharmaceutical, Tokyo, Japan) was added to 750 mL of ‘Diluent A’ (KH₂PO₄ (4.4 g), Na₂HPO₄ (6.0 g), l-cysteine hydrochloride (0.5 g), Tween 80 (0.5 g), and agar (1.0 g) in 1,000 mL of distilled water). Subsequently, 5 mL of GAM1/4 broth containing 3% (w/v) soy protein (Fujipro-F; Fuji Oil Co., Izumisano, Japan) without (control) or with 3.2% (w/v) KP was stored overnight in an AnaeroPouch (Mitsubishi Gas Chemical, Tokyo, Japan) and used as the culture broth.

Fresh faecal samples were collected from five healthy female donors aged 22–26 years who did not have large bowel disease before and had not taken antibiotics for at least 3 months. Volunteers collected 2 g of fresh stool samples, placed them into an aluminium pouch with AnaeroKeep (Mitsubishi Gas Chemical), and transferred them to our laboratory with ice packs for 2 h. The sample was homogenised in 18 mL of GAM1/4 broth; the resulting faecal slurry was inoculated into the culture broth and incubated at 37 °C for 24 h under anaerobic conditions using an AnaeroPouch with shaking (60 rpm).

Chemical analysis of human faecal cultures

The culture pH was measured using a pH metre (LAQUA Twin B-711, Horiba, Kyoto, Japan). We centrifuged the samples at 5,000 × g for 10 min at 4 °C, collected the supernatant, and carried out chemical analysis. The ammonia and total phenolic contents of human faecal cultures were determined using reagent sets for water analysis (Nos. 7 and 53, respectively; Kyoritsu Chemical-Check, Tokyo, Japan) and a grating microplate reader (SH-1000 Lab; Corona Electric, Hitachinaka, Japan). Indole content was determined using a colourimetric assay performed with Kovac's reagent (Nakata et al. 2017). The organic acid content was determined as follows (Nakata et al. 2017): 0.4 mL of the sample was mixed with 0.1 mL of 1 mol/L H₂SO₄ and centrifuged at 12,000 × g for 10 min at 4 °C; the obtained supernatant was passed through a membrane filter (pore size: 0.45 μm) and injected into a high-performance liquid chromatography system under the following conditions: column, ICSep ORH-801 (Tokyo Chemical Industry, Tokyo, Japan); operating temperature, 35 °C; elution solution, 0.005 mol/L H₂SO₄; flow rate, 0.8 mL/min. The eluted compounds were detected using a refractive-index detector.

Microbiota analysis

Bacterial cell counts of mouse caecal and fermented human faecal samples were directly determined according to a previously described protocol (Shikano et al. 2019) using dielectrophoretic impedance measurement (DEPIM) and a bacterium counter (PHC, Tokyo, Japan) (Hirota et al. 2014).

16S rRNA (V4) amplicon sequencing was performed by Fasmac Co. Ltd. (Atsugi, Japan). Briefly, DNA was extracted from 0.1 g of the caecal content and 1 mL of faecal culture suspensions using an MPure bacterial DNA extraction kit (MP Bio Japan, Tokyo, Japan). The DNA library was prepared using a two-step PCR method (Sinclair et al. 2015). The V4 region was amplified using the 515f forward and 806r reverse primers and ExTaq HS DNA polymerase (Takara Bio, Kusatsu, Japan) in the first PCR (94 °C for 2 min, followed by 20 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s; and then a final extension at 72 °C for 5 min). After purification of the products using an AMPure XP kit (Beckman Coulter Life Science, Tokyo, Japan), individual DNA fragments were tagged using a second PCR (94 °C for 2 min, followed by eight cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min) performed with the same polymerase kit. The DNA libraries were multiplexed and loaded onto an Illumina MiSeq system (Illumina, San Diego, CA, USA). Reads harbouring a mismatched sequence at the start region were filtered using the FASTX Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/); 235–260 bp reads were selected. Chimeras in the selected reads were identified and omitted using the QIIME2 bioinformatics pipeline (https://qiime2.org/). A feature table was generated using the dada2 denoise-paired option in the QIIME2 plugin; the sequences were clustered into amplicon sequence variants (ASVs) using the SILVA 138 database (https://www.arb-silva.de/). The sequences obtained from the animal experiment and human faecal cultures were deposited in the DNA Data Bank of Japan Sequence Read Archive under accession number DRA014587 (https://ddbj.nig.ac.jp/) and Sequence Read Archive in NCBI under accession numbers SRR22406027-36 (https://www.ncbi.nlm.nih.gov/sra).

