Abstract
To clarify the effects of the koji amazake and its lactic acid fermentation product amazake by Lactobacillus sakei UONUMA (LAF-amazake) on defecation status and intestinal microflora, a double-blind parallel group comparison test in healthy volunteers with relatively low stool frequency was conducted. Subjects ingested koji amazake or LAF-amazake for 1 week one bottle (108 mL, 118 g) once a day every morning. Subjects filled out daily questionnaires concerning their defecation conditions for the entire 3 weeks. First, second and third periods were the pre-test non-intake, the test food intake and the washout periods, respectively. Although there were no significant changes in intestinal microflora, it was found that defecation frequencies in the koji amazake group in the second and third periods were significantly higher when compared with those in the first period. These results indicated that the koji amazake may be a food material with constipation-relieving effects.
Introduction
Constipation is a condition in which the frequency of defecation is twice a week or less. This results in stool remaining in the colon for longer periods (Urao., 2014). The causes of constipation are the obstruction of stool by stenosis of the gastrointestinal tract, and subjective symptoms include bloody stools, abdominal pain, nausea, recto depressed comfort, abdominal bloating sensation, lower abdominal pain, anorexia, and dizziness.
Prebiotics are typically the non-digestible fiber compounds. Non-digestible fiber compounds can pass through the upper part of the gastrointestinal tract and stimulate the growth or activity of beneficial microorganisms such as Bifidobacteria and Lactobacillus in the colon, and improve host health (Hutkins et al., 2016). Insufficient intake of non-digestible dietary fiber is thought to be the major cause of constipation in young students (Ikegami et al., 1996). Probiotics are microorganisms (foods containing them) that are claimed to provide health benefits (Hill et al., 2014). It has been demonstrated that probiotics have preventative and curative effects on antibiotic-associated diarrhea and traveler's diarrhea (Hayes et al., 2016; McFarland., 2007). Moreover, it has been shown that the beneficial effects of probiotics are the normalization of the gut function, such as transit time, frequency of bowel movements and quality of stools (Rijkers et al., 2011). Synbiotics refer to food ingredients or dietary supplements combining probiotics and prebiotics. Synbiotic treatment has been shown to be promising for maintaining and repairing gut microbiota and the gut environment (Shimizu et al., 2013).
Koji amazake is a traditional sweet Japanese beverage produced from rice koji, water, and steamed rice. The main ingredient is glucose generated from the starch in rice koji. This rice koji is digested by saccharification enzymes such as α-amylase and glucoamylase produced by Aspergillus oryzae. It has been reported that koji amazake includes over 350 ingredients, including several kinds of oligosaccharides, vitamin B complex, amino acids, and ergothioneine (Oguro et al., 2017). In particular, oligosaccharides in koji amazake have been studied in detail (Oguro et al., 2018). In the functional analyses, it has been reported that koji amazake improved the subjective symptoms of patients with liver cirrhosis (Nagao et al., 2013).
It has been reported that the flavor and ingredients of the koji amazake could be reformed by lactic acid fermentation (LAF-amazake) using Lactobacillus sakei UONUMA strains (Oguro et al., 2017). These Lactobacillus sakei UONUMA strains were isolated from Japanese pickles in snow caverns in the Uonuma region, Niigata, Japan (Nishiwaki et al., 2014). The lactic acid bacteria and its fermentation products were assessed for their health beneficial effects, such as improving lipid metabolism (Kajimoto et al., 2002), decreasing blood pressure (Yamamoto et al., 1994), and immunomodulation (Kanzato et al., 2008). Lactobacillus sakei is a psychrotrophic lactic acid bacteria and is important for not only sake brewing, but also production of dry sausage and meat/fish products. Furthermore, Lactobacillus sakei has biotechnological potential for biopreservation and immunomodulation (Bredholt et al., 2001). It has been reported that the Lactobacillus sakei LK-117 strain induced a sustained increase in IL-12p40 production and reduced the development of atopic dermatitis-like skin lesions in a manner independent of IgE plasma levels in the NC/Nga mouse (Masuda et al., 2011; Masuda et al., 2012). As koji amazake and LAF-amazake are considered to be the probiotics, it can be expected to have the normalizing effects of gut function such as transit time, frequency of bowel movements and quality of stools.
The aim of this study was to clarify the effects of koji amazake and LAF-amazake on defecation status and intestinal microflora using a double-blind parallel group comparison test in healthy volunteers with relatively low stool frequency.
