Ingestion of dark chocolate improves constipation and alters the intestinal microbiota in Japanese women

Abstract

In a previous study, we found that proteins from cacao beans (cacao proteins) were resistant to digestive enzymes and that ingestion of the indigestible cacao proteins promoted defecation and altered the intestinal microbiota in mice. Therefore, we investigated whether ingestion of dark chocolate containing high amounts of cacao proteins improves constipation and alters the intestinal microbiota in Japanese women. Bowel movement frequency and stool amount after dark chocolate ingestion were significantly higher than before dark chocolate ingestion and significantly higher than after ingestion of white chocolate with no cacao proteins. Next, stool samples were collected, and the intestinal microbiota was analyzed by next-generation sequencing-based 16S rRNA. There was no significant difference in the α-diversity index of the gut microbiota between before and after ingestion of white chocolate, but the α-diversity index of the gut microbiota after ingestion of dark chocolate was significantly higher than before ingestion. The relative abundances of Faecalibacterium and Megamonas in the fecal microbiota after dark chocolate ingestion were significantly higher than before dark chocolate ingestion and significantly higher than after white chocolate ingestion. The relative abundances of Anaerostipes, Butyricicoccus, and Roseburia in the fecal microbiota after dark chocolate ingestion were significantly higher than before ingestion. Spearman correlation analysis revealed a correlation between the stool amount and relative abundances of Megamonas and Roseburia in the dark chocolate ingestion group. These results indicate that ingestion of dark chocolate improved constipation in humans and promoted increase of the relative abundances of butyrate producing bacteria such as Faecalibacterium, Megamonas, Anaerostipes, Butyricicoccus, and Roseburia in the intestinal microbiota.

INTRODUCTION

Constipation is one of the most common bowel disorders worldwide. Due to recent changes in dietary habits and the effects of psychological and social factors, the incidence of constipation is clearly on the rise, seriously affecting health and quality of life [1]. Laxatives can improve bowel movements but have side effects [2].

Functional food components called prebiotics have also attracted attention as ingredients to improve constipation by increasing the growth of beneficial bacteria in the colon [3, 4]. Prebiotics are not degraded by digestive enzymes, so they pass undigested into the colon, where they are fermented by intestinal bacteria. Among them, oligosaccharides such as fructo-oligosaccharides and galacto-oligosaccharides are fermented by intestinal bacteria such as Bifidobacterium and Lactobacillus, which leads to an increase in the number of these bacteria in the colon [5,6,7,8,9,10,11,12]. Bifidobacterium and Lactobacillus produce acetic acid and lactic acid that decrease the intestinal pH. This lower pH stimulates peristalsis and decreases the colonic transit time, thus leading to improved constipation [4, 13].

Chocolate and cocoa are made from fermented and roasted cacao beans (Theobroma cacao L.). They contain large amounts of polyphenols that have several potential effects such as prevention of cardiovascular and inflammatory diseases, metabolic disorders, and cancer [14,15,16,17,18,19,20,21]. However, the health effects of proteins from cacao beans (cacao proteins) were unknown because the proteins had been difficult to extract from cacao beans and purify. Therefore, in a previous study, we established a new extraction and purification method for cacao proteins [22]. We then found that cacao proteins are resistant to digestive enzymes and that ingestion of the proteins promoted defecation in mice [22]. Furthermore, next-generation sequencing (NGS)-based 16S rRNA microbial profiling revealed that the ingestion of cacao proteins led to a significant increase in the relative abundances of Lactococcus and Mucispirillum but a significant decrease in the relative abundances of Anaerotruncus, Oscillospira, and Roseburia in the mouse intestinal microbiota [22].

Ingestion of cacao husk supplements tended to increase the number of bowel movements in children with idiopathic chronic constipation [23]. Similarly, ingestion of cacao bran cereals increased the stool amount and bowel movement frequency in healthy normolipidemic subjects [24]. Although such defecation effects of ingestion of cacao materials have been reported, to the best of our knowledge, no clinical studies have demonstrated that chocolate or cocoa consumption improves constipation. We thus investigated whether ingestion of dark chocolate containing high amounts of cacao proteins promotes bowel movement and alters the intestinal microbiota in humans with constipation.

