Effect of short‐term consumption of yellow peas as noodles on the intestinal environment: A single‐armed pre‐post comparative pilot study

Abstract Legumes contain dietary fiber and resistant starch, which are beneficial to the intestinal environment. Here, we investigated the effects of yellow pea noodle consumption on the gut microbiota and fecal metabolome of healthy individuals. This single‐armed pre‐post comparative pilot study evaluated eight healthy female participants who consumed yellow pea noodles for 4 weeks. The gut microbiota composition and fecal metabolomic profile of each participant were evaluated before (2 weeks), during (4 weeks), and after (4 weeks) daily yellow pea noodle consumption. 16S rRNA gene sequencing was performed on stool samples, followed by clustering of operational taxonomic units using the Cluster Database at High Identity with Tolerance and integrated QIIME pipeline to elucidate the gut microbiota composition. The fecal metabolites were analyzed using capillary electrophoresis time‐of‐flight mass spectrometry and liquid chromatography time‐of‐flight mass spectrometry. Compared to day 0, the relative abundances of five bacterial genera (Bacteroides, Bilophila, Hungatella, Parabacteroides, and Streptococcus) in the intestinal microbiota significantly decreased, wherein those of Bifidobacterium longum and Ruminococcus bromii were increased on day 29 and decreased to the basal level (day 0) on day 57. Fecal metabolomic analysis identified 11 compounds showing significant fluctuations in participants on day 29 compared to day 0. Although the average levels of short‐chain fatty acids in participants did not differ significantly on day 29 compared to those on day 0, the levels tended to increase in individual participants with >8% relative abundance of R. bromii in their gut microbiota. In conclusion, incorporating yellow peas as a daily staple may confer human health benefits by favorably altering the intestinal environment.