Materials and Methods
Preparation of koji amazake and LAF-amazake The koji amazake sample was a commercial product (Hakkaisan Brewery Co., Ltd., Minamiuonuma, Japan). It was sterilized at 85 °C for 30 min. The LAF-amazake sample was prepared by mixing 1×106 cfu/g of Lactobacillus sakei UONUMA strain 1 and the koji amazake, which was then incubated at 30 °C for 16 h. The sample reached 2.0×108 cfu/mL and lactic acid levels increased to 164.7 mg/mL. The LAF-amazake was sterilized at 85 °C for 30 min after lactic acid fermentation. The nutritional composition of the koji amazake and the LAF-amazake are shown in Table 1. Measurement of food ingredients in the koji amazake and the LAF-amazake were carried out at Niigata Environment Hygiene Central Laboratory Company (NEHCLC, Nagaoka, Japan).
Table 1.
The nutritional composition of the
koji amazake and the LAF-
amazake
| Food ingredients |
koji amazake |
LAF-amazake |
| Protein (g/100 g) |
1.2 |
1.2 |
| Fat (g/100 g) |
0.2 |
0.2 |
| Ash (g/100 g) |
0.1 |
0.1 |
| Carbohydrate (g/100 g) |
25.9 |
26.1 |
| Dietary fiber (g/100 g) |
0.2 |
0.3 |
| Moisture (g/100 g) |
72.7 |
72.5 |
| Sodium (mg/100 g) |
1.0 |
1.0 |
| Calories (kcal/100 g) |
110 |
110 |
Subjects Twenty-eight healthy volunteers with relatively low stool frequency (age: 20–63 years) were randomly divided into 2 groups: those receiving koji amazake (n = 14) or LAF-amazake (n = 14). Baseline characteristics of subjects are shown in Table 2. Subjects agreed to participate in this examination, and submitted a written informed consent form, and did not have diabetes, cerebrovascular disease, pregnancy, gastrointestinal illness or any other significant disease. All examinations were performed in accordance with the guidelines of the Helsinki Declaration and the Ethics Committee of Niigata Bio-Research Park, Inc., Japan, which approved this study (Permission No. IRB2017-BPD-004, December 1, 2017), and were registered in the UMIN Clinical Trials Registry (Registration No. UMIN000030711).
Table 2.
The baseline characteristics of the subjects
| Characteristics |
koji amazake |
LAF-amazake |
| Age (years) |
30.6±12.9 |
28.1±11.6 |
| Gender |
|
|
| Male |
5 |
4 |
| Female |
9 |
10 |
| Height (cm) |
166.1±9.0 |
161.5±7.4 |
| Body weight (kg) |
55.0±8.7 |
54.5±7.2 |
| BMI (kg/m2) |
19.8±1.9 |
20.8±1.4 |
The values represent mean ± standard deviation.
Study Schedule A double blind parallel group comparison test in healthy volunteers with relatively low stool frequency was conducted to clarify the effects of koji amazake and LAF-amazake on defecation status and intestinal microflora (Fig. 1). Throughout the experimental period, subjects regulated their diet to avoid eating products containing probiotics and synbiotics. Observations were made for 1 week (first period) before the intake period to obtain baseline values for the test. During the test food intake period (second period), subjects ingested koji amazake or LAF-amazake for 1 week in the form of one bottle (108 mL, 118 g) every morning. This was followed by a 1-week washout period (third period). Feces were collected 3 times: on the last days of the pre-test first period, the test food intake second period, and the washout third period.
Defecation records and fecal characteristics Throughout the first, second, and third periods, subjects recorded their defecation frequency and quantity [defecation frequency (times/week), days of defecation (days/week), and defecation index (pieces/week)], and assessed their fecal characteristics (shape, color, odor, and sensation) using a score sheet. The defecation index was expressed in terms of the volume of medium eggs, one of which was given to each subject as a reference. Fecal shape was determined in reference to the Bristol stool form scale (Lewis et al., 1997). Fecal shape was described as 1 of 5 types, ranging among “1: very hard”, “2: hard”, “3: smooth (banana shape)”, “4: soft”, and “5: watery”. Fecal color was described as 1 of 5 colors, ranging among “1: black”, “2: dark brown”, “3: brown”, “4: ocher”, and “5: yellow”. Fecal odor was described as 1 of 5 types, ranging among “1: very strong”, “2: strong”, “3: general”, “4: almost no odor”, and “5: odorless”. Sensation after defecation was described as 1 of 5 types, ranging among “1: very un-refreshing”, “2: un-refreshing”, “3: general”, “4: refreshing”, and “5: very refreshing”.