Culturable bacteria were isolated and identified from human faecal cultures using the 16S rDNA sequence and National Center for Biotechnology Information BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), similar to a previous study (Takei et al. 2022).

In vitro antioxidant properties and effect on nitric oxide (NO) secretion in murine macrophage-like RAW264.7 cells of human faecal cultures

To estimate the antioxidant properties of human faecal cultures, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide anion (O₂⁻) radical-scavenging capacity and ferric ion (Fe³⁺)-reducing power of the sample cultures were measured using previously described methods (Kaga et al. 2021). To estimate immunomodulatory properties in vitro, NO secretion in murine macrophage-like RAW264.7 cells (TIB-71; American Type Culture Collection, Manassas, VA, USA) was determined as previously reported (Harada et al. 2021). Briefly, RAW264.7 cells (2 × 10⁶/mL) were suspended in 50 mL of foetal bovine serum-Dulbecco's modified Eagle’s medium (Nissui Pharmaceuticals) and seeded into 96-well microplates (0.1 mL/well). After incubation at 37 °C for 2 h in an atmosphere of 5% CO₂, 0.1 mL of a medium containing the appropriately diluted sample was added to each well; after incubation for 20 h, the NO concentration in the culture medium was determined using 100 g/L Griess-Romijn nitrite reagent (Fujifilm Wako Pure Chemical).

Statistical analysis

Data were analysed using Student's t-test or ordinary one-way ANOVA with post hoc Tukey’s test. Statistical significance was defined as p < 0.05. The α-diversity of the microbiota was determined using Shannon–Wiener (H') and Simpson (D) indices (Kim et al. 2017). The β-diversity was expressed using principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), and the values were calculated according to the number of ASVs using MetaboAnalyst (https://www.metaboanalyst.ca/). The variable importance in projection scores of PLS-DA was used to detect specific bacterial groups (KP-RIB) in human faecal samples.

Results and discussion

Body, faecal, and organ weights of mice

No disease symptoms or abnormalities were observed in the mice during the feeding period. Faecal frequency and weight were significantly higher in KP-fed mice than in NF-fed mice (p < 0.01, Table 2). No significant differences were evident in body weight gain and liver weight, although these were tended to be lower in the KP group. The spleen weight of mice fed KP was lower than that of mice fed NF.

Table 2.

Body, organ, and faecal weights, plasma lipid and gluicose levels, and and spleen pro-inflammatory cytokines in Institute of Cancer Research (ICR) mice fed a diet containing no fibre (NF) or 5% (w/w) kanpyo (KP) for 14 days

	NF	KP
Body weight (g)
Initial	36.5 ± 2.0	37.1 ± 0.5
After 14 days	47.4 ± 3.3	46.0 ± 1.4
Gain	10.9 ± 1.9	8.8 ± 1.0
Organ weight (g)
Liver	2.972 ± 0.170	2.537 ± 0.192
Kidneys	0.693 ± 0.032	0.657 ± 0.044
Spleen	0.135 ± 0.011	0.104 ± 0.006*
Epididymal fat pads	2.044 ± 0.310	2.013 ± 0.149
Caecum	0.442 ± 0.022	0.382 ± 0.032
Colon length (cm)	8.25 ± 0.59	8.80 ± 0.47
Faeces
Frequency (n/day/mouse)	19 ± 1	34 ± 1**
Weight (g/day/mouse)	0.236 ± 0.017	0.294 ± 0.017*
Plasma lipids and glucose (mg/100 mL)
Triacylglycerol	123 ± 20	102 ± 11
Total-cholesterol	155 ± 12	134 ± 5
Glucose	310 ± 16	270 ± 21
Spleen pro-inflammatory cytokines (pg/g spleen tissue)
Tumor necrosis factor-α	517 ± 25	281 ± 13**
Interleukin-6	964 ± 98	831 ± 58

Values are mean and SEM (n = 6)

*,**There are significant differences between NF and KP groups

*p < 0.05, **p < 0.01

Plasma lipid and glucose levels and spleen pro-inflammatory cytokines in mice

Plasma triacylglycerol, total cholesterol, and glucose levels were tended to be low with the KP diet, although the difference was insignificant (Table 2). Among the splenic pro-inflammatory cytokines, TNF-α was significantly suppressed by KP.