| INTRODUC TI ON
The human intestinal tract is a complex consortium of over 300 bacterial species and trillions of individual bacteria, which comprise the intestinal microbiota and form a unique ecosystem adapted to the intestinal environment (Quigley, 2013;Sender et al., 2016). The gut microbiome modulates gastrointestinal development, confers enhanced metabolic capabilities, and protects against pathogens (Bäckhed et al., 2005). Several studies have revealed the crucial role of diet in determining the composition of the human gut microbiota. Strategic dietary interventions have been shown to modulate the abundance of specific species and have gained interest in the development of novel therapeutic methods for disease control and prevention (David et al., 2014;Kolodziejczyk et al., 2019;Walker et al., 2011).
Recent studies have clarified that intestinal bacterial metabolites, such as organic acids, short-chain fatty acids (SCFAs), and branched amino acids, contribute to health maintenance. SCFAs produced by intestinal bacteria impart various health effects, such as serving as a source of energy for intestinal epithelial cells, lowering intestinal pH, suppressing the growth of harmful bacteria, preventing obesity, reducing intestinal inflammation, and regulating immune functions (Ríos-Covián et al., 2016). Species and occupancy rates of intestinal bacteria, as well as diet composition, significantly affect the production of these metabolites (Green et al., 2016;Li et al., 2018). In particular, dietary fiber and resistant starch (RS) are important components that contribute to maintaining the intestinal environment and normal defecation (Bird et al., 2010).
Legumes, rich in proteins, minerals, and phytochemicals such as polyphenols, are recognized as highly nutritious foods (Marinangeli et al., 2020). Consumption of legumes leads to various beneficial effects, such as sustained satiety, body weight improvement, metabolic syndrome prevention, and extension of healthy life expectancy (Darmadi-Blackberry et al., 2004;Polak et al., 2015). Legumes are also high in dietary fiber and RS, which are beneficial for reducing postprandial blood glucose levels (Clemente & Olias, 2017;Mayengbam et al., 2019). Several clinical trials have explored the favorable role of legume consumption in the modulation of the human gut microbiota and metabolite profiles (Baxter et al., 2018;Fernando et al., 2010;Finley et al., 2007;Kadyan et al., 2022;Sheflin et al., 2017). For example, the consumption of canned chickpeas (200 g/day) for 3 weeks reduced the abundance of pathogenic and putrefactive gut bacterial species in healthy adults (Fernando et al., 2010). Similarly, consuming cooked navy beans at 35 g/day for 28 days increased gut bacterial diversity and altered gut microbial composition compared to the baseline in colorectal cancer survivors (Sheflin et al., 2017). Another study showed that navy beans significantly altered the stool metabolome and metabolic pathways involved in maintaining colon health in cancer survivors (Baxter et al., 2018). Owing to their health and environmental benefits, legumes are increasingly consumed not only as unprocessed foods but also as processed foods such as noodles, plant-based meat, and milk alternatives. Despite these benefits, the actual intake of legumes is lower than optimal (~60 g) in many regions worldwide. A diet low in legumes increases the risk of heart disease, and this risk is higher in younger age groups than in older age groups (GBD 2017Diet Collaborators, 2019. In Japan, the intake of legumes, a rich source of dietary fiber, is low among younger people. The dietary fiber intake is low, particularly in females aged 20-50 years (<16 g/day vs. ≥24 g/day [recommended limit]; Committee for Development of the "Dietary Reference Intakes for Japanese" & Ministry of Health, Labour and Welfare, Japan, 2020; Ministry of Health, Labour, and Welfare, Japan, 2020).
Dietary fiber and RS are fermented by intestinal bacteria and exert favorable effects on the intestinal environment (Baxter et al., 2018;Mayengbam et al., 2019;Sheflin et al., 2017), which is dependent on the structure of dietary fiber and RS in food. For example, SCFA production during in vitro fecal fermentation with pinto bean cells as a substrate increased after enzymatic treatment of beans compared to intact beans, indicating the effects of cell wall integrity of pinto beans on the modulation of the microbiota composition (Guan et al., 2020). However, it remains unclear whether processed legumes can consistently provide the health benefits of unprocessed legumes. Moreover, few studies have explored the effects of legume consumption as a staple food source on the intestinal environment. In addition, inculcating the habit of eating legumes in people who have not been consuming them daily can be challenging. Like other legumes, yellow peas are enriched with protein and dietary fiber, thus attracting attention as a plant-based meat alternative (Ferawati et al., 2021;Smith et al., 2012). We previously reported that yellow pea noodles are palatable and showed the possibility of their continuous consumption as a staple food (Yoshimoto et al., 2020). However, knowledge of the effects of continuous consumption of such foods on the gut microbiota is limited.
Therefore, the present pilot study aimed to investigate the effects of consuming yellow pea noodles continuously for 28 days on the gut microbiota composition and fecal metabolomic profiles of healthy individuals.

| Participants
The participants in this study were recruited through an advertisement sent to users of the UNLOG app in Japan. A total of 2524 individuals were recruited after responding to the advertisement.
Individuals were screened based on the following inclusion criteria: (1) women aged 20 to less than 50 years, (2) defecation frequency 3-5 times per week, (3) body mass index (BMI) between 23.0 and 25.0, (4) answered that they were feeling a little concern over their physical condition, (5) answered that they were feeling a little stress, and (6) experienced a cold or flu once within the last year.
A total of 21 candidates fulfilled the above criteria, among whom 18 agreed to participate. Ten participants were excluded after deviating from the study conditions, leaving eight participants in the study ( Figure 1).

| Test material
Yellow pea noodles made exclusively from unshelled yellow pea flour were obtained from ZENB JAPAN Co., Ltd. (Aichi, Japan). The prepared boiled yellow pea noodles (100 g) contained 65 g water, 7.1 g protein, 26.2 g carbohydrates, 7.4 g total dietary fiber, and 2.1 g RS.