MATERIALS AND METHODS

Study design

This study was designed as a randomized, investigated-blind study to evaluate the effects of ingestion of dark chocolate on bowel movement and the intestinal microbiota. The study was conducted at Beauty & Health Research Inc. (Tokyo, Japan) between June 2015 and July 2015 according to the guidelines laid down in the Declaration of Helsinki. The study protocol was approved by the Shinkokai Institutional Review Board, Medical Research Department, Beauty & Health Research Inc. (Tokyo, Japan), and by the Meiji Institutional Review Board, R&D Division, Meiji Co., Ltd. (Tokyo, Japan). All of the study procedures including its explanation, written informed consent acquisition, questionnaire, and testing were conducted in Japanese. Written informed consent was obtained from all subjects before their participation in the study. This study has been registered with the UMIN (University Hospital Medical Information Network in Japan) under UMIN ID UMIN000049960.

Participants

Healthy women were eligible to participate in this study if they were between 20 and 49 years old and met the criteria of having no more than 4 bowel movements per week, as constipation is defined in Rome III as having stools less than 3 times per week [25]. Candidates were excluded if they 1) had a chronic disease that was being treated with medicine; 2) had an allergy to chocolate, milk, or soy; 3) had a headache by ingestion of chocolate; 4) were lactose intolerant; 5) habitually consumed cacao products, natto, yogurt, lactic acid fermenting beverages, or foods that contained Lactobacillus more than 4 times per week within 3 months before this study; 6) habitually ingested medicine, supplements, or functional foods before this study or would be during the study period; 7) participated in other clinical trials or monitoring studies within 1 month before this study or would be participating in other clinical trials or monitoring studies after agreeing to participate in this study; 8) were pregnant or planning to become pregnant during this study; 9) were lactating; 10) consumed more than 60 g alcohol per day; or 11) were judged as ineligible by their primary care physician.

Test foods and intervention

The test foods comprised dark chocolate (Chocolate Kouka, Cacao 72%) and white chocolate (Meiji White Chocolate Stick Pack) were manufactured by Meiji Co., Ltd. (Tokyo, Japan). The dark chocolate contains 2.7 g cacao proteins per 25 g of chocolate, and the white chocolate contains no cacao proteins. The amount of cacao proteins was calculated from the protein amount of cacao beans contained in the dark chocolate. The protein, lipid, and carbohydrate contents and calories in the dark chocolate were designed to be almost the same as those in the white chocolate (Table 1).

Table 1. Composition of the dark and white chocolates.

Component	White chocolate/25 g	Dark chocolate/25 g
Energy (kcal)	148.2	142.3
Total fat (g)	10.0	10.3
Total carbohydrate (g)	12.4	11.3
Protein (g)	2.1	2.7
Cacao proteins (g)	0.0	2.7
Milk proteins (g)	2.1	0.0

Experimental protocol of human study

Physicians asked the subjects about their bowel movements to exclude as many irritable bowel syndrome patients as possible. Lawyers were responsible for the security of identifiable data. Participants were requested not to eat any fermented dairy products, foods, or beverages containing viable lactobacilli or oligosaccharides for at least 2 weeks before the start of the study. Antibiotics and cathartics were restricted from screening to the end of the study. Otherwise, the participants were instructed to continue their normal eating habits. After receiving a written explanation of the purpose of the study, all participants provided written informed consent to participate in this study for 3 weeks: 1 week with no chocolate (pre-ingestion period) and 2 weeks of chocolate ingestion. Participants were randomly divided into either the dark chocolate group or the white chocolate group so that the groups had almost the same average ages, heights, body weights, body mass indices (BMIs), percentages of chocolate intake, and bowel movement frequencies in the first week (Fig. 1). Because of the parallel-group design, the subjects only received either dark or white chocolate, so there is a low risk of bias due to color or shape. During the 2-week ingestion period, participants freely ate five pieces (total 25 g) of the provided chocolate per day. Participants recorded the frequency of bowel movement, amount of stool, and intake of the test food in a diary for 1 week before ingestion of the chocolate and for the last week during the 2-week ingestion period. The intake rate of the test food was calculated by dividing the number of consumed chocolates by the number of days on which the chocolate was consumed, and the average intake rate for each group was calculated. The amount of stool was visually estimated in comparison with the volume of eggs and recorded, assuming that 1 egg weighs 50 g as described previously [26]. Participants collected a portion of their stools with a plastic spoon in their home toilet and placed the stools in a plastic tube. Stool samples were refrigerated to cool as quickly as possible and sent to Beauty & Health Research under refrigeration by courier on the day of stool collection. Stool samples that arrived the day after delivery were stored at −80°C. All stools in each group collected for 1 week before ingestion and for the last week during the ingestion period were mixed and subjected to NGS-based 16S rRNA microbial profiling.