Gourd fruit has been widely used not only as KP, a traditional processed food in Japan (Lowry 2005), but also for ethnic therapeutic applications such as anti-inflammatory, anti-hyperlipidaemic, anti-hypertensive, and cardioprotective activities (Turroni et al. 2022). The defecation and lack of effect on the caecal weight of KP might coincide with insoluble dietary fibres (Isken et al. 2010) rich in KP. Additionally, low spleen weight suggests that KP acts on the immune system (Kim et al. 2013). However, additional feeding experiments are required to obtain more evident results.

Caecal microbiota in tested mice

Total bacterial counts and α-diversity

There were no significant differences in the direct total bacterial cell count, measured using DEPIM, in the caecum (approximately 11 log cells/g) and the total read number of the 16S rDNA amplicons (89000–112000) between the NF and KP groups (Table 3). On the other hand, the number of ASVs was significantly higher in the KP group (311) than in the NF group (189). In addition, the α-diversity indices, Shannon index, and Simpson diversity were higher in the KP group than in the NF group.

Table 3.

Direct cell count and alpha-diversity of caecal microbiota of ICR mice fed a diet containing no fibre (NF) or 5% (w/w) kanpyo (KP) for 14 days

	NF	KP
Direct cell count (Log cells/mL)	11.20 ± 0.08	11.08 ± 0.05
16S rDNA (V4) amplicon sequence analysis
Total read number	111,661 ± 22,339	88,994 ± 7890
Number of ASV	189 ± 22	311 ± 16**
Shannon index (H')	2.719 ± 0.103	3.944 ± 0.172**
Simpson diversity (D)	0.852 ± 0.011	0.943 ± 0.010**

Values are mean and SEM (n = 6)

*,**There are significant differences between NF and KP groups

*p < 0.05, ** p < 0.01

Greater diversity might affect host health, and a loss in species richness in the gut is a common finding in several disease states, such as obesity, type 2 diabetes, and inflammatory bowel diseases (Heiman and Greenway 2016). The abundance and richness of diversity of these microbes can be improved by adding dietary fibre to the diet (Mayengbam et al. 2019). However, their diversity is reduced by feeding on some soluble and fermentable dietary fibres, such as inulin (Zhang et al. 2017).

Relative abundances at phylum, genus, and ASV levels

PCA performed on the number of reads for each ASV (Fig. 1A) revealed differences in the gut microbiota composition between the NF and KP groups. According to the caecal microbiota profile at the phylum level, expressed as the relative abundance of phyla (Fig. 1B), the dominant phyla in the NF group were Firmicutes (57%), Actinobacteria (30%), and Bacteroidetes (10%). Compared to the NF group, the abundance of Bacteroidetes was significantly increased in the KP group (26%) and that of Actinobacteria was decreased in the KP group (15%). The Firmicutes-to-Bacteroidetes ratio was low in the KP group (Fig. 1C).

Fig. 1.

Principal component analysis (PCA) (A), abundances at phylum (B), and (D) genus levels in caecum of Institute of Cancer Research (ICR) mice fed a diet containing NF or 5% (w/w) kanpyo (KP) for 14 days. C The ratio of Firmicutes to Bacteroidota is significantly different from that in the NF group: * p < 0.05, ** p < 0.01

In the phylum Firmicutes, the dominant genus in the NF group was Allobaculum (25%), followed by Faecalibaculum (7.7%), which belongs to the family Erysipelotrichaceae (Fig. 1D). Among these genera, the abundance of Allobaculum was significantly lower in the KP group (8.0%) than in the NF group. In contrast, Lachnospiraceae g. (11%) and Clostridium sensu stricto 1 (4.0%), belonging to the Clostridiaceae family, were significantly higher in the KP group. In the phylum Bacteroidota, Muribaculaceae g. was dominant (5.3%) in the NF group, and the abundance in the KP group was higher (12%). Additionally, the abundance of Alistipes, which belongs to Rikenellaceae, was higher in the KP group (3.1%) than in the NF group (1.1%). Most of the abundant Actinobacteriota in the NF group were divided into Bifidobacterium (25%, Bifidobacteriaceae) and Coriobacteriaceae (5.0%). The abundance of the genera in the KP group (13.9% and 0.40%, respectively) was lower than that in the NF group.