| Study schedule
This study employed a single-arm pre-post comparison strategy.
The total study period was 10 weeks (Figure 2), comprising a preobservation period of 2 weeks, an experimental period of 4 weeks, and a post-observation period of 4 weeks. During the experimental period, the participants consumed boiled yellow pea noodles (200 g per serving) once daily, with a seasoning of their choice. Participants were not allowed to consume yellow pea noodles during the pre-and post-observation periods. Additionally, participants were not allowed to consume new medications, supplements, or foods that could affect the intestinal microbiota during the entire study period, such as probiotics, food products fortified with oligosaccharides or dietary fiber, or health foods that improve constipation. However, participants already taking such substances were allowed to continue taking them during the entire study period, maintaining a consistent intake quantity.
During the total study period, participants recorded their defecation schedules and responded to online questionnaires using the UNLOG app. DialBetics, a smartphone-based application, was used to record the participants' meals (Waki et al., 2014). The participants were instructed to take photographs of all meals and send them to the research account. Subsequently, meal images were analyzed using DialBetics to quantify the energy and nutrient intake. Stool samples were collected on days 0, 15, 29, and 57 for further analysis. The study protocol was registered in the University Hospital F I G U R E 1 Flowchart of screening and recruitment of participants for the study. · Women aged 20 to less than 50 years · Defecation frequency of 3-5 times a week · BMI between 23.0 and 25.0 · In the questionnaire survey: · Answered that they were feeling a little concern over their physical condition · Answered that they were feeling a little stress · Had a cold or flu once within the last year Excluded (n = 6) · Pregnant or breastfeeding (n = 3) · History of gastrointestinal illness (n = 1) · Food allergies (n = 1) · Judged as inappropriate for other reasons (n = 1) Excluded (n = 4) · Consumed yellow pea noodles during the pre-yellow pea noodle consumption period (n = 2) · Took antibacterial substances (n = 1) · Could not be contacted (n = 1) F I G U R E 2 Study schedule for 10 weeks, including 2-week preobservation period (days −13 to 0), 4 weeks of yellow pea noodle consumption period (days 1 to 28), and 2-week post-observation period (days 29 to 56). The start of yellow pea noodle consumption was day 1.   (Sugimoto et al., 2010).
Signal peaks corresponding to isotopomers, adduct ions, and other product ions of known metabolites were excluded.

| Sample collection and DNA extraction
Rice grain-sized fecal samples were collected on days 0, 15, 29, and 57, using a fecal collection kit (Techno Suruga Laboratory, Shizuoka, Japan

| Data analysis
Taxonomic identification was performed by clustering operational taxonomic units (OTUs) using the Cluster Database at High Identity with Tolerance (CD-HIT-OTUs) and integrated QIIME pipeline for 16S rRNA (Caporaso et al., 2010;Li et al., 2012). Briefly, sequence reads 1 and 2 were assembled and clustered using CD-HIT-OTUs, and representative sequences were extracted. The QIIME pipeline was used to assign phylogenetic nomenclature to the representative sequences.

| Statistical analysis
Statistical analysis of the metabolomic data was conducted using Welch's t-test. Paired t-tests were used to compare other data using the bell curve in Excel (Social Survey Research Information Co., Ltd., Tokyo, Japan). p-Values <.05 were considered to be statistically significant.

| Effect of yellow pea noodle consumption on body weight and defecation frequency
All participants consumed at least 26 servings of boiled yellow pea noodles (200 g/day/serving). The average body weights of participants did not differ significantly (p > .05) before (day 0, 58.9 ± 3.4 kg) and after daily consumption (day 29, 58.9 ± 4.0 kg) of yellow pea noodles. Additionally, the average body mass index (BMI) values of participants on day 0 (23.5 ± 0.7) and day 29 (23.5 ± 0.8) and the number of defecations between days −13 to 0 (10.5 ± 2.9) and days 15 to 28 (11.0 ± 2.9) did not differ significantly (p > .05; Table S1).

| Effect of yellow pea noodle consumption on intestinal metabolites
Fecal metabolomic analysis identified 11 compounds that displayed significant fluctuations in participants on day 29 compared to day 0 (Table S2). As shown in Table S3, the average acetic acid levels in the feces of participants did not differ significantly (p > .05) between day 0 (38.26 ± 13.92 μmol/g) and day 29 (41.00 ± 21.63 μmol/g). Similarly, propionic acid and butyric acid levels in feces did not differ significantly on day 29 compared to those on day 0 (Tables S4 and S5).