Microbial sampling and DNA extraction Feces samples for microbial community analysis were collected and stored using a feces collection kit (Techno Suruga Laboratory Co., Ltd., Japan). Sampling and storage were carried out according to the manufacturer's instructions. For whole DNA extraction, the bead-beating technique with guanidine isothiocyanate solution was conducted (Hosomi et al., 2017).
Analysis of 16S rRNA gene amplicon sequences For microbial community structure analysis of fecal samples, amplicon sequencing analysis targeting 16S rRNA gene was performed. Extracted DNA was used as a template and the V4 region of prokaryotic 16S rRNA gene was specifically amplified using an AmpliTaqGold 360 Master Mix (ThermoFisher Scientific, USA). As a primer pair used for PCR, 515F-806R (Caporaso et al., 2012) with an adapter sequence was used. PCR conditions were: 1) Initial denaturation: 95 °C for 10 min, 2) Denaturation: 95 °C for 1 min, 3) Annealing: 50 °C for 1 min, 4) Extension: 72 °C for 1 min, 5) Final Extension: 72 °C for 7 min, steps from 2) to 4) were repeated 30 times. Obtained PCR products were purified using Agencourt AMPure XP (Beckman Coulter, USA). Then, 2nd PCR was performed to add the index sequence using Nextera XT Index Kit v2 (Illumina, USA). DNA sequencing was performed using the Miseq (Illumina, USA) sequencer, and the obtained DNA sequences were analyzed using QIIME2 pipeline (Caporaso et al., 2010). Sequences that passed the quality check and chimeric check were assembled and phylogenetically classified.
Statistical analysis The results regarding defecation frequency and quantity (defecation frequency, days of defecation, and defecation index), and their fecal characteristics (shape, color, odor, and sensation) were statistically analyzed using two-way repeated measures ANOVA, followed by Tukey test. Levels of significance were set at p < 0.05 and p < 0.01. The average abundance changes of intestinal bacterial flora were statistically analyzed using one-way repeated measures ANOVA, followed by Bonferroni test. Levels of significance were set at p < 0.05. Statistical analysis was conducted using BellCurve for Excel (Social Survey Research Information Co., Ltd, Japan)
Results
Subjects This test was conducted from January to March 2018. Twenty-eight healthy volunteers were enrolled in this study and no subjects dropped out.
Effects of koji amazake and LAF-amazake on human defecation frequency, and quantity The koji amazake and the LAF-amazake were prepared and given to healthy volunteers in double-blind crossover tests. The values of defecation frequency and quantity in the koji amazake and LAF-amazake groups are shown in Table 3. In the first period, in all healthy volunteers with relatively low stool frequency, the mean values for defecation frequency in the koji amazake and LAF-amazake groups were 4.36 and 4.43 times/week, respectively. In the koji amazake group, the defecation frequency during the second and third periods was significantly greater than that during the first period (first vs. second; p < 0.001, first vs. third; p < 0.001, second vs. third; p = 0.58). However, in the LAF-amazake group, this value increased during the second period, there were no significant differences (first vs. second; p = 0.13, first vs. third; p = 0.97, second vs. third; p = 0.07). In the third period, the defecation frequency in the koji amazake group was significantly greater than that in the LAF-amazake group ( p < 0.05). Although there was a significant difference during the trial period (p < 0.05), the days of defecation had no significant difference between the koji amazake and LAF-amazake groups (p = 0.29). The values for days of defecation in the LAF-amazake group were unaltered during all periods. Although the values for defecation index in both groups increased during the second and third periods, these values showed no significant differences between the koji and LAF-amazake groups (p = 0.17). The defecation index only showed a significant difference in the trial period (p < 0.01), similarly to days of defection.
Table 3.