Fig. 1.

Subject flow diagram.

Outcome

The primary efficacy endpoint was the bowel movement frequency, and the secondary efficacy endpoints were the stool amount and human intestinal microbiota.

NGS-based 16S rRNA microbial profiling

Bacterial DNA extraction from the feces was performed at the TechnoSuruga Laboratory (Shizuoka, Japan) [27]. The V4 region of 16S rRNA was amplified using region-specific primers (forward, 515F; reverse, 806rcbc91–150) and TaKaRa Ex Taq (Takara Bio Inc., Osaka, Japan) [28]. The polymerase chain reaction (PCR) amplicon was purified using a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA). The DNA libraries were quantified using a GenNext NGS Library Quantification Kit (Illumina, San Diago, CA, USA).

The sequence data were analyzed using Quantitative Insights Into Microbial Ecology (QIIME) version 1.8.1 software [28]. When the quality score fell below 20 as determined using the PRINSEQ software, the sequences were removed [29]. After filtering, a total of 8,677,888 reads were obtained from 30 samples, with an average of 289,263 reads per sample. The sequences were normalized by rarefaction using QIIME. The remaining samples were grouped into operational taxonomic units (OTUs) at a minimum of 97% similarity using QIIME’s UCLUST method based on the Greengenes database.

The Shannon index was used to assess the α-diversity of gut microbiota in groups that ingested either the dark or white chocolate. The correlation coefficient between the stool amount and relative abundances of bacteria in the dark chocolate ingestion group were analyzed by Spearman’s rank correlation test.

Statistical analysis

All data are expressed as means ± SEM (standard error of the mean) and were analyzed with IBM SPSS Statistics ver. 29.0.1.0 (IBM, Armonk, NY, USA). The exact significance probability was analyzed with the Exact Tests option. A p-value <0.05 was considered statistically significant. Differences in height, body weight, BMI, percentage intake of test food, and stool amount between before and after intake of chocolate in each group were determined by paired t-test. Differences between the dark and white chocolate groups for these evaluation items were determined by unpaired t-test. Differences in bowel movement frequency and relative abundances of fecal bacteria between before and after ingestion in each group were determined by Wilcoxon signed-rank sum test. Differences in bowel movement frequency and relative abundances of fecal bacteria between the groups were determined by Mann–Whitney U test. Differences in α-diversity (Shannon index) between before and after ingestion in each group were assessed by Wilcoxon signed-rank test, and differences between the groups were assessed by Mann–Whitney U test.

RESULTS

Characteristics of participants

A total of 48 healthy Japanese women were recruited and screened by evaluation of medical history, multidimensional assessment, laboratory variables, and assessment of individual habitual diet (Fig. 1). As a result, four participants did not meet the eligibility requirements, and two participants declined to participate, leaving 42 participants eligible. Their primary care physician selected 40 of the 42 participants who met the requirements for bowel movement frequency and age. Among the 40 participants, four women in the dark chocolate group and five women in the white chocolate group were dropped from the study due to poor health or protocol violations (Fig. 1). The poor health of these participants was not found to be causally related to the intervention. The final study population consisted of 31 female participants with a mean age 39.9 years (26–49 years range) and a mean of 2.6 bowel movements per week. Body weight, height, BMI, bowel movement frequency, and percentage of test sample intake did not differ significantly between the dark and white chocolate groups (Table 2). Body weight and BMI did not differ significantly between before and after ingestion of the dark chocolate for two weeks (Table 2).