| Effect of yellow pea noodle consumption on intestinal microbiota
The relative abundances (percentage of reads) of five bacterial genera (Bacteroides, Bilophila, Hungatella, Parabacteroides, and Streptococcus) in the intestinal microbiota of participants significantly decreased on day 29 compared to those on day 0 ( Table 1).
The average relative abundances of eight OTUs were significantly changed in all participants on day 29 compared to those on day 0. In particular, the relative abundances of OTU6 (Bifidobacterium longum) and OTU8 (Ruminococcus bromii) increased significantly on day 29 compared with those on day 0. The relative abundances of the remaining six OTUs decreased significantly on day 29 compared with those on day 0 ( Table 2). Analyzing individual differences in intestinal microbiota composition revealed that in seven participants (no. 2, 4, 5, 6, 7, 8, and 11), the relative abundance of OTU6 (Bifidobacterium longum) increased clearly on day 29 compared to that on day 0, but was reduced to the basal level on day 57. However, this bacterium was not detected in participant no. 3 during the entire experimental period, likely because levels were below the detection limit ( Figure 3). Similarly, OTU8 (R. bromii) was detected in six participants

| DISCUSS ION
The present study evaluated the effects of yellow pea noodle consumption on the gut microbiota and fecal metabolome of healthy adults. The study findings suggest that the consumption of yellow pea noodles clearly and favorably altered individual compositions of the gut microbiota ( Figure 5).
Yellow pea noodle consumption did not significantly affect the average body weight, BMI, or defecation frequency of participants.
Several studies have reported the beneficial effects of peas or high-fiber-containing foods on weight gain (Abete et al., 2009;Ito et al., 2023;Jarrar et al., 2019;Lambert et al., 2017). No fluctuations in body weight were observed in a trial in which participants with BMIs between 18.5 and 30 consumed a high-fiber cereal (Jarrar et al., 2019). Furthermore, the potential of yellow pea fiber to induce weight loss in obese individuals has been previously reported (Lambert et al., 2017). Taken together, these studies suggest that fiber-rich yellow pea noodles could provide a food commodity to help reduce body weight fluctuations in individuals with high BMIs.
Nevertheless, the average BMI of participants in the current study remained stable at 23.5, indicating that all participants maintained their healthy body weight throughout the study period. Further studies are required to explore the benefits of yellow pea noodle consumption in obese individuals.
The intestinal microbiota analysis in the present study revealed that the relative abundances of Bifidobacterium longum and R. bromii significantly increased on day 29 compared to those on day 0, which TA B L E 2 Significantly fluctuating operational taxonomic units during yellow pea noodle consumption. Note: All values are the mean ± standard deviation (SD) of eight participants; Day 0, before consumption; Day 29, after consumption of yellow pea noodles.

F I G U R E 3 Fluctuations in the relative abundance of
Bifidobacterium longum (OTU6) among participants.