The ASV heat map is shown in Fig. 2 for genera, where differences in abundance were observed between the two groups. Among the ASVs that were higher in the KP group than in the NE group, two Lachnospiraceae ASVs were estimated to be Kineothrix alysoides (similarity 97.2%)- and Eubacteriales sp. (92.8%)-like bacteria. Cl. sensu stricto_1 ASVs were estimated to be Cl. saudiense/disporicum (99.2%)-like bacteria. Muribaculaceae ASVs were estimated to be Muribaculum intestinale (90.9%-94.8%)-like bacteria. Alistipes ASV was estimated to be A. onderdonkii (95.7%)-like bacteria. Two ASVs belonging to Allobaculum and Bifidobacterium were significantly lower in the KP group than in the NF group and were estimated to be A. stercoricanis (92.9%)- and B. pseudolongum (100%)-like bacteria, respectively.

Fig. 2.

Heatmap of ASVs for kanpyo (KP)-responsive indigenous genera in caecum of ICR mice fed a diet containing NF or 5% (w/w) kanpyo (KP) for 14 days, shown in Fig. 1D. *, ** Significantly different from control cultures: * p < 0.05, ** p < 0.01

Among the identified KP-RIB groups, Lachnospiraceae family members have been reported as butyrate-producing desirable symbionts that affect their hosts by producing SCFAs, converting primary-to-secondary bile acids, and facilitating colonisation resistance against intestinal pathogens (Sorbara et al. 2020). However, the opposite effects have also been reported and should be considered at the genus and species levels (Abdugheni et al. 2022). The relative abundance of K. alysoides, a spore-forming anaerobe, in the gut of these mice was associated with reduced anxiety-like behaviours (Liddicoat et al. 2020). Cl. disporicum produces lactic acid and ursodeoxycholic acid (Tawtep et al. 2017) and is increased in mice fed fermented green loofah, a fibre-rich vegetable (Shikano et al. 2019). Lee et al. (2022b) reported that the culture supernatant of A. onderdonkii reduced the viability of primary pancreatic cancer cells. M. intestinale-like bacteria in murine caecum are also increased by dietary fibres and polyphenol-rich food materials, such as chilli pepper and brown algae Sargassum horneri (Lee et al. 2022b; Xia et al. 2022b).

Among the suppressed KP-RIB groups, Allobaculum and B. pseudolongum were found in the gut of mice fed a high-sucrose-low-fibre diet rather than a regular chow diet (Kuda et al. 2017). Furthermore, Allobaculum in mice fed a high-fat diet led to the aggravation of enteritis and a positive correlation with body weight and blood glucose levels in diabetic nephropathy (Li et al. 2020; Zheng et al. 2021; Rice et al. 2022). In the present study, the increased and suppressed KP-RIB groups may be correlated with pro-inflammatory cytokines and blood lipid and glucose levels, as shown in Table 2.

Chemical compounds in human faecal cultures

Before fermentation, the pH values of the control and KP broths were approximately 6.5 and 6.4, respectively. During the 24 h incubation, although individual differences were present, the pH drop was more significant in the KP cultures than in the control cultures (Fig. 3A, p < 0.05). Among the organic acids, n-butyrate and lactate were tended to be higher in KP cultures than in control cultures. However, the difference was insignificant (Fig. 3B). Indole and ammonia generation were significantly suppressed in KP cultures (Fig. 3C). Phenol was suppressed in KP cultures but was insignificant due to individual differences in the control cultures.

Fig. 3.

Measurement values of pH (A), organic acids (B), and putative putrefactive compounds (C) of human faecal culture microbiota without (control) or with 2% (w/v) kanpyo (KP) supplementation incubated at 37 °C for 24 h anaerobically. Faecal inoculates were obtained from five volunteers, represented by open circles, open triangles, open squares, closed circles, and closed triangles. ^a,bValues with different superscript letters are significantly different (p < 0.05). *, ** Significantly different from control cultures: * p < 0.05, ** p < 0.01

Among the SCFAs, butyrate in the gut serves as a primary nutrient that provides energy to colonocytes and as a cellular mediator. Butyrate also has health functions; for example, it has gut barrier, immunomodulation, and anti-inflammatory properties (Bedford and Gong 2018). In addition, the overproduction of protein-related putrefactive compounds, such as ammonia, phenol, and indole, is a putative risk factor for tumour and cancer development (Abu-Ghazaleh et al. 2021). Furthermore, indole reportedly produced in the gut is involved in the expression of virulence genes in pathogenic E. coli and Citrobacter rodentium (Kumar and Sperandio 2019).