Table 2. Characteristics of the constipated Japanese women in the study.

Characteristic	White chocolate	Dark chocolate	p-value1
Number of participants	15	16
Gender	female	female
Age (years)	39.5 ± 1.8	40.2 ± 1.8	0.798
Height (cm)	159.8 ± 1.3	158.0 ± 1.1	0.280
Body weight before intake (kg)	51.8 ± 1.9	50.5 ± 1.5	0.583
Body weight after intake (kg)	51.5 ± 1.9	50.2 ± 1.4	0.200
BMI before intake (kg/m2)	20.3 ± 0.7	20.2 ± 0.5	0.922
BMI after intake (kg/m2)	20.1 ± 0.7	20.1 ± 0.5	0.175
Bowel movement frequency before intake (times/week)	2.53 ± 0.19	2.75 ± 0.32	0.569
Percentage of chocolate intake (%)	99.5 ± 0.36	99.6 ± 0.42	0.856

Values are means ± SEM (n=15 or 16).

¹p-values indicate significant differences between dark and white chocolate groups in t-test. Only p-values for body weight and body mass index (BMI) after intake indicate significant differences between before and after intake of the dark chocolate.

Effect of ingestion of dark chocolate on bowel movement in Japanese women with constipation

In a previous study, we found that cacao proteins were resistant to digestive enzymes and that ingestion of the indigestible proteins promoted defecation in mice [22]. We thus investigated whether ingestion of dark chocolate containing high amounts of cacao proteins could promote bowel movement in humans. The dark chocolate and white chocolate were made to be almost the same in terms of protein, carbohydrate, lipid contents and calories (Table 1). The dark chocolate contained cacao proteins, whereas the white chocolate contained milk proteins. Thus, the white chocolate with no cacao proteins was ingested in the control group (placebo).

First, we examined the effect of dark chocolate ingestion on bowel movement frequency in participants with constipation. The bowel movement frequency after dark chocolate ingestion (4.94 ± 0.59 times per week) was significantly higher than before ingestion (2.75 ± 0.32 times per week; p=0.001), whereas a marginal increase in the bowel movement frequency was also found after ingestion of the white chocolate (p=0.031; Fig. 2). However, the bowel movement frequency after dark chocolate ingestion (4.94 ± 0.59 times per week) was significantly higher than after white chocolate ingestion (3.33 ± 0.40 times per week; p=0.049; Fig. 2). These results indicate that an increase in the bowel movement frequency was observed after ingestion of both dark and white chocolate and that ingestion of dark chocolate increased the bowel movement frequency more than white chocolate ingestion in participants with constipation.

Fig. 2.

Effect of ingestion of dark chocolate on bowel movement frequency in constipated Japanese women.

Bowel movement frequency per week in the groups that ingested either the dark or white chocolate. Values are means ± SEM (n=15 or 16).

*p<0.05, **p<0.01.

Next, we examined the effect of dark chocolate ingestion on stool amount in participants with constipation. The stool amount after dark chocolate ingestion (694.5 ± 137.5 g per week) was significantly higher than before ingestion (396.2 ± 75.5 g per week; p=0.001), whereas this increase in stool amount was not found after ingestion of the white chocolate (p=0.481). Furthermore, the stool amount after dark chocolate ingestion (694.5 ± 137.5 g per week) was significantly higher than after white chocolate ingestion (342.2 ± 50.6 g per week; p=0.027). These results indicate that ingestion of dark chocolate increased the stool amount as well as bowel movement frequency in participants with constipation.