Effects of
koji amazake and LAF-
amazake on defecation frequency, quantity and fecal
|
Amazake |
First period
Pre-test
(Week 1) |
Second period
Test food intake
(Week 2) |
Third period
Washout
(Week 3) |
p-value |
|
Amazake |
Period |
Interaction |
Defecation frequency a),b)
(times/week) |
koji
LAF |
4.36±1.60A
4.43±1.60 |
6.07±1.94B
5.00±1.88 |
5.79±1.42B
4.36±1.65* |
0.15 |
<0.001 |
<0.05 |
Days of defecation a), c)
(days/week) |
koji
LAF |
4.00±1.52
4.00±1.52 |
5.00±1.52
4.07±1.69 |
4.93±1.44
4.14±1.51 |
0.29 |
<0.05 |
0.07 |
Defecation index a), d)
(pieces/week) |
koji
LAF |
11.61±5.71
9.31±4.50 |
14.54±5.34
11.86±5.41 |
13.86±5.56
11.01±6.77 |
0.17 |
<0.01 |
0.95 |
| Fecal shape a), e) |
koji
LAF |
2.68±0.78
2.58±0.58 |
2.98±0.42
2.99±0.65 |
3.08±0.43
2.58±0.61 |
0.20 |
0.06 |
0.19 |
| Fecal color a), e) |
koji
LAF |
2.78±0.37
2.71±0.47 |
2.91±0.44
3.10±0.41 |
2.85±0.36
2.85±0.36 |
0.74 |
<0.05 |
0.28 |
| Fecal odor a), e) |
koji
LAF |
2.75±0.50
2.69±0.55 |
2.95±0.38
2.77±0.53 |
2.94±0.49
2.74±0.46 |
0.36 |
0.21 |
0.67 |
| Sensation after defecation a), e) |
koji
LAF |
3.12±0.87
2.96±0.94 |
2.95±0.64
3.00±1.05 |
3.15±0.56
2.87±0.72 |
0.62 |
0.91 |
0.52 |
| Incidence rate of banana shape (%) f) |
koji
LAF |
52.46
30.65 |
63.10
54.29 |
72.84
50.82 |
|
|
|
Results were analyzed by two-way repeated measures ANOVA followed by the Tukey test. Different superscript and asterisk indicate significant difference among different period (p< 0.01) and amazakes (p< 08.05), respectively. Amazake, period, and interaction in p-value column represents the comparison between koji amazake and LAF-amazake group, among first, second and third period, and interaction effect (amazake type൷period), respectively.
a) The values represent mean ± standard deviation.
b) The defecation frequency in one week (times/week).
c) The defecation days in one week (days/week).
d) The defecation index was expressed by a volume of medium eggs as one unit (pieces/week).
e) The fecal shape, color, odor, and the sensation after defecation was described as 1 of 5 types as written in the text.
f) The incidence rate of banana shape (smooth, No.3) (%).
Effects of koji amazake and LAF-amazake on fecal characteristics The values for fecal characteristics in the koji amazake and LAF-amazake groups are also shown in Table 3. As the subjects had relatively low stool frequency, the values for fecal shape, color, odor were between 2 and 3 in the first period. There were no significant differences in fecal shape, odor and sensation after defecation in both groups during all periods. The values for fecal shape in the koji amazake group during the second and third periods were close to 3. However, this value in the LAF-amazake group during the third period returned to the value of the first period. The values for fecal color had significant differences during the trial period (p < 0.05). The values for fecal color were close to 3 in both groups during the second period. The values for fecal odor in the koji amazake group during the second and third periods were close to 3. However, these values in the LAF-amazake group were unchanged during all periods. The values for sensation after defecation in both groups were unaltered during all periods, and these values were almost 3. The value for the incidence rate of banana shape in the koji amazake group during the test food intake second period was greater than that during the first period, and this greater value continued during the washout third period. Although the value of the incidence rate of the banana shape in the LAF-amazake group (30.65%) was smaller than that in the koji amazake group (52.46%) during the first period, this value in the LAF-amazake group during the second period was also greater than that during the first period, and this greater value continued during the third period.
