The gut microbiome plays an important role in the development of both irritable bowel syndrome (IBS) and obesity. In the case of IBS, research has shown that alterations in the composition and diversity of the gut microbiome can contribute to the development and symptoms of IBS.¹ Imbalances in microbiome can lead to increased gut permeability, inflammation, and visceral hypersensitivity, all of which are associated with symptoms of IBS.^2,3

Similarly, the gut microbiome has been implicated in the development of obesity. Studies have revealed differences in the composition of the gut microbiome between obese and non-obese individuals.⁴ Certain compositional changes in the gut microbiome have been found to be associated with weight gain and obesity, while others are associated with weight loss. Additionally, the gut microbiome can affect appetite by producing certain hormones and neurotransmitters that influence food intake.⁵

While there is evidence suggesting a link between the gut microbiome, IBS, and obesity,⁶ the exact mechanisms and causality are still being actively studied. Factors such as diet, lifestyle, and genetics also play significant roles in these conditions.⁷ Nonetheless, manipulating the gut microbiome through approaches like probiotics, prebiotics, and fecal microbiota transplantation shows promise in managing symptoms of IBS and potentially influencing weight regulation in obesity.⁸

Probiotics are commonly used as gut microbiome-based therapy for IBS and are often consumed through fermented dairy food. However, since dairy itself has the potential to cause digestive symptoms, fermented rice products are attracting attention as an alternative delivery route. A fermented rice drink with Lactiplantibacillus plantarum JSA22 strain was developed by the National Institute of Food Science, Korea. L. plantarum JSA22 has probiotic properties and can inhibit Salmonella Typhimurium infection of intestinal epithelial cells.⁹ A fermented rice drink with L. plantarum JSA22 showed antioxidant activity and produced higher lysine content than fermented rice drink with Lacticaseibacillus rhamnosus GG. Also, fermented rice drink with L. plantarum JSA 22 significantly increased butyrate compared to dairy yogurt in the in vitro fecal fermentation experiment. Mice consumed fermented rice drink with L. plantarum JSA 22 showed a decrease in B cell population while stimulating concentrations of IFN-γ and IL-2, indicating potential immune-modulating effects.¹⁰ Our group showed that consuming fermented rice drink with L. plantarum JSA22 decreased disease activity and inflammatory cytokines, such as IL-6 and TNF-α in the dextran sodium sulfate-induced colitis mice model.¹¹ This study aims to investigate the effect of fermented rice drink on symptoms, blood tests for metabolic disorders, fecal and salivary microbiome, and fecal metabolites in overweight patients with IBS.

Subjects

We conducted this study targeting overweight¹² (body mass index [BMI] ≥ 23 kg/m²) adults aged 20 years to less than 65 years residing in South Korea who were diagnosed with IBS. Exclusion criteria included individuals diagnosed with and undergoing treatment for cancer, patients with inflammatory bowel diseases, those diagnosed with organ conditions within the past 6 months that could induce abdominal symptoms (such as peptic ulcers, acute gastritis, cholecystitis, pancreatitis, appendicitis, liver abscess, and acute hepatitis), individuals incapable of providing voluntary informed consent, cases where research participation by the subjects was deemed inappropriate by the investigators, and individuals regularly taking medications that could significantly affect gastrointestinal (GI) motility and microbiome (such as antibiotics, anticholinergics, antipsychotics, antiparkinsonian agents, or probiotics) or those whose participation was discontinued or excluded by the investigator due to factors affecting GI motility. This study was conducted following approval from the Institutional Review Board of Kangbuk Samsung Hospital (IRB No. 2022-07-008).

Study Design

The study was a single-center, randomized, double-blind, controlled, parallel-group design in which the effect of daily consumption of a fermented rice drink with L. plantarum JSA22 was compared with a nonfermented rice drink without probiotics (placebo control drink group) in patients with IBS and metabolic syndrome.

Study Products

The fermented rice drink used in this study was produced using the patented strain of L. plantarum JSA22 obtained from the National Institute of Food Science and Technology, Korea. Its primary ingredients consist of rice, water, and rice bran, with a liquid volume ranging from 180-220 mL and a solid content of 15-20%. Compared to regular dairy fermented products, it contains 10 times more lysine, 4.6 times more gamma aminobutyric acid, and 2.2 times more dietary fiber. Both the fermented rice drinks with L. plantarum JSA22 and nonfermented rice drinks without probiotics were manufactured by a specialized fermentation company with Hazard Analysis Critical Control Point-certified facilities.

Protocol

Upon obtaining the patient’s consent, a screening period of 2 weeks was observed, followed by a random allocation to either the fermented rice drink or nonfermented rice drink group (Fig. 1). After allocation, participants’ baseline dietary information was obtained through a mobile app and phone interviews with a dietitian (Gut & Food Healthcare Co, Ltd, Seongnam, Korea). Questionnaire surveys, blood samples, and saliva and stool samples were collected before and after the month-long of the rice drink administration. Additionally, a follow-up telephone survey was conducted 1 month after the end of the consumption period to assess any changes in the patient’s symptoms. Any adverse symptoms that occurred during the consumption period were checked at each visit.

Figure 1. Flow diagram of the hospital visit protocol.

