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Effect of a Nutritionally Balanced Diet Comprising Whole Grains and Vegetables Alone or in Combination with Probiotic Supplementation on the Gut Microbiota
1Pulmuone Co., Ltd., Seoul 06367, Korea
2Department of Food and Nutrition, and Research Institute of Human Ecology, Seoul National University, Seoul 08826, Korea
3Department of Food and Nutrition, The University of Suwon, Gyeonggi 18323, Korea
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Prev Nutr Food Sci 2021; 26(2): 121-131
Published June 30, 2021 https://doi.org/10.3746/pnf.2021.26.2.121
Copyright © The Korean Society of Food Science and Nutrition.
Abstract
Keywords
INTRODUCTION
The intestinal ecosystem is closely related to human health. Intestinal dysbiosis is associated with various diseases, such as inflammatory bowel disease (IBD), obesity, non-alcoholic liver disease, and high blood pressure (Singh et al., 2017). The composition of the gut microbiota is dependent on internal factors, such as sex, race, and age, and is affected by external environmental factors, such as antibiotics, drugs, stress, and diet. Diet is a source of nutrients for microorganisms and is considered the most important factor influencing the gut microbiota (Shin et al., 2019). Arumugam et al. (2011) introduced the following three enterotypes of the human gut microbiome according to dietary habits: the
Modern society is exposed to the “Western-style diet”, which is characterized by excessive intake of energy-rich foods, such as fat, cholesterol, and animal protein. This type of diet causes inflammatory conditions, and sustained inflammatory reactions can lead to various diseases, such as obesity, metabolic syndrome, and colon cancer (Cândido et al., 2018). Specifically, chronic inflammation is caused by lipopolysaccharides, a component of the cell wall of Gram-negative bacteria that is closely related to dietary factors, such as animal fat intake (Schoeler and Caesar, 2019). Therefore, the composition of the gut microbiota has been well studied based on investigations of dietary control for preventing chronic diseases (Chae, 2016; Barrett et al., 2018; Garcia-Mantrana et al., 2018; Losasso et al., 2018). Compared to the regular diet, the Mediterranean diet, which comprises olive oil, whole grains, nuts, vegetables, and fruits, among other foods, increases the levels of
Based on recent findings of probiotic function, studies are being conducted on the potential of probiotics for the treatment and prevention of chronic diseases (Shokryazdan et al., 2017). Probiotics are defined as living microorganisms that have a beneficial effect on the host when consumed in adequate amounts. Many of their beneficial effects, such as normalization of intestinal microbiota, competitive exclusion of pathogens, regulation of the intestinal transit, and modulation of the immune response, have been reported (FAO/WHO, 2001; Lee et al., 2018). This study included
This study aimed to evaluate the effect of a nutritionally balanced diet consisting of whole grains and vegetables alone or in combination with probiotics on the composition of gut microbiota in healthy adults. Participants were provided with one nutritionally balanced meal per day in the form of a lunch meal box for 2 weeks, and the abundance of major intestinal microorganisms was compared before and after the intervention. Furthermore,
MATERIALS AND METHODS
Subjects and experiment design
This study enrolled healthy adult volunteers aged 19∼65 years from October 8, 2019 to October 22, 2019. All participants provided written informed consent. Participants were randomly assigned to one of three groups and administered either a nutritionally balanced meal (B-diet group), a probiotic capsule (probiotics group), or a nutritionally balanced meal with a probiotic capsule (symbiotic group) once per day for 2 weeks following a 2-week washout period. Feces were collected before and after intake of the experimental diet, and all participants completed questionnaires related to defecation and dietary habits. Anthropometric biomarkers such as the body weight (kg), body fat percentage (%), and body mass index (BMI; m/kg2) were measured at the beginning and end of the intervention periods using a Body Composition Analyzer (InBody 230; InBody Co., Ltd., Seoul, Korea). The overall clinical study design is shown in Fig. 1. This study was conducted with approval of the Institutional Review Board of Suwon University (approval number: 1910-045-01).
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Figure 1. Schematic outline of the study protocol for the B-diet, probiotics, and synbiotics groups.