Microbiota in human faecal cultures

Total bacterial counts and α-diversity

The direct total bacterial cell count in the faecal cultures was 9.6–10.0 Log cells/mL (Fig. 4A). The total read number for ASVs in each faecal culture ranged from 73,000 to 115,000 (Fig. 4B), and the ASV number was 110–230 (Fig. 4C). The α-diversity indices in KP cultures were lower than those in control cultures (Fig. 4D, E).

Fig. 4.

Direct bacterial cell count (A), total read number (B), ASV number (C), Shannon–Wiener H′ index (D), Simpson D index (E), PCA (F), and partial least squares-discriminant analysis (PLS-DA) (G) of human faecal cultures without (control) or with 3.2% (w/v) kanpyo (KP) incubated at 37 °C for 24 h anaerobically. Faecal inoculates were obtained from five volunteers, represented by open circles, open triangles, open squares, closed circles, and closed triangles. *, ** Significantly different from control cultures: * p < 0.05, ** p < 0.01

Phylum, genus, and ASV levels

The gut microbiota varied widely across individuals, and the grouping in the PCA plot was by individual rather than KP addition (Fig. 4F). PLS-DA revealed that each faecal microbiota was affected by KP in the same direction (Fig. 4G).

Figure 5 shows the microbiota of the human faecal cultures. The dominant phyla in the control cultures were Firmicutes (61%), Bacteroidota (15%), Proteobacteria (15%), and Actinobacteria (8.7%) (Fig. 5A). In KP cultures, Actinobacteria (19%) was higher, and Proteobacteria (8.2%) was lower than in the control cultures.

Fig. 5.

Microbiome composition at phylum (A) and genus (B) levels in human faecal culture microbiota without (control) or with 3.2% (w/v) kanpyo (KP) incubated at 37 °C for 24 h anaerobically. *, ** Significantly different from control cultures: * p < 0.05, ** p < 0.01

In the phylum Firmicutes, Faecalbacterium (5.9%), Lachnospira (3.7%), and Anaerostipes (3.2%) in KP cultures were significantly higher than those in control cultures (Fig. 5B). In the phylum Proteobacteria, the dominant genus in the control culture was Escherichia (7.0%), and that in the KP culture was suppressed (2.0%). Almost all Bacteroidota and Actinobacteria were defined as the genera Bacteroides and Bifidobacterium.

Figure 6 shows a heatmap of ASVs in the top 30 abundance. Even in genera that showed significant differences (Fig. 5), individual differences in ASV levels did not reach statistical significance. For example, an increased KP-RIB genus Faecalibacterium was divided into two ASVs of F. prausnitzii (98.8–99.6%)-like bacteria. Bifidobacterium was divided into three ASVs: B. adolescentis/stercosis (100%)-, B. catenulatum/pseudocatenulatum (100%)-, and B. longum/breve (100%)-like bacteria. From these cultures, B. adolescentis, B. pseudocatenulatum, and B. longum were isolated and identified. Among the three Bifidobacterium ASVs, B. adolescentis was detected as a KP-RIB in two volunteers. B. pseudocatenulatum was detected as a KP-RIB in the other two volunteers. The other volunteers had KP-RIB B. longum. In contrast, KP suppressed Escherichia sp. (100%)- and Anaerotignum faecicola (100%)-like bacteria, irrespective of individual differences.

Fig. 6.

Heatmap of typical ASVs in human faecal culture microbiota without (control) or with 3.2% (w/v) kanpyo (KP) incubated at 37 °C for 24 h anaerobically. * Significantly different from control cultures: * p < 0.05

Among the species of KP-RIB groups with increased numbers besides Bifidobacterium, F. prausnitzii is associated with the capacity to produce beneficial metabolites, mainly the SCFA butyrate, which is known to have several health-promoting effects and has been regarded as a next-generation probiotic (De Filippis et al. 2022). Gut Lachnospira abundance is low in patients with immune-mediated inflammatory diseases (Forbes et al. 2018). Anaerostipes convert lactate to butyrate and inositol to propionate and acetate and are regarded as desirable gut commensals (Bui et al. 2021).