Questionnaires

The demographic questionnaire included age, sex, height, weight, place of residence, smoking status, alcohol consumption, medication usage, and past medical history. IBS symptoms were evaluated using Rome IV questionnaire-Korean version.¹³ As the primary efficacy variables, a 7-point Likert scale was used to assess the global IBS symptoms impacting daily life for the subjects at 4 weeks and 8 weeks. This question asked, “Considering these symptoms, how much have they affected your daily life in the past 24 hours?” Possible response options were 0 (none at all), 1 (very mild), 2 (mild), 3 (moderate), 4 (strong), 5 (very strong), and 6 (unbearable). As secondary efficacy variables, a 7-point Likert scale was also used to assess each of the following symptoms: abdominal pain/discomfort, distension/bloating, urgency, and bowel habit. Furthermore, the change of participants’ overall health status, abdominal discomfort/pain, and bowel habits during the 4-week treatment period were assessed using the following categories: (1) completely improved, (2) significantly improved, (3) somewhat improved, (4) no change, or (5) worsened. Both “completely improved” and “significantly improved” were defined as “optimal improvement.” Additionally, changes in stool form using Bristol stool form scale,¹⁴ changes in stress index using Brief Encounter Psychosocial Instrument-Korean version (BEPSI-K),¹⁵ and changes in psychological comorbidity using Hospital Anxiety and Depression Scale (HADS) were measured before and after treatment.¹⁶

Blood Metabolic Syndrome Indicators

All blood tests were conducted after a fasting period of at least 12 hours. Total cholesterol, triglycerides, high-density lipoprotein-C, and low-density lipoprotein-C were measured using an automated analyzer (Advia 1650; Siemens, Berlin, Germany). Total cholesterol and triglycerides were analyzed using an enzymatic calorimetric test. High-density lipoprotein-C measurement used the selective inhibition method, and low-density lipoprotein-C measurement was analyzed using a homogenous enzymatic calorimetric test. Fasting blood glucose levels were measured using an automated analyzer (Advia 1650) through the hexokinase method, and fasting insulin levels were measured using an immunoradiometric assay (Biosource, Nivelles, Belgium). The intra-assay coefficients of variation ranged from 2.1% to 4.5%, while the inter-assay coefficients of variation for quality control ranged from 4.7% to 12.2%. The degree of insulin resistance was assessed using the Homeostatic Model Assessment for Insulin Resistance according to the following formula: (fasting insulin [μU/mL] × fasting glucose [mmol/L])/22.5.

Fecal and Salivary Microbiome Analysis

Stool and saliva samples collected at Kangbuk Samsung Hospital were sent to a laboratory for next-generation sequencing 16S ribosomal RNA analysis (Endomix, Inc., Seongnam, Korea) within 2 days of collection. Total DNA was extracted from 300 μL of fecal solution using the QIAamp PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany). For the analysis of the gut microbiome, the Variable regions V3 and V4 within the 16S ribosomal RNA gene were amplified through polymerase chain reaction (PCR). Subsequently, a 2-step PCR was employed to prepare the library suitable for the MiSeq platform (300 bp × 2). The first-step PCR was performed using the KAPA HiFi HotStart ReadyMix PCR kit (Roche, Wilmington, MA, USA). The PCR reaction conditions included an initial denaturation at 95℃ for 3 minutes, followed by 30 cycles of denaturation at 95℃ for 30 seconds, annealing at 55℃ for 30 seconds, extension at 72℃ for 30 seconds, and a final extension at 72℃ for 5 minutes. The PCR products were purified using HiAccuBead (AccuGene, Incheon, Korea). The purified PCR products were subjected to barcode sequencing for sample identification using a second round of PCR. The PCR products generated after the second round of PCR were purified using the same method as previously described. The final PCR products were quantified using a Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA). These products, with consistent concentrations, were then sent to Macrogen (Seoul, Korea) for MiSeq sequencing. Sequence data obtained through MiSeq sequencing were processed for error removal and statistical analysis using the Qiime2 software. Denoising and error removal were performed using the dada2 algorithm within the Qiime2 program. Operational taxonomic unit (OTU) clustering was carried out using Qiime2’s vsearch, with a 97% similarity threshold based on the open-reference approach. Each OTU was classified using a pre-trained sklearn classifier from the Silva reference database (silva-138-99-nb-classifier). This classifier was used to classify OTUs into a total of 7 taxonomic levels (species, genus, family, order, class, phylum, and kingdom). Data summarization in Qiime2 was performed using the taxa and krona commands, while other visualizations were created using Python’s matplotlib and R’s ggplot2. Various diversity metrics were used for alpha diversity analysis within samples, including ace, chao1, chao1_ci, gini_index, shannon, and simpson metrics. Bray-Curtis distance and Jaccard distance were used for beta diversity analysis between samples.