Experimental diet
The nutritionally balanced diet used in the study was a modified version of the 211 diet, a balanced meal plan developed by Pulmuone (Seoul, Korea). The 211 diet emphasizes selection of protein foods with low saturated fats, vegetables, and whole grains to prevent excessive carbohydrate consumption (Ha et al., 2020). Nutritionally balanced meals are rich in whole grains (e.g., oats, barley, quinoa, lentil, and chickpeas), vegetables (e.g., broccoli, carrot, cabbage, tomato, and mushroom), nuts and beans, and have a low content of red meat. The nutritionally balanced diet was high in fiber and low in saturated fats, and was provided in commercialized lunch boxes produced by the Pulmuone Green Juice Co., Ltd. (Chungbuk, Korea). On weekdays, a refrigerated lunch box containing a nutritionally balanced diet was delivered each day, and two frozen lunch boxes were provided on Friday for the weekend. Table 1 shows the nutritional content of each product and a list of the food ingredients. The probiotic capsule (400 mg) containing 10 billion colony forming unit (CFU) of
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Table 1 . Nutrient content of the balanced diet1)
Weekday (Mon∼Fri) Weekend (Sat∼Sun) Energy (kcal) 561 592 Carbohydrate (g) 88 (63%)2) 84 (57%) Protein (g) 23 (16%) 28 (19%) Fat (g) 13 (21%) 16 (24%) Dietary fiber (g) 14 4 1)Consists of the following: whole grains (oats, sorghum, adlay, quinoa, and brown rice), beans (kidney beans, black beans, lentils, lima beans, chickpeas, pea, and corn), vegetables (egg-plant, potato, sweet potato, red pepper, sweet pumpkin, carrot, wild parsley, romaine lettuce, garlic, Japanese apricot, white radish, broccoli, black olive, king oyster mushroom, celery, asparagus, cabbage, lettuce, button mushroom, onion, lotus root, cucumber, red onion, red cabbage, cheongyang chili, red pepper, green pepper, cauliflower, tomato, paprika, zucchini, and fragrant mushroom).
2)Percent of calories from each nutrient.
Assessment of defecation habits
Defecation habits were evaluated using the Bristol Stool Scale (Lewis and Heaton, 1997) and constipation diagnosis questionnaires (Longstreth et al., 2006), which include stool frequency and regularity, gut transit time, feeling of incomplete evacuation, difficulty in defecation (the act of pushing and squeezing), and bloating, assessed on a four-point scale. Average scores before and after the intervention period were compared.
Analysis of gut microbiota composition
Fecal samples before and after the intervention period were collected using a stool collection kit (Noble Biosciences, Inc., Gyeonggi, Korea) provided by Green Cross Genome Corporation (Gyeonggi, Korea). Collection kits containing fecal samples collected by the subjects were collected and sent to Green Cross Genome Corporation for analysis.
DNA extraction, amplification, and sequencing
DNA extraction from feces was performed using the Chemagic DNA Stool Kit (PerkinElmer, Inc., Waltham, MA, USA). A NEXTFLEX 16S V4 Amplicon-Seq kit (Bioo Scientific Corp., Austin, TX, USA) was used to create a library of the extracted DNA, which was evaluated for size and quality using the 2200 TapeStation (Agilent Technologies, Inc., Santa Clara, CA, USA). Sequencing was performed using a MiSeq Reagent Kit v2 (2×150 bp) and the MiSeq Sequencing System (Illumina, Inc., San Diego, CA, USA).
Bioinformatics analysis
Pre-processing and amplicon sequence variant clustering of the results file from the Illumina MiSeq Platform were performed using QIIME 2 (https://dev.qiime2.org). For microbial identification, BLCA software (https://github.om/qunfengdong/BLCA) and the NCBI RefSeq database (http://www.ncbi.nlm.nih.gov/refseq) were used. The final processed data were calculated as the number of reads per layer, and each calculated number of reads was converted into relative abundance for analysis.
Statistical analysis
All results were analyzed using SPSS, version 21.0 software (SPSS Inc., Chicago, IL, USA). Results were expressed as the mean±standard deviation (SD) or standard error of mean (SEM). Defecation habits and the relative abundances of gut microbiota before and after the intervention period were verified by paired Student’s
RESULTS
General characteristics of participants
Table 2 provides information regarding the study participants. A total of 65 individuals were recruited, of which 63 were included in the analysis. The participants had an average age of 36.8±8.0 years, average weight of 65.0±2.6 kg, average BMI of 23.7±0.8 mg/kg2, and average body fat percentage of 30.2±1.4%. There were no significant differences between groups.
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Table 2 . General characteristics of study participants
B-diet group Probiotics group Synbiotics group P -valueSex [n (%)] Male 8 (38.1) 7 (33.3) 7 (33.3) Female 13 (61.9) 14 (66.7) 14 (33.3) Age (yrs) 36.9±7.9 36.7±7.60 36.7±8.4 0.995 Body weight (kg) 64.8±2.60 65.1±2.56 65.3±2.78 0.992 BMI (kg/m2) 23.9±0.84 23.4±0.64 23.6±0.78 0.898 Body fat (%) 30.6±1.22 30.5±1.41 29.7±1.56 0.881 Values are expressed as the mean±SD (n=21).