Effect of ingestion of dark chocolate on human intestinal microbiota

Because ingestion of dark chocolate increased the bowel movement frequency and stool amount in the participants, we examined the alteration in their intestinal microbiota. One participant in the dark chocolate group was unable to collect stools for NGS analysis, and thus the NGS analysis was performed with 30 participants (Fig. 1).

The Shannon index was used to assess the α-diversity of the gut microbiota in groups that ingested either the dark or white chocolate. There was no significant difference in the α-diversity index of the gut microbiota between before and after ingestion of white chocolate, but the α-diversity index of the gut microbiota after ingestion of dark chocolate was significantly higher than before ingestion (Fig. 3, p=0.013). This result indicates that ingestion of dark chocolate increased the diversity of the gut microbiota.

Fig. 3.

Effect of ingestion of dark chocolate on the α-diversity of the gut microbiota.

The Shannon index was used to assess the α-diversity of the gut microbiota in the groups that ingested either the dark or white chocolate (n=15). *p<0.05.

In the NGS-based 16S rRNA microbial profiling of the bacteria in the fecal samples, 28 bacterial genera were mainly identified (Table 3). First, we selected the bacterial genera with significant changes in relative abundance in the fecal microbiota both between the dark and white chocolate groups after intake and between before and after intake of the dark chocolate. After dark chocolate ingestion, the relative abundances of Faecalibacterium and Megamonas in the fecal microbiota were significantly higher than before ingestion (p<0.001 for Faecalibacterium and p=0.031 for Megamonas), but the same changes were not observed in the participants who ate the white chocolate (Fig. 4A and 4B). Furthermore, the relative abundances of Faecalibacterium and Megamonas in the fecal microbiota after dark chocolate ingestion were significantly higher than after white chocolate ingestion (p=0.047 for Faecalibacterium and p=0.049 for Megamonas; Fig. 4A and 4B). These results show that the ingestion of the dark chocolate increased the relative abundances of Faecalibacterium and Megamonas in the human intestinal microbiota.

Table 3. Relative abundances of bacterial genera in the groups that ingested either the dark or white chocolate.

Bacterial genera1	White chocolate		Dark chocolate
	Before intake	After intake	Before intake	After intake
	(Relative abundance [%])
Akkermansia	1.16 ± 0.69	0.49 ± 0.28	0.74 ± 0.30	0.66 ± 0.28
Anaerostipes	0.11 ± 0.04	0.12 ± 0.05	0.16 ± 0.05*2	0.35 ± 0.12
Bifidobacterium	24.40 ± 5.57	26.07 ± 5.79	18.72 ± 3.69	15.49 ± 4.66
Bacteroides	2.73 ± 0.67	4.77 ± 1.16	2.74 ± 0.85	4.49 ± 0.83
Bacillus	0.48 ± 0.25	0.20 ± 0.11	0.35 ± 0.23	0.08 ± 0.06
Blautia	19.04 ± 2.44	17.99 ± 2.96	21.25 ± 2.83	21.51 ± 2.64
Butyricicoccus	0.14 ± 0.04	0.13 ± 0.05	0.13 ± 0.04**2	0.26 ± 0.07
Catenibacterium	0.00 ± 0.00	0.00 ± 0.00	1.28 ± 0.95	1.01 ± 0.71
Collinsella	1.19 ± 0.45	1.03 ± 0.38	1.92 ± 0.34	1.57 ± 0.27
Coprobacillus	0.22 ± 0.16	0.17 ± 0.10	0.06 ± 0.02	0.02 ± 0.00*3
Coprococcus	8.50 ± 2.24	7.04 ± 1.89	6.39 ± 1.01	6.33 ± 0.90
Dialister	0.18 ± 0.08	0.16 ± 0.08	0.33 ± 0.22	0.43 ± 0.22
Dorea	1.65 ± 0.51	1.49 ± 0.32	1.75 ± 0.22	1.87 ± 0.27
Eggerthella	0.23 ± 0.06	0.27 ± 0.09	0.14 ± 0.04	0.17 ± 0.05
Enterococcus	2.45 ± 2.17	0.18 ± 0.15	1.08 ± 0.79	0.18 ± 0.11
Faecalibacterium	3.34 ± 0.54	4.31 ± 1.00	3.79 ± 0.73**2	8.08 ± 1.37*3
Klebsiella	0.65 ± 0.64	0.07 ± 0.05	0.03 ± 0.02	0.35 ± 0.31
Lactobacillus	0.17 ± 0.11	0.30 ± 0.18	0.22 ± 0.14	0.19 ± 0.15
Megamonas	0.02 ± 0.02	0.01 ± 0.00	0.01 ± 0.01*2	0.40 ± 0.18*3
Megasphaera	1.17 ± 0.60	1.22 ± 0.56	0.01 ± 0.00	0.07 ± 0.04*3
Methanobrevibacter	0.13 ± 0.13	0.10 ± 0.10	0.25 ± 0.19	0.07 ± 0.05
Oscillospira	0.95 ± 0.34	1.04 ± 0.26	0.99 ± 0.17**2	1.41 ± 0.26
Parabacteroides	0.26 ± 0.11	0.47 ± 0.19	0.17 ± 0.05**2	0.51 ± 0.20
Prevotella	0.01 ± 0.01	0.01 ± 0.01	0.04 ± 0.02	0.44 ± 0.33
Roseburia	1.92 ± 0.79	2.84 ± 1.06	1.57 ± 0.41*2	2.58 ± 0.65
Ruminococcus	2.72 ± 1.20	5.60 ± 1.60	4.97 ± 1.66	5.18 ± 1.59
Streptococcus	3.22 ± 0.98	5.20 ± 1.85	6.05 ± 1.53	4.73 ± 2.04
Turicibacter	0.24 ± 0.11	0.19 ± 0.08	0.73 ± 0.48	0.32 ± 0.15