Fecal Metabolite Analysis

After microbiome analysis, stool samples were sent to a laboratory for metabolite analysis (Chungbuk National University, Cheongju, Korea). The changes in various metabolites after consuming fermented or nonfermented rice drink were investigated using proton nuclear magnetic resonance (¹H-NMR), following the method of Bo et al¹⁷ (Fig. 2). The stool samples obtained from IBS patients before and after fermented or nonfermented rice drink consumption were diluted to 10% (weight/volume) in distilled water. Subsequently, the samples were centrifuged at 16 000 × g for 10 minutes, and the supernatant was collected. The obtained supernatant was then mixed with an equal volume of deionized water containing 10% deuterium oxide and 1 mM sodium 2,2-dimethyl-4-silapentane-1-sulfonic acid (DSS) to achieve a final DSS concentration of 0.5 mM. The pH of the mixture was adjusted to 6 ± 0.01 using 2 M HCl or NaOH. The mixture (700 µL) was transferred into 0.5 mm NMR tubes, and ¹H-NMR spectra were acquired on a Varian INOVA 500 MHz NMR spectrometer (Varian Inc, Palo Alto, CA, USA). Identification and quantification of individual spectra were performed using the Processor and Profiler modules of the Chenomx NMR suite, V.6.1 (Chenomx, Inc, Edmonton, Alberta, Canada). Each sample was analyzed in triplicate. Data on fecal metabolites were missing (samples not obtained or incorrectly processed) in 3 participants who consumed nonfermented rice drink and 2 who consumed fermented rice drink.

Figure 2. Quantitative analysis of metabolite in stool from patients with irritable bowel syndrome. H+-NMR, proton nuclear magnetic resonance; PBS, phosphate buffered saline; DSS, sodium 2,2-dimethyl-4-silapentane-1-sulfonic acid.

Statistical Methods

In this study, assuming a 15% placebo response rate when using two-sided tests and the chi-squared test and aiming for an 80% statistical power at alpha = 0.05, we calculated the sample size required to detect a 33% difference in the response rate between the fermented rice and the nonfermented rice drink groups. The calculated sample size was n = 60, and considering a dropout rate of 10%, we planned to recruit 66 participants.

Subjects’ overall assessments were calculated as the arithmetic mean across the baseline and treatment period for each subject. The primary analysis was based on the intention-to-treat population, which included all patients successfully randomized. Descriptive analysis of secondary objective criteria was based on available data. Treatment differences were tested using the non-parametric Wilcoxon test for continuous variables or the Fisher’s exact test for binary variables. All P-values are 2-sided. All statistical analyses were performed using SAS version 26.0 for SAS Institute Inc. (SAS Institute Inc, Cary, NC, USA).

The non-parametric Wilcoxon signed-rank test was used to compare the differences in overall microbiome before and after the administration of fermented and nonfermented rice drinks for each participant. The analysis of similarities test was used with the Bray-Curtis distance as a parameter to evaluate the dissimilarity in microbiome composition between groups before and after administration. The beta diversity results were visualized through nonmetric multidimensional scaling analysis.

Baseline Characteristics

A total of 62 IBS patients considered overweight entered the trial, of whom 60 were randomized after the 2-week run-in phase, with 60 (100%) completing the trial. All subjects were randomized evenly to the fermented and nonfermented rice drink groups. The treatment groups were comparable with regard to age, sex, smoking, alcohol, BMI, BEPSI-K, HADS, and IBS symptom characteristics (Table 1). Both groups were predominantly female (80.0% to 83.3% female), and all findings on blood tests taken at screening were evaluated for their clinical significance in relation to the inclusion and exclusion criteria.

Table 1 . Baseline Demographic, Symptomatic, and Psychological Data in Subjects

Demographic	Fermented rice drink group (n = 30)	Nonfermented rice drink group (n = 30)	P-value
Age (yr)	39.17 ± 12.8	37.40 ± 8.30	0.528
Gender (F/M)	24/6	25/5	0.739
BMI (kg/m2)	25.5 ± 1.1	24.5 ± 0.7	0.123
Smokinga	25/2/3	25/3/2	0.819
Alcoholb	8/19/3	8/21/1	0.781
Abdominal symptoms
Overall IBS	3.5 ± 0.4	3.2 ± 0.4	0.250
Abdominal pain	3.3 ± 0.5	3.0 ± 0.5	0.400
Bloating	3.7 ± 0.5	3.1 ± 0.4	0.051
Urgency	2.5 ± 0.6	2.4 ± 0.6	0.738
Bowel habit
IBS-C/D/M	9/19/2	8/21/1	0.266
BEPSI-K	2.5 ± 0.4	2.3 ± 0.3	0.585
HADS
Anxiety	12.8 ± 0.9	13.2 ± 1.2	0.616
Depression	17.9 ± 1.1	17.5 ± 1.2	0.607

^aNonsmoker/exsmoker/current smoker.

^bNondrinker/exdrinker/current drinker.

F, female; M, male; BMI, body mass index; IBS, irritable bowel syndrome; IBS-C, constipation predominant IBS; IBS-D, diarrhea predominant IBS; IBS-M, mixed IBS; BEPSI-K, Brief Encounter Psychosocial Instrument Korean version; HADS, Hospital Anxiety and Depression Scale.

Data are presented as mean ± SD or n.

Baseline Dietary Information

The analysis of baseline dietary information for participants revealed that, for most nutrients, there were no significant differences between the 2 groups. However, mean dietary fiber intake is slightly but significantly higher in participants allocated to the fermented rice group than those allocated to the nonfermented rice group. Since random allocation had already been performed and some participants had started administration of test drinks, no reassignment based on dietary information was carried out (Table 2).