B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotic capsule containing
Lactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08.BMI, body mass index.
Microbial composition at baseline
The main phyla analyzed in the feces of all subjects participating in the experiment are shown in Table 3. The gut microbiome of the participants was composed of the phyla Bacteroidetes (49.53±3.84%), Firmicutes (41.87±0.37%), Proteobacteria (3.58±0.65%), Actinobacteria (1.03±0.38%), Verrucomicrobia (1.35±0.98%), and Fusobacteria (1.21±0.97%), with the top six most abundant phyla accounting for 98.5% of the microbiota. The ratio of Firmicutes-to-Bacteroidetes (F/B) ratio was 1.15±0.23, and there were no significant differences between groups.
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Table 3 . Relative abundances of baseline gut microbiota communities at the phylum level in all participants
All subjects (n=63) Test groupNS B-diet group Probiotics group Synbiotics group Bacteroidetes 49.53±3.84 44.42±4.23 55.33±3.25 48.84±4.05 Firmicutes 41.87±3.37 45.17±3.51 37.45±2.85 42.98±3.75 Proteobacteria 3.58±0.65 4.01±0.68 4.14±0.97 2.60±0.32 Actinobacteria 1.03±0.38 0.87±0.20 0.89±0.28 1.32±0.65 Fusobacteria 1.21±0.97 2.67±2.30 0.32±0.25 0.63±0.35 Verrucomicrobia 1.35±0.98 1.47±0.98 0.44±0.32 2.13±1.64 Others 1.36±0.50 0.99±0.44 1.43±0.49 1.66±0.55 F/B ratio 1.15±0.23 1.34±0.23 0.80±0.12 1.31±0.29 Values are expressed as the mean±SEM (n=21).
F/B ratio, Firmicutes-to-Bacteroidetes ratio. B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotic capsule containing
Lactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08. NS, no significant difference between groups.
Changes in gut microbiota induced by dietary intake
Changes in the main phyla according to dietary intake are shown in Fig. 2. The B-diet group increased the abundance of Bacteroidetes by 113% (
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Figure 2. Relative abundance of bacteria constituting the gut microbiome at the phylum level. T0, baseline value; T2, after 2 weeks of intervention. B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotics capsule containing
Lactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08. *P <0.05 compared with baseline (T0), calculated using pairedt -tests.
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Figure 3. Effect of each diet on changes in the Firmicutes-to-Bacteroidetes (F/B) ratio. T0, baseline value; T2, after 2 weeks of intervention. Values are expressed as the mean±SEM. B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotics capsule containing
Lactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08. *P <0.05 compared with baseline (T0), calculated using pairedt -tests.
Changes in the gut microbiota according to dietary intake at the family level are shown in Fig. 4. The B-diet group showed a significant increase in the abundance of Bacteroidaceae (
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Figure 4. Effect of each diet on relative bacterial abundances at the family level. T0, baseline value; T2, after 2 weeks of consumption. B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotics capsule containing
Lactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08. *P < 0.05, **P <0.01, and ***P <0.001 compared with baseline (T0), calculated using pairedt -tests.
Changes in the gut microbiota according to dietary intake at the genus level are shown in Fig. 5 and Fig. 6. The B-diet group showed an increase in the abundance of
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Figure 5. Effect of each diet on relative abundances of bacteria in the major microbiota at the genus level. Relative abundances of the top five genera are shown. T0, baseline value; T2, after 2 weeks of consumption. Values are expressed as the mean±SEM. B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotics capsule containing
Lactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08. *P <0.05 and **P <0.01 compared with values at baseline (T0), calculated by pairedt -tests.
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Figure 6. Effect of each diet on relative abundances of bacteria in the microbiota at the genus level. Genera with significant differences in even one of the groups are shown. (A) Relative abundances of bacterial taxa accounting for more than 1%. (B) Relative abundances of bacterial taxa accounting for less than 1%. T0, baseline value; T2, after 2 weeks of consumption. Values are expressed as the mean±SEM. B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotics capsule containing
Lactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08. *P <0.05 and **P <0.01 compared with values at baseline (T0), calculated by pairedt -tests.