F I G U R E 4 Fluctuations in the relative abundance of
Ruminococcus bromii (OTU8) among participants.
suggested the benefits of yellow pea noodle consumption. However, these levels returned to pre-consumption levels when yellow pea noodle consumption was stopped, suggesting that the relative abundances of these bacteria were reversible and caused by the continuous consumption of yellow pea noodles. Bifidobacterium longum ferments dietary fiber (Iwata et al., 2009). R. bromii quickly degrades RS and is particularly important for the fermentation of aged starch with a native granular structure (RS2) and retrograded starch after heating and cooling (RS3) (Ze et al., 2012). Boiled yellow pea noodles are rich in total dietary fiber (7.4%) and RS (2.1%). The meal records revealed a significant increase in the average daily intake of dietary fiber in participants during the yellow pea noodle consumption period (20.03 g) compared to that during the pre-observation (10.42 g) and post-observation (9.40 g) periods. However, energy intake and other major nutrients (protein, fat, and salt equivalents) did not change significantly. Therefore, we speculate that the dietary fiber from yellow pea noodles contributed to the increased relative abundance of these bacteria during the consumption period. Notably, bifidobacteria are predominant in the Japanese intestinal flora. In particular, Bifidobacterium longum is the only bifidobacterium reportedly present in both infants and adults over long periods. An estimated 87% of the total Japanese population harbors this bacterium (Odamaki et al., 2018). Furthermore, the gut enterotype of the Japanese population is classified as Ruminococcus type, with a high relative abundance of Ruminococcus bacteria (Arumugam et al., 2011). Therefore, the favorable effects of yellow pea noodle consumption on the intestinal microbiota could be adaptable for Japanese.
Acetic acid is a major metabolite of Bifidobacterium longum, whereas acetic acid, butyric acid, and propionic acid are the major metabolites of R. bromii (Bianchi et al., 2019). However, we found no significant changes in these metabolites (Tables S2-S5). We found an increased abundance of Bifidobacterium longum ( Table 2), whereas that of Bacteroides and Parabacteroides, whose major metabolite is acetic acid, decreased during yellow pea noodle consumption (Table 1). These results suggest that changes in acetic acid may have been counterpoised by alterations in the microbial composition. On the contrary, participants with ≥8% relative abundance of R. bromii in the gut microbiota on day 29 (nos. 3, 4, 6, and 7) tended to produce higher levels of SCFAs compared to other participants (Tables S3-S5). This suggests that the effects of yellow pea noodles on metabolites may vary depending on the composition of the intestinal microbiota of an individual. These findings align with previous reports in which RS fermentation was enhanced in individuals harboring R. bromii, but not in those with undetectable R. bromii levels (Louis et al., 2007;Walker et al., 2011). Moreover, this may explain the nonincrease in the relative abundances of Bifidobacterium longum and R. bromii on day 29 in individuals with undetectable levels of these bacteria on day 0.
Bifidobacterium longum is a probiotic that benefits the intestinal environment and suppresses inflammation (Yao et al., 2021). R.
bromii is a keystone species that degrades RS and favorably alters the composition of the gut microbiota (Scott et al., 2015). Moreover, several studies have reported the health benefits of SCFAs produced by the intestinal microbiota (Rauf et al., 2022). For example, acetic acid produced by Bifidobacterium longum protects against enteropathogenic infections (Fukuda et al., 2011). Taken together, the study findings suggest that consuming yellow peas as a daily staple may confer human health benefits.