Table 2 . Nutrient Differences Between Fermented and Nonfermented Rice Drink Groups

Calorie and nutrient	Fermented rice drink group (n = 29)	Nonfermented rice drink group (n = 28)	P-value
Calorie (kcal)	1880.8 ± 346.70	1744.7 ± 405.11	0.179
Carbohydrate (g)	220.6 ± 55.07	197.7 ± 59.00	0.137
Lipid (g)	69.9 ± 20.54	60.8 ± 20.18	0.095
Protein (g)	76.0 ± 16.16	77.2 ± 19.44	0.794
Dietary fiber (g)	18.9 ± 5.78	15.5 ± 4.33	0.017
Soluble dietary fiber (g)	2.6 ± 1.14	12.3 ± 56.59	0.359
Vitamin A (μg RAE)	661.1 ± 1250.41	310.5 ± 140.32	0.146
Vitamin D (μg)	3.6 ± 6.65	2.7 ± 4.43	0.550
Vitamin E (mg)	15.5 ± 6.04	13.4 ± 5.29	0.160
Vitamin K (μg)	152.0 ± 97.10	108.6 ± 79.47	0.069
Vitamin C (mg)	57.2 ± 37.72	42.7 ± 29.25	0.110
Thiamin (mg)	1.6 ± 0.62	1.5 ± 0.65	0.394
Riboflavin (mg)	1.3 ± 0.55	1.2 ± 0.35	0.468
Niacin (mg)	12.5 ± 5.22	11.3 ± 3.85	0.324
Vitamin B6 (mg)	1.8 ± 1.34	1.4 ± 0.51	0.129
Folic acid (μg)	368.0 ± 171.45	323.0 ± 106.37	0.238
Vitamin B12 (μg)	11.4 ± 15.18	7.9 ± 8.69	0.298
Calcium (mg)	369.0 ± 179.10	308.6 ± 113.21	0.133
Phosphate (mg)	923.3 ± 309.31	870.7 ± 258.25	0.489
Sodium (mg)	4084.0 ± 1200.69	3519.8 ± 1166.84	0.078
Potassium (mg)	2184.9 ± 667.73	1961.1 ± 522.57	0.164
Iron (mg)	14.8 ± 5.97	12.7 ± 5.04	0.157

Data are presented as mean ± SD.

Baseline Symptoms and Symptom Changes After Consuming Rice Drink

In both groups, global IBS symptoms and individual symptoms, including abdominal pain, urgency, and abdominal bloating, improved significantly after consuming rice drinks for a month (P < 0.01). There was no difference between the 2 groups in the improvement of these symptoms except the abdominal bloating, which was more significantly improved in the fermented rice drink group than in the nonfermented rice drink group (P < 0.05; Fig. 3). Bowel habit did not change after consuming rice drink for 1 month.

Figure 3. Comparison of symptom changes between fermented and nonfermented rice drinks consumption. The improvement in abdominal bloating was greater in the fermented rice drink group than in the nonfermented rice drink group (*P < 0.05). FR, fermented rice group; NFR, nonfermented rice group; ns, not significant.

Metabolic Syndrome-related Blood Tests

As a result of metabolic syndrome-related blood tests, no significant changes were observed before and after the consumption of rice drinks in either group (Table 3).

Table 3 . Changes in Blood Metabolic Syndrome Indicators Before and After Consuming Fermented and Nonfermented Rice Drinks

Blood metabolic indicator	Fermented rice drink group (n = 30)		P-value	Nonfermented rice drink group (n = 30)		P-value
	Pre	Post		Pre	Post
Glucose (mg/dL)	93.03 ± 11.7	91.23 ± 11.90	0.216	94.03 ± 10.73	93.1 ± 12.92	0.616
Total cholesterol (mg/dL)	186.6 ± 28.24	189.6 ± 27.25	0.468	194.2 ± 22.74	191 ± 30.76	0.117
LDL (mg/dL)	110 ± 25.85	111.2 ± 26.49	0.713	120.4 ± 31.82	114.7 ± 32.70	0.066
HDL (mg/dL)	66.87 ± 19.13	66.27 ± 17.67	0.714	69.40 ± 15.57	67.08 ± 17.09	0.114
TG (mg/dL)	106.2 ± 86.82	111.5 ± 92.22	0.558	87 ± 39.78	88.83 ± 38.89	0.766
Insulin (μU/mL)	8.913 ± 4.37	9.811 ± 7.40	0.461	8.742 ± 3.81	8.814 ± 4.07	0.913
HOMA-IR	2.095 ± 1.104	2.301 ± 1.991	0.518	2.057 ± 1.006	2.072 ± 1.190	0.936

Pre, before consumption of rice drink; Post, after consumption of rice drink; LDL, low-density lipoprotein; HDL, high-density lipoprotein; TG, triglyceride; HOMA-IR, Homeostatic Model Assessment for Insulin Resistance.

Data are presented as mean ± SD.

Fecal Microbiome Analysis

A total of 47 out of 60 subjects provided stool samples before and after the consumption of fermented and nonfermented rice drinks. As shown in Figure 4, there was no significant difference in OTU or Shannon diversity index before and after consumption in the fermented (Fig. 4A and 4B) and the nonfermented rice drink group (Fig. 4C and 4D). In comparing the composition of stool microbiome at the phylum level, Bacillota, Actinomycetota, and Bacteroidota were observed as the main phyla in both the fermented rice group and the nonfermented rice group. Among the top 3 major phyla, a slight increase in Bacillota was observed after consuming fermented rice drink (P = 0.064). However, there was no significant difference in the relative abundance of all 3 phyla before and after consuming fermented and nonfermented rice drinks. When comparing stool microbiome composition at the genus level, 23 major genera were found in at least 1% of all samples. When comparing gut microbiome before and after rice drink consumption at the genus level, Blautia was significantly increased in the fermented rice drink group (P = 0.020). However, there was no significant before and after difference in the nonfermented rice drink group (Fig. 4E and 4F). In the beta-diversity analysis using the Bray-Curtis distance index, neither the fermented nor nonfermented rice drink groups showed any significant differences before and after consumption.