Microbial community analysis for Intestinal flora To evaluate the relationship between the increased defecation frequencies found in the koji amazake group and intestinal flora, the microbial communities were analyzed. We performed amplicon sequence analysis targeting the prokaryotic 16S rRNA gene V4 region. A total of 6 groups (14 samples/group) and 84 samples (total 1,777,595 sequence-reads) were analyzed, and the average number of sequence-reads per sample was 21,162 (min; 8,950, max; 40,587). Table 4 summarizes the alpha diversities of intestinal flora in fecal samples. As a result of the analysis by QIIME2, chao1, an index of species richness, fluctuated in an average range of 434.2 to 495.5 in each period, and no bias was found in specific samples. The Shannon index, an index of species evenness, was 6.879–7.008, which was almost the same in all periods. As beta diversity, Principal Coordinate Analysis (PCoA) did not show any clustering due to differences in experimental groups or periods (data not shown). As a taxonomy group at the phylum level, the Firmicutes, Bacteroidetes and Actinobacteria accounted for 90% or more of the total Prokaryotes. It is known that bacteria belonging to the Firmicutes, Bacteroidetes and Actinobacteria are universally present in the actual human intestinal environment (The Human Microbiome Project Consortium, 2012). Based on these results, it was shown in the present study that these are major intestinal bacterial communities. Abundance of the amplicon sequences belonging to the above three phyla and the other phyla was shown by a box-and-whisker plot (Fig. 2). The average abundance of the Firmicutes, Bacteroidetes, Actinobacteria and others in each group were in the range of 48.7 to 55.4%, 26.7 to 31.2%, 11.4 to 17.2% and 2.6 to 5.2%, respectively. These average abundances of the intestinal bacterial flora were not significantly changed during the first, second and third periods in both the Koji amazake and LAF-amazake groups. Bifidobacterium spp., Enterococcus spp. and Lactobacillus spp. are generally known to have beneficial effects on the intestinal environment, and Clostridium spp. is known to be adversely affected. These bacterial genera are regarded as indicators of probiotics, and their abundance was investigated by focusing on these microorganisms. The abundance of these microorganisms in each group are shown Fig. 3. The abundance of Enterococcus, Lactobacillus and Clostridium were low, averaging less than 0.5% in each group, which was considered unsuitable for statistical analysis. On the other hand, Bifidobacterium spp. was detected with high frequency from all samples.
Table 4.
Alpha diversities of intestinal flora in fecal samples
| Ingestion and period |
No. of Samples |
Sequence counts |
Sequence counts/sample (Mean) |
Observed features |
Observed features/sample (Mean) |
Diversity indices |
| Chao1 |
Shannon indes |
| Mean ± SD |
Mean ± SD |
| All |
84 |
1,777,595 |
21,162 |
3,856 |
471 |
464.4 ± 19.4 |
7.003 ± 0.019 |
| Koji_1w |
14 |
266,245 |
19,018 |
2,430 |
436 |
441.3 ± 18.7 |
6.879 ± 0.019 |
| Koji_2w |
14 |
310,202 |
22,157 |
2,443 |
442 |
434.2 ± 17.0 |
6.888 ± 0.020 |
| Koji_3w |
14 |
292,229 |
20,874 |
2,447 |
460 |
459.3 ± 21.0 |
7.058 ± 0.019 |
| LAF_1w |
14 |
302,056 |
21,575 |
2,698 |
492 |
479.5 ± 19.0 |
7.021 ± 0.019 |
| LAF_2w |
14 |
345,622 |
24,687 |
2,794 |
513 |
495.5 ± 20.4 |
7.083 ± 0.021 |
| LAF_3w |
14 |
261,241 |
18,660 |
2,703 |
480 |
476.7 ± 20.3 |
7.088 ± 0.019 |
Koji and LAF indicate the type of amazake ingested, and 1w, 2w and 3w indicate that the samples were collected in 1st, 2nd and 3rd period, respectively.
The abundance of Bifidobacterium spp. was in the range of 7.9 to 10.1%. Although outliers occurred in Koji_1w (first) and Koji_3w (third), no significant differences were observed between the groups in the results of multiple comparison test by one-way analysis of variance. These results showed that the apparent fluctuation of intestinal bacterial flora among groups did not occur, regardless of koji amazake and/or LAF-amazake intake.
Discussion
Koji amazake is a traditional sweet Japanese beverage that is produced from rice koji, which is made by Aspergillus oryzae growing on steamed rice, water, and cocked rice in some cases. In recent years, the flavor and ingredients of koji amazake were reformed by lactic acid fermentation using Lactobacillus sakei UONUMA strains. The lactic acid bacteria and its fermentation products have been assessed for their health beneficial effects. Species such as Lactobacillus paracasei, Lactobacillus casei, and Lactobacillus rhamnosus have been commonly used as probiotics of lactic acid bacteria (Collins et al., 1991). The intake of fermented milk beverages containing the Lactobacillus casei strain Shirota has been reported to increase the frequency of bowel movements and stool quality in healthy volunteers with a low defecation frequency (Matsumoto et al., 2006). Therefore, this study was performed to clarify the effects of koji amazake and LAF-amazake on defecation status using a double-blind parallel group comparison test in healthy volunteers with relatively low stool frequency.