F I G U R E 5
Effects of yellow pea noodle consumption on gut microbiota and fecal metabolomic profile of healthy adults. Yellow pea noodle consumption did not induce major changes in the metabolomic profile in overall data but favorably altered individual gut microbiota compositions, suggesting that long-term intake of yellow pea noodles may confer health-promoting effects. Consumption of yellow pea noodles caused significant changes in the average relative abundances of five bacterial genera (Bacteroides, Bilophila, Hungatella, Parabacteroides, and Streptococcus) in the intestinal microbiota. Of these, the average relative abundance of Bilophila decreased below 50% on day 29 compared to day 0 ( Table 1).
Bilophila wadsworthia is the only species reported in Bilophila, and the abundance of this species was shown to be increased by an animal-based diet (Odamaki et al., 2016). Studies have indicated that it destroys the intestinal mucosa to produce hydrogen sulfide, a toxic substance, and is involved in the induction of inflammation in the intestinal tract (David et al., 2014;Devkota et al., 2012). Bilophila wadsworthia comprises approximately <0.01%-0.2% of normal human intestinal microbiota (Baron, 1997;Dostal Webster et al., 2019;Vandeputte et al., 2017). In the present study, the average relative abundance of Bilophila was less than 0.1% on day 29. This finding indicates that dietary habits of consuming yellow pea noodles as a staple food promote not only the intake of RS and dietary fiber in yellow peas but also reduce health risks derived from animalbased diets and improve the intestinal environment. Furthermore, Odamaki et al. (2016) showed that the animal-diet-induced increase in the abundance of Bilophila was restored by the intake of yogurt supplemented with Bifidobacterium longum, which could be attributed to the bile salt hydrolase activity of bifidobacteria (Begley et al., 2006). Our study showed that the yellow pea noodles consumption resulted in a reduction in Bilophila despite no restrictions on the intake of animal-based diets. Therefore, we inferred that the abundance of bifidobacteria increased by fermentation of RS and dietary fiber in yellow pea noodles could have reduced the growth of bile-using bacteria, Bilophila. Moreover, the dietary habit of eating yellow pea noodles could have made participants feel full, leading to reduced consumption of animal-based diets and, therefore, a reduced abundance of Bilophila. Taken together, these results suggest that dietary habits with a high degree of freedom to consume yellow pea noodles once daily with any seasoning at any time and without restrictions on intake of animal-based diets contribute to reducing long-term health risks and can be continued with little patience.
This study revealed a significant decrease in the relative abundance of five bacterial genera ( Table 1) and significant changes in 11 metabolites in the feces (Table S2) during the yellow pea noodle consumption period. We showed that lysophosphatidylcholine (LPC; 12:0) and the genera Bilophila and Bacteroides in the feces were significantly reduced during yellow pea noodle consumption.
These findings are consistent with those of previous animal studies (Tang et al., 2021;Yang et al., 2022). To our knowledge, this is the first study to reveal a new correlation between the human intestinal microbiota and the fecal metabolome. It has been suggested that LPC abundance promotes the release of inflammatory cytokines and is associated with intestinal inflammation (Tagesson et al., 1985;Tang et al., 2021) and epithelial barrier function. In vitro and in vivo assessments have shown that LPC treatment damages the tight junctions of colonic epithelial cells. Therefore, we speculate that the consumption of yellow pea noodles reduces LPC production and suppresses inflammation and epithelial barrier damage in the human intestine. However, these findings need to be validated in future studies. Furthermore, because the molecular structures of the LPCs detected in previous studies and in this study could be different, further studies are necessary to confirm the effects of yellow pea noodles on LPC abundance.
The present study had several limitations. First, the study was only conducted with Japanese participants. Gut enterotypes are known to vary by race and ethnicity; hence, the findings may not be generalizable to other populations. Second, the study duration was short. As the effects of yellow pea noodles on the intestinal microbiota composition were reversed upon cessation of consumption, evaluating its effects should be conducted over a longer duration. Third, the results of this study indicated that Bifidobacterium longum and R.
bromii degrade the dietary fiber and RS present in yellow pea noodles; however, the mechanism of decomposition remains unknown.
Further in vitro studies are required to clarify how these bacteria degrade the dietary fiber and RS in yellow peas. Fourth, this study was limited to evaluating changes in the intestinal microbiota and associated metabolites. Evaluating the effects of yellow pea noodle consumption on other factors, such as obesity and inflammatory markers, could further strengthen its applicability as a daily staple.
In conclusion, this pilot study revealed the favorable effects of yellow pea noodle consumption on the composition of the gut microbiota, providing evidence that yellow pea noodle consumption is beneficial for human health. Furthermore, this study used an app that recorded the participants' diets, including yellow pea noodles, and their physical conditions. The benefit of such apps in helping participants become aware of healthy dietary lifestyles and providing motivation for long-term continuation should be evaluated.

ACK N OWLED G M ENTS
This research was funded by Mizkan Holdings Co. Ltd. We thank the participants and everyone involved at UNLOG K.K for their cooperation in conducting this study. We also thank Editage (www.edita ge.com) for the English language editing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The nucleotide sequence data obtained in this study are available in the DDBJ Sequence Read Archive under accession numbers DRR353683-DRR353714. Data supporting the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
This study protocol was approved by the ethics committees of Mizkan Holdings Co., Ltd., Aichi, Japan, and UNLOG K.K, Tokyo, Japan.

I N FO R M ED CO N S ENT
The participants provided informed consent prior to the study in accordance with the Declaration of Helsinki.