Figure 4. Fecal microbiome analysis. Comparison of α-diversity before and after consumption of fermented (A, B) and nonfermented rice drink (C, D) based on operational taxonomic unit (OTU) and Shannon indices. Comparison of relative abundance of fecal microbiome before and after consumption of fermented (E) and nonfermented rice drink (F) at the genus level. Pre, before consumption of rice drink; Post, after consumption of rice drink.

Salivary Microbiome Analysis

A total of 42 out of 60 subjects provided saliva samples before and after the consumption of fermented rice drink and nonfermented rice drink. Figure 5 shows that there was no significant difference in OTU or Shannon diversity index before and after consumption in the fermented (Fig. 5A and 5B) and the nonfermented rice drink groups (Fig. 5C and 5D). In comparing the composition of gut microbiome at the phylum level, Bacillota, Actinomycetota, Pseudomonadota, Bacteroidota, and Fusobacteriota were observed as the main phyla in both the fermented rice group and the nonfermented rice group. Among the top 5 major phyla, a slight increase in Bacteroidota was observed after consuming fermented rice drink (P = 0.053). However, there was no significant difference in the relative abundance of all 5 phyla before and after consuming fermented and nonfermented rice drinks. When comparing gut microbiome composition at the genus level, 17 major genera were found in at least 1% of all samples. When comparing salivary microbiome before and after rice drink consumption at the genus level, Prevotella (P = 0.017) and Oribacterium (P = 0.018) were significantly increased in the fermentation group; however, there was no significant before and after difference in the non-fermentation group (Fig. 5E and 5F). In the beta-diversity analysis using the Bray-Curtis distance index, neither the fermented nor nonfermented rice drink groups showed any significant difference before and after consumption.

Figure 5. Salivary microbiome analysis. Comparison of α-diversity before and after consumption of fermented (A, B) and nonfermented rice drink (C, D) based on operational taxonomic unit (OTU) and Shannon indices. Comparison of relative abundance of salivary microbiome before and after consumption of fermented (E) and nonfermented rice drink (F) at the genus level. Pre, before consumption of rice drink; Post, after consumption of rice drink.

Fecal Metabolite Analysis

In the fermented rice drink group, butyrate showed a decrease, and acetate and fumarate exhibited slight increases after consumption of 1 month compared to their respective pre-consumption levels. Conversely, in the nonfermented rice drink group, acetate showed a decrease, and fumarate exhibited an increase after consumption of 1 month compared to pre-consumption levels. However, none of these changes were significant. In contrast to the fermented rice drink group, butyrate, propionate, and valerate were significantly decreased in the nonfermented rice drink group. These results suggested that fermented rice drink exhibited relatively more beneficial effects than nonfermented rice drink regarding health-beneficial fatty acid change (Table 4).

Table 4 . Changes in Fecal Metabolites of Irritable Bowel Syndrome Patients With Consumption of Fermented Rice Drink and Nonfermented Rice Drink