Data are represented as mean ± SEM (n=15).

¹Bacterial genera mainly identified in NGS-based 16S rRNA microbial profiling were listed in the table.

²Asterisks attached to the value of relative abundance before intake of the dark chocolate indicate a significant difference between before and after intake of the dark chocolate in Wilcoxon signed rank sum test (*p<0.05, **p<0.01).

³Asterisks attached to the value of relative abundance after intake of the dark chocolate indicates a significant difference after intake of the white and dark chocolates in Mann–Whitney U test (*p<0.05).

Fig. 4.

Effect of ingestion of dark chocolate on the intestinal microbiota in constipated Japanese women.

Relative abundances of (A) Faecalibacterium, (B) Megamonas, (C) Anaerostipes, (D) Butyricicoccus, (E) Roseburia, and (F) Oscillospira in the groups that ingested either the dark or white chocolate. Values are means ± SEM (n=15).*p<0.05, **p<0.01, ***p<0.001.

Next, we selected the bacterial genera with significant changes in relative abundance in the fecal microbiota between before and after intake of the dark chocolate. After dark chocolate ingestion, the relative abundances of Anaerostipes, Butyricicoccus, Roseburia, Oscillospira, and Parabacteroides in the fecal microbiota were significantly higher than before ingestion (p=0.020 for Anaerostipes, p=0.006 for Butyricicoccus, p=0.042 for Roseburia, p=0.004 for Oscillospira, and p=0.004 for Parabacteroides), but the same changes were not observed in the participants who ate the white chocolate (Fig. 4C–4F, Table 3).

Finally, the correlation coefficients between the stool amount and relative abundances of bacteria in the dark chocolate ingestion group were analyzed by Spearman’s rank correlation test. Among the bacteria, a correlation was found between the stool amount and relative abundances of Megamonas and Roseburia (r=0.403, p=0.027 for Megamonas; r=0.465, p=0.010 for Roseburia) in the dark chocolate ingestion group (Fig. 5A and 5B).

Fig. 5.

Correlation between the stool amount and relative abundances of Megamonas and Roseburia in the dark chocolate ingestion group.

The correlation coefficients between the stool amount and relative abundances of (A) Megamonas (r=0.403, p=0.027) and (B) Roseburia (r=0.465, p=0.010) in the dark chocolate ingestion group were analyzed by Spearman’s rank correlation test (n=30).