Antioxidant and immunomodulatory activities in human faecal cultures

In KP cultures, the DPPH radical-scavenging capacity increased during fermentation; however, the increase was not significant (Fig. 7A). Before incubation, the O₂⁻ radical-scavenging capacity of the control and KP broths was difficult to detect; during incubation, the scavenging capacity in KP cultures increased (Fig. 7B). The Fe^3±reducing power in KP broth was higher than that of the control, and it was increased in broth cultures, particularly in KP cultures (Fig. 7C). Before incubation, the NO secretion-promoting effect of the broths in RAW264.7 cells, which is recognised as an immunomodulatory effect, was not detected, and it was increased by fermentation in both cultures (Fig. 7D).

Fig. 7.

DPPH radical-(A) and superoxide anion radical-(B) scavenging capacities, Fe³⁺-reducing power (C), and promotion of NO secretion in RAW264.7 cell culture medium (D) of human faecal cultures without (control) or with 3.2% (w/v) kanpyo (KP) incubated at 37 °C for 24 h anaerobically. ^a,bDifferent letters indicate significant differences: p < 0.05

The antioxidant and immunomodulatory effects of KP were low in the present study. However, these activities are induced by fermentation, which may occur in the gut. Similar results have been reported in human culture studies on water fermentable dietary fibres in brown algae (alginate, laminaran), soy protein, red chilli pepper, and turmeric (Lee et al. 2022a; Xia et al. 2022a, 2022b).

In this study, the abundance of butyrate-producing and other SCFA-producing bacteria increased in the caecal contents of KP-fed mice and human faecal cultures supplemented with KP. In the mouse experiment, both the increased and suppressed KP-RIB groups might be correlated with the suppression of pro-inflammatory cytokines, blood lipid, and glucose levels. In the human faecal culture experiment, Bifidobacterium spp. and F. prausnitzii-like bacteria were detected as the KP-RIB in cultures prepared using samples from all volunteers. Our results suggest that KP and KP-RIB have a combined effect on host health. Further studies on the mechanisms of the interaction between KP components and desirable KP-RIB, particularly the mechanisms in butyrate-producing human gut commensals, should be the focus of future research.

Conclusion

We detected KP-RIB by performing 16S rRNA (V4) amplicon sequencing analysis of the caecal microbiota of ICR mice fed a diet containing 5% KP for 14 days and on human faecal cultures supplemented with 3.2% KP for 24 h. The abundance of butyrate-producing and other SCFA-producing bacteria increased in the caecal content of KP-fed mice and the KP-supplemented human faecal cultures. In the mouse experiment, both the increased and suppressed KP-RIB groups may be correlated with the suppression of pro-inflammatory cytokines. In the human faecal culture experiment, Bifidobacterium spp. and F. prausnitzii-like bacteria were detected as the KP-RIB in cultures prepared using samples from all volunteers. Moreover, fermentation increased O₂⁻ radical-scavenging capacity and Fe³⁺-reducing power more notably in KP cultures than in control cultures. These results suggest that KP-RIB and KP intake may synergistically affect host health. However, further studies are required to clarify the synergistic effects of KP and KP-RIB.

Authors contribution

MY and HO: conceptualisation, methodology, validation, formal analysis, investigation, resources, data curation, writing—original draft, and visualisation. TK: conceptualisation, methodology, validation, formal analysis, resources, data curation, writing—review and editing, visualisation, supervision, and project administration. YX and AN: conceptualisation, methodology, validation, formal analysis, investigation, resources, data curation, writing—original draft, and visualisation. HT, JI, and ST: conceptualisation, methodology, and supervision.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

Ethical approval

This study was approved by the Animal Experiment Committee of the Tokyo University of Marine Science and Technology (Approval No. R3-1), and the Committee for Research Involving Human Subjects of the Tokyo University of Marine Science and Technology approved the study protocol (Approval No. R03-001).

Informed consent

All volunteers provided written informed consent before participating in the study.