Metabolites	Fermented rice drink group (n = 23)					Nonfermented rice drink group (n= 24)				Relative ratio between the FR and NFR
	Pre (mM)	Post (mM)	P-value	Relative ratio		Pre (mM)	Post (mM)	P-value	Relative ratio
Fatty acid
Acetate	90.63 ± 61.37	95.23 ± 88.94	0.827	1.05	82.85 ± 36.86		67.16 ± 50.39	0.280	0.81	1.29
Butyrate	19.05 ± 19.12	12.21 ± 8.72	0.060	0.64	17.39 ± 12.02		9.89 ± 4.50	0.019	0.56	1.12
Propionate	106 ± 209.44	40.11 ± 53.28	0.090	0.37	69.43 ± 71.96		30.21 ± 18.26	0.023	0.43	0.86
Valerate	20.22 ± 22.36	9.18 ± 6.99	0.028	0.45	15.01 ± 8.26		9.21 ± 6.59	0.021	0.61	0.73
Lactate	28.6 ± 53.05	18.84 ± 17.37	0.303	0.65	22.92 ± 17.86		21.3 ± 15.62	0.744	0.92	0.70
Citrate	6.97 ± 8.41	5.93 ± 4.39	0.513	0.85	5.16 ± 4.06		6.47 ± 5.34	0.325	1.25	0.67
Fumarate	2.26 ± 2.07	2.42 ± 1.90	0.594	1.07	2.33 ± 2.34		3.06 ± 2.31	0.302	1.31	0.81
Malate	23.57 ± 34.78	16.04 ± 11.48	0.275	0.68	16.05 ± 9.24		16.48 ± 12.68	0.887	1.02	0.66
Indole derivatives
5-hydroxyindole-3-acetate	4.71 ± 5.78	4.11 ± 2.89	0.531	0.87	4.17 ± 3.26		5.03 ± 3.17	0.288	1.20	0.72
Indole-3-acetate	4.31 ± 3.69	4.53 ± 3.80	0.620	1.05	5.19 ± 4.62		5.03 ± 3.2	0.891	0.96	1.08
Indole-3-lactate	5.33 ± 5.87	4.03 ± 2.99	0.166	0.75	4.61 ± 2.85		5.17 ± 3.6	0.528	1.12	0.67
Choline metabolites
Betaine	10.88 ± 12.16	5.97 ± 5.47	0.039	0.54	7.41 ± 4.72		5.53 ± 4.16	0.187	0.74	0.73
Choline	24.13 ± 36.5	18.16 ± 17.75	0.330	0.75	20.88 ± 21.58		20.04 ± 23.13	0.902	0.96	0.78
Trimethylamine	16.4 ± 15.48	16.86 ± 19.85	0.930	1.02	9.59 ± 5.25		8.43 ± 5.28	0.474	0.87	1.16
Trimethylamine N-oxide	19.29 ± 17.25	21.34 ± 31.52	0.785	1.10	10.11 ± 4.86		10.57 ± 8.28	0.808	1.04	1.05
Cadaverine	4.31 ± 5.06	5.02 ± 6.12	0.664	1.16	4.09 ± 2.44		3.20 ± 1.61	0.134	0.78	1.49
Chenodeoxycholic acid	28.59 ± 54.87	14.01 ± 10.92	0.219	0.49	13.67 ± 9.26		14.34 ± 11.05	0.830	1.04	0.46
Cholate	3.30 ± 4.31	2.56 ± 1.87	0.318	0.77	2.58 ± 2.10		3.52 ± 2.34	0.135	1.36	0.56
Glycocholate	5.01 ± 6.07	3.33 ± 2.33	0.166	0.66	3.12 ± 1.61		3.92 ± 2.27	0.092	1.25	0.53
Amino acids
Alanine	23.74 ± 39.15	27.47 ± 52.95	0.782	1.15	16.30 ± 9.58		12.11 ± 10.72	0.140	0.74	1.55
Carnitine	35.81 ± 68.91	14.26 ± 17.10	0.122	0.39	18.98 ± 16.57		18.25 ± 18.05	0.901	0.96	0.41
Cysteine	12.58 ± 17.53	8.44 ± 6.59	0.248	0.67	10.41 ± 6.43		10.03 ± 7.01	0.846	0.96	0.69
Glutamate	18.11 ± 23.11	15.39 ± 21.14	0.667	0.85	14.31 ± 7.9		11.80 ± 5.88	0.268	0.82	1.03
Histidine	5.43 ± 4.38	4.13 ± 2.89	0.087	0.76	5.83 ± 5.21		6.00 ± 3.73	0.892	1.02	0.73
Isoleucine	10.96 ± 16.22	6.32 ± 8.01	0.252	0.57	6.90 ± 4.32		5.52 ± 3.26	0.261	0.79	0.72
Leucine	11.13 ± 12.61	7.29 ± 7.36	0.208	0.65	7.81 ± 5.84		5.63 ± 3.25	0.099	0.72	0.90
Lysine	8.25 ± 11.78	6.36 ± 4.09	0.461	0.77	7.08 ± 4.28		5.06 ± 3.16	0.103	0.71	1.07
Methionine	15.76 ± 35.25	9.22 ± 8.54	0.323	0.58	9.55 ± 6.01		11.12 ± 8.84	0.504	1.16	0.50
Phenylalanine	4.50 ± 5.26	4.45 ± 3.65	0.956	0.98	4.74 ± 3.93		4.70 ± 3.99	0.974	0.99	0.99
Serine	14.61 ± 22.80	7.73 ± 4.84	0.139	0.52	13.40 ± 12.24		9.88 ± 6.78	0.265	0.73	0.71
Tyrosine	5.33 ± 5.35	4.24 ± 2.9	0.210	0.79	4.95 ± 3.94		4.53 ± 2.59	0.675	0.91	0.86
Taurine	39.98 ± 51.86	24.19 ± 21.73	0.109	0.60	28.63 ± 24.82		30.22 ± 24.34	0.833	1.05	0.57
β-Alanine	824.59 ± 1054.74	734.86 ± 781.50	0.419	0.89	676.62 ± 488.10		887.87 ± 790.57	0.249	1.31	0.67
Tryptophan	4.37 ± 5.19	3.59 ± 3.39	0.396	0.82	3.88 ± 2.41		3.93 ± 2.55	0.943	1.01	0.80
Arginine	22.90 ± 36.05	16.02 ± 14.50	0.422	0.69	21.5 ± 21.21		14.60 ± 8.77	0.157	0.67	1.02

Pre, before consumption of rice drink; Post, after consumption of rice drink; FR, fermented rice drink; NFR, nonfermented rice drink.

Relative ratio in fecal metabolites before and after consumption of FR or NFR. Relative ratios between the FR and NFR represent the comparative changes in fecal metabolites between FRand NFR, denoted as comparative ratios. A P-value of less than < 0.05 was considered statistically significant.