DISCUSSION

In the present study, ingestion of dark chocolate containing high amounts of cacao proteins facilitated bowel movement and altered the intestinal microbiota in Japanese women with constipation. This study is the first to show that ingestion of dark chocolate can improve constipation in clinical human studies.

When white chocolate containing milk powder was consumed by the control group (placebo), the bowel movement frequency after white chocolate ingestion was higher than before ingestion (Fig. 2). We speculate that this increased frequency is due to lactose from the milk powder, as described previously [30]. However, the bowel movement frequency after dark chocolate ingestion was significantly higher than after white chocolate ingestion (Fig. 2), indicating that ingestion of dark chocolate has a strong defecation effect. Ingestion of dark chocolate not only increased the bowel movement frequency but also increased the stool amount in the constipated participants. However, the weight of the stools in this study was measured by a method of visual estimation, so it should be confirmed by measuring the total weight of stools in the future.

Dark chocolate is mainly composed of proteins, dietary fiber, and fats from cacao beans and sugar. On the other hand, white chocolate is mainly composed of fats from cacao beans, sugar, and milk powder. During the ingestion period, the participants ate 25 g of dark chocolates daily that contained about 2.7 g of proteins and 3 g of dietary fiber from cacao beans. Ingestion of 6 or 7 g of dietary fiber per day did not improve constipation in previous clinical studies [31], so it is unlikely that the promotion of bowel movement by the dark chocolate is due to dietary fiber alone. In a previous study, we revealed that proteins from cacao beans (cacao proteins) were resistant to digestive enzymes and that ingestion of the indigestible cacao proteins promoted defecation in mice [22]. Taking into account that cacao proteins are present in dark chocolate but not in white chocolate, ingestion of dark chocolate is likely to improve constipation in humans by the same mechanism that mediated the cacao protein-mediated defecation effect in mice.

Among the 28 bacterial genera mainly identified in the NGS-based 16S rRNA microbial profiling of bacterial communities in the stools (Table 3), we focused most on the increase in Faecalibacterium, Megamonas, and Roseburia for several reasons. First, the relative abundances of Faecalibacterium and Megamonas increased after ingestion of the dark chocolate but not after ingestion of the white chocolate (Fig. 4A and 4B). Furthermore, the relative abundances of Faecalibacterium and Megamonas after dark chocolate ingestion were significantly higher than after white chocolate ingestion (Fig. 4A and 4B). Second, the Spearman correlation analysis revealed a correlation between the stool amount and relative abundances of Megamonas and Roseburia in the dark chocolate ingestion group (Fig. 5A and 5B). Finally, Faecalibacterium, Megamonas, and Roseburia produce butyrate [32,33,34], which stimulates bowel peristalsis and decreases the colonic transit time, leading to improved constipation [35]. Anaerostipes and Butyricicoccus, which were increased by ingestion of the dark chocolate in this study (Fig. 4C and 4D), are also known to produce butyrate [36, 37]. Thus, ingestion of dark chocolate might improve constipation by stimulating bowel peristalsis through the action of butyrate produced by these intestinal bacteria. It has been reported that butyrate is produced from dietary fiber in the colon [38], so not only cacao proteins but also dietary fiber derived from cacao beans might play a role in improving constipation.

Our previous study showed that ingestion of cacao proteins might facilitate defecation in mice in part by reducing the population of Oscillospira [22]. However, the relative abundance of Oscillospira after dark chocolate ingestion was significantly higher than before ingestion in this study (Fig. 4F), suggesting that Oscillospira likely does not contribute to improvement of constipation in humans.

To reveal whether the ingestion of dark chocolate improves constipation by stimulating bowel peristalsis through the action of butyrate produced by Faecalibacterium, Megamonas, Anaerostipes, Butyricicoccus, and Roseburia, it is necessary to investigate whether the ingestion of dark chocolate promotes butyrate production in the human intestine.

DATA AVAILABILITY

All datasets generated for this study are included in the article.

CONFLICT OF INTEREST

There are no conflicts of interest.