The defecation frequencies during the second and third periods were significantly greater than those during the first period (first vs. second; p < 0.001, first vs. third; p < 0.001, second vs. third; p = 0.58) in the koji amazake group. In the third period, the defecation frequency in the koji amazake group was significantly greater than that in the LAF-amazake group ( p < 0.05). These results indicated that the defecation frequency in healthy volunteers with a tendency for constipation might increase after intake of koji amazake, and that koji amazake might be a food material with constipation relieving effects. This is the first report on the evaluation of the effectiveness of koji amazake on the state of bowel movements. LAF-amazake was produced by lactic acid fermentation using Lactobacillus sakei UONUMA strains. As the intake of foods containing Lactobacillus strain have been reported to increase the frequency of bowel movements and stool quality in healthy volunteers with a low defecation frequency, LAF-amazake may have an effect on bowel movements. However, the values for defecation frequency, days of defecation and defecation index in the LAF-amazake group were unaltered during all periods.
The values of the fecal shape in the koji amazake group during the second and third periods were close to 3. However, the values for fecal shape in the LAF-amazake group during the third period returned to the value of the first period. The banana shape is the ideal fecal shape (Takiguchi et al., 1997). The value of the incidence rate of banana shape in the LAF-amazake group was smaller than that in the koji amazake group during the first period. This suggests that the bowel condition in the koji amazake group is better than that in the LAF-amazake group. The values for the incidence of banana shape in both groups during the second and third periods were greater than those during the first period. These results indicated that both the koji amazake and the LAF-amazake have an effect on bowel movements. It has been reported that the acidification of intestinal conditions due to the activity of Bifidobacterium might lead to brighter fecal color and the alkalification of the intestinal conditions due to the activity of ammonia producing bacteria might lead to darker fecal color (Seki et al., 2004). The values of fecal color were close to 3 in both groups during the second period. These results suggest that both the koji amazake and the LAF-amazake have acidification activity in the intestinal environment. It has been reported that fecal odor, concentrations of intestinal ammonia, indole and p-cresol decrease during the intake of the lactic acid forming bacteria (Ara et al., 2002). The values for fecal odor in the koji amazake group during the second and third periods were close to 3. These results indicate that the koji amazake have suppressing activity of the concentrations of intestinal ammonia, indole and p-cresol.
In order to clarify the effects of koji and LAF-amazake intake on the intestinal bacterial community, microbial community structures were analyzed. There were no apparent changes in intestinal bacterial flora in relation to intake of either amazake. From these findings, the overall intestinal bacterial flora would not significantly change under the present experimental conditions. The genus level analysis targeting the members used as indicators of probiotics also showed no apparent differences, regardless of intake group and period. In general, it is known that a bowel movement improving effect occurs by fluctuating intestinal flora. In a previous study on improvement of bowel movement and its effect on intestinal flora, intake of fermented milk and fructooligosaccharides was found to improve bowel movements. In addition, the number of bacteria of the genus Bifidobacterium and the occupancy rate of total bacteria in feces were significantly increased (Kinumaki et al., 2001, Tokunaga et al., 1993). However, the results obtained in this study suggest that the increased defecation frequencies found in the group with koji amazake intake was not caused by the intake effect on fecal bacterial community, but was likely caused by other unknown mechanisms.
Conclusion
The effects of koji amazake and LAF-amazake on defecation status were examined using a double blind parallel group comparison test in healthy volunteers with relatively low stool frequency. The defecation frequency in healthy volunteers with a tendency for constipation increased after intake of koji amazake. Koji amazake is expected to be useful as a food material with constipation relieving effects. Although LAF-amazake contains sterilized lactic acid bacteria and lactic acid, LAF-amazake has no significant effect on improving constipation.
Acknowledgments The authors wish to thank the staff of the Niigata Bio-Research Park, Inc., for their support in this study and the registration of the UMIN Clinical Trials Registry. This research was partly supported by the regional revitalization project of the Cabinet Office, Government of Japan (A3007).
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