Despite these metabolic changes before and after consumption within each experimental group, no significant differences were observed when comparing the value of fecal fatty acids, indole derivatives, and choline metabolites after 1 month of consumption between fermented and nonfermented rice drink groups (Fig. 6; P > 0.05). This suggests that while there were alterations in specific metabolites, the overall composition of these vital metabolic markers did not significantly differ between the fermented and nonfermented rice drink groups. Therefore, these findings suggest that the impact of fermented rice drink consumption on the fecal metabolite involves subtle changes, including a non-significant yet observable trend of elevated acetate within the short-chain fatty acids (SCFAs) profile compared to nonfermented rice drink.

Figure 6. Fecal metabolites change in irritable bowel syndrome (IBS) patients with fermented and nonfermented rice drink. (A) Violin plot showed comparison of short-chain fatty acids (SCFAs) between after consumption of fermented and nonfermented rice drink. Parameters of inspection behavior for the different treatments presented as boxplots, indicating the median and quartiles with whiskers reaching up to 1.5 times the interquartile range. The average was plotted concentration of SCFAs (red squares) in the same diagram. (B) Volcano plot analysis of fecal metabolites in IBS patients comparing the consumption of fermented rice drink and nonfermented rice drink.

We aimed to determine whether consuming a fermented rice drink with L. plantarum JSA22 for 1 month would control the symptoms of IBS and improve objective indicators related to being overweight. In both the fermented and nonfermented rice drink groups, symptoms such as overall symptoms and abdominal pain significantly improved after consumption of the rice drink compared to before taking it; however, the improvement in abdominal bloating significantly improved in the fermented rice drink group compared to the nonfermented rice drink group. Additionally, there was no difference in metabolism-related blood tests and fecal metabolites between the 2 groups before and after consumption of rice drink. However, an increase in gut microbiome that reduces intestinal gas and inhibits body fat accumulation was observed after fermented rice drink consumption. In contrast, there was no significant change in gut microbiome in the nonfermented rice drink group.

The fermented rice drink in this study contained a single-strain probiotic. It has been reported that single-strain probiotics are less effective in controlling symptoms than multi-strain probiotics.¹⁸ However, we thought that fermented rice drink might be able to overcome this limitation because it contains several metabolites and dietary fiber in addition to probiotics. In previous studies, the fermented milk product containing Bifidobacterium lactis DN-173 reduced abdominal girth and increased GI transit,¹⁹ and the fermented milk product containing Lactobacillus acidophilus La-5 and Bifidobacterium BB-12, although it contained 2 probiotics, improved the patient’s IBS quality of life by 18% and severity of bloating.²⁰ However, since 34.7% of Korean IBS patients complain of milk-related intolerance, it is difficult to prescribe fermented milk products to Korean IBS patients.^21,22 Since the fermented rice drink used in this study does not have lactose to trigger any lactose intolerance, it may be appropriate to prescribe it to IBS patients with bloating.

Diet is important in causing symptoms of IBS. In particular, the FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) carbohydrates, a gluten-containing diet, and a high-fat diet are the main factors causing symptoms in patients with IBS.^22,23 In healthy humans, rice is completely absorbed in the small bowel and produces less intestinal gas after ingestion compared to wheat and other sources of carbohydrates.²⁴ The rice-based exclusion diet may improve symptoms in patients with IBS by reducing intestinal gas production.²⁵ When comparing the respective protein, carbohydrate, and fat ratios, rice at 8%, 91%, and 2%, is significantly lower in fat compared to dairy-based yogurt at 22%, 30%, and 48%.²⁶ Therefore, fermented rice drinks will be a good substitute for fermented milk for IBS patients suffering from lactose intolerance.

In this study, we observed changes in the microbiome in saliva and stool before and after the consumption of a fermented rice drink. Before taking the rice drink, the most abundant genera of the stool were Blautia, Bifidobacterium, and Prevotella. Among these, Blautia and Prevotella were found to be the most abundant genera in IBS patients in other studies.^27,28 In the fermented rice drink group, Blautia significantly increased by 1.5 times after consumption of the drink compared to baseline, while there was no difference in the nonfermented rice drink group. Blautia is a genus of anaerobic bacteria with probiotic characteristics found widely in the mammalian stool.²⁹ Some species of Blautia can use CO, H₂/CO₂, and carbohydrates as energy sources. Based on animal studies, Blautia hydrogenotrophica was developed as a live biotherapeutic product for treating IBS despite its higher abundance in IBS patients.³⁰ Its potential mechanisms of action include competition with sulfate-reducing bacteria, a reduction in the production of the gases H, H₂S, and methane, and a normalization of the gut microbiome.³⁰ Although we could not confirm the species level, the significant increase in Blautia may be one of the mechanisms for the improved bloating in the current study. In addition, Blautia is significantly and inversely associated with visceral fat accumulation, regardless of sex,³¹ and produces butyric acid and acetic acid,³² which decrease obesity by regulating G-protein coupled receptors.³³ Therefore, the gut microbiota profile changes by consuming fermented rice drink with L. plantarum JSA22 seems to be beneficial for both IBS and obesity.

Additionally, in the case of saliva, 17 major genera were observed. Few studies report the relationship between oral microbiome and symptoms of IBS, and Veillonella was associated with abdominal pain in IBS patients in a previous study.³⁴ In this study, Prevotella and Oribacterium significantly increased by 1.7 times and 1.8 times, respectively, after taking fermented rice drink for one month. Oribacterium is a strictly anaerobic and non-spore-forming bacterial genus, and its relationship with human metabolism is unclear. However, Prevotella bacteria are known to be related to fat loss. Participants with a high abundance of Prevotella lost more weight than participants with low abundance of Prevotella after 6 weeks of whole grain consumption,³⁵ and baseline Prevotella abundance was associated with greater fat loss in response to ad libitum wholegrain consumption among participants with low salivary amylase (AMY1) gene copy number, but not among participants with high AMY1 copy number.³⁶ These changes in the salivary microbiome, along with changes in Blautia in the stool, are expected to increase body fat loss and improve blood test values related to obesity. However, the reason these results did not appear as changes in metabolic syndrome indicators in fecal metabolites or blood tests is likely because the fermented rice drink consumption period was short. Nevertheless, considering that it took 4 months to 10 months for the beneficial gut microbiome to change in previous studies,³⁷ it is very interesting that an increase of beneficial microbiome in the stool and saliva was observed after consuming fermented rice drink for just 1 month. The fermented food-rich diet resulted in an increase in the α-diversity of the gut microbiome³⁸ and provided nutrients to promote the growth of indigenous gut microbes.³⁹ Therefore, fermented foods can be presented as a good way to grow various beneficial microbes.

Metabolites produced by the gut microbiome include bile acids, SCFA, amino acids, tryptophan, vitamins, and neurotransmitters known to play an important role in developing digestive diseases such as IBS.⁴⁰ Ninety-five percent of SCFAs are acetate, propionate, and butyrate, which are generated by carbohydrate metabolism in the colon and play an important role in the integrity of the intestinal mucosa, glucose and lipid metabolism, immune system, and inflammatory response.⁴¹ The observed non-significant yet observable trend of increase in acetate after consuming fermented rice drink in our study is anticipated to play a similar role. The change in SCFA exhibited beneficial trends in the fermented rice group compared to the nonfermented drink group; however, it was a subtle change. Therefore, we can speculate that other factors, such as increased Blautia, have a major role in improving symptoms in this study. Although we did not check the colonic mucosal immune status in the current study, the immune-modulating effect of fermented rice drink demonstrated in the preclinical study is another plausible mechanism for reducing IBS symptoms.¹¹ An interesting part of the results of this study is that the propionate and butyrate decreased, though it was not significant, after taking a fermented rice drink. This result contradicts our previous study that showed the fermented rice drink with L. plantarum JSA22 increased butyrate.¹⁰ However, it is difficult to directly compare because the previous study resulted from 24-hour fermentation using an in vitro fecal fermenter and the comparator was dairy yogurt. The comparator in this study was nonfermented rice, which may be why there is no significant difference in metabolites, unlike the previous study. Nevertheless, there is a possibility that the butyrate initially increased and then decreased back to baseline over time in the fermented rice drink group because the current study was conducted by consuming for 1 month. Conversely, in human studies exposed to diverse confounding factors such as various diets, a month-long period may be relatively short to induce metabolite changes.⁴² Therefore, future studies are warranted to analyze metabolites at various time points from days after administration to long-term as 12 weeks.

Probiotics have been used for a long time and are believed to have health benefits, but the level of evidence is not high. This is because studies so far have limitations, such as not being randomized controlled trials or being unable to control various confounding factors. The strength of this study is that it was performed with a parallel, double-blind design by following a recent expert recommendation to be cautious in IBS studies using probiotics,⁴³ and several variables were analyzed to reduce factors that could affect the study results. Gender, age, drinking, smoking, depression, anxiety, stress, and IBS subgroup proportion, all of which can affect the patient’s IBS symptoms, were not different between the fermented and nonfermented rice drink groups. In addition, subjects who took drugs that could affect symptoms and intestinal microorganisms were excluded from the study. The difference in fiber intake between groups was small, but it was a significant difference, which is a limitation of this study. On the other hand, considering that fiber generates gas and can cause bloating in IBS patients, our results could be interpreted more positively because fermented rice drinks decreased bloating symptoms in subjects who consumed more fiber than in the control drink group, who consumed less fiber.

In this study, we used a nonfermented rice drink as a control drink for the fermented rice drink with L. plantarum JSA22 to investigate the effect of probiotics and fermentation. However, rice germ itself is rich in bioactive factors such as phytosterol, orizanol, tocopherol, and tocotrienol,⁴⁴ so differences in symptom improvement in the fermentation group may have been underestimated in this study. Therefore, if a control drink with no bioactive effect was used instead of nonfermented rice, the effect of fermented drinks on symptoms and other parameters could be more significant.

In conclusion, a fermented rice drink significantly improved abdominal bloating in IBS patients. The stool and saliva of patients who consumed the fermented rice drink showed an increase in bacteria that reduced visceral fat accumulation. However, no changes in blood tests or stool metabolites associated with being overweight were seen. A one-month period of drinking fermented rice may be too short to detect changes in blood and fecal metabolites; longer-term research is needed in the future.

This study was supported by the research program (RS-2022-RD010340) of the Rural Development Administration in South Korea.

None.

Conceptualization, funding acquisition, and review and editing: Jung Ho Park and Yong Sung Kim; investigation and data curation: Moon Young Lee, Hyunbin Seong, Nam Soo Han, and Hae-Jin Hu; writing of original draft: Nam-Hee Kim and Hye Sun Choi; and methodology and supervision: Jung Ho Park and Yong Sung Kim. All authors approved the final manuscript.