Relative abundances of Lactobacillus and Bifidobacterium
The relative abundances of the genus
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Figure 7. Effect of each diet on (A)
Lactobacillus and (B)Bifidobacterium composition of the microbiota. T0, baseline value; T2, after 2 weeks of consumption. Values are expressed as the mean±SEM. B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotics capsule containingLactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08. *P <0.05 compared with values at baseline (T0), calculated by pairedt -tests.
Changes in defecation habits induced by dietary intake
Defecation habits of the subjects are shown in Table 4. Before the dietary intervention, the average Bristol score of the subjects was 3.9±1.20 points, and 55.6% of the subjects scored 4 points with a normal fecal shape. An evaluation of each item (including frequency, regularity, transit time, the difficulty of defecation, incomplete evacuation, and bloating) showed that there were no significant differences between the groups at baseline (T0). The significance of dietary intake was evaluated using paired
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Table 4 . Defecation habits and fecal symptoms of study participants
Items Periods B-diet group P -valueProbiotics group P -valueSynbiotics group P -valueBristol score T0 4.24±0.23 0.590 4.00±0.26 0.760 3.48±0.28 0.162 T2 4.10±0.23 4.10±0.27 3.86±0.21 Stool frequency T0 3.14±0.20 0.649 3.10±0.19 1.000 3.19±0.19 0.329 T2 3.05±0.20 3.10±0.22 3.33±0.19 Stool regularity T0 2.05±0.13 0.666 2.19±0.15 0.493 2.14±0.13 0.055 T2 2.00±0.14 2.10±0.15 1.86±0.14 Gut transit time T0 1.57±0.18 0.493 1.48±0.11 1.000 1.81±0.19 0.379 T2 1.48±0.15 1.48±0.15 1.67±0.21 Difficult of defecation T0 1.81±0.18 0.540 1.86±0.16 0.104 2.05±0.16 0.030 T2 1.90±0.14 1.67±0.16 1.76±0.15 Incomplete evacuation T0 2.29±0.18 0.025 1.86±0.16 0.056 2.29±0.12 0.004 T2 1.86±0.14 1.62±0.16 1.81±0.16 Bloating T0 2.71±0.18 0.009 2.43±0.19 0.042 2.71±0.18 0.038 T2 2.10±0.18 2.05±0.21 2.24±0.15 Values are expressed as the mean±SD (n=21).
Significant differences were measured using paired
t -tests.The seven types of Bristol stool scale are as follows: type 1, separate hard lumps; type 2, lumpy and sausage-like; type 3, sausage-shape with cracks on the surface; type 4, like a smooth, soft sausage or snake; type 5, soft blobs with clear-cut edges; type 6, mushy consistency with ragged edges; type 7, liquid consistency with no solid pieces.
Constipated, including type 1 and type 2 on the Bristol stool scale; normal, including type 3, type 4, and type 5 on the Bristol stool scale; inflammation, including type 6 and type 7 on the Bristol stool scale.
B-diet group, whole grains, and vegetable-enriched diet group; probiotics group,
Lactobacillus plantarum PMO 08-administered group; synbiotics group, whole grains, and vegetable-enriched diet group combined withL. plantarum PMO 08.
Dietary compliance
Through surveying intake of the diet provided during the experiment and any side effects, all participants were found to have complied with the provided diets and probiotic supplements during the study period, and no participants experienced any side effects (data not shown).
DISCUSSION
This study was conducted to evaluate the effect of a nutritionally balanced diet enriched in whole grains and vegetables alone or in combination with probiotics on the composition of the gut microbiota. Nutritionally balanced diets were provided to participants in lunch boxes. Meals provided on weekdays contained 561 kcal, with a ratio (%) of carbohydrate to protein to fat of 63:16:21 and 14 g of dietary fiber. Meals provided on weekends contained 592 kcal, with a ratio (%) of carbohydrates to protein to fat of 57:19:24 with 4 g of dietary fiber. We evaluated the synergistic effect of a nutritionally balanced diet in combination with a probiotic supplement containing
Firmicutes and Bacteroidetes account for more than 90% of the gut microbiota, and a high F/B ratio is associated with IBD, type 2 diabetes, and obesity (Magne et al., 2020). The F/B ratio is an important indicator of body weight control as it is higher in patients with obesity and decreases with reduced caloric intake (Turnbaugh et al., 2006). Ley et al. (2006) first identified a causal relationship between the microbiome and obesity, which was confirmed by inducing obesity in lean mice via transplanting the feces of obese mice, showing that the ability to extract energy from food could be affected by intestinal microorganisms. Therefore, the nutritionally balanced diet used in the current study is expected to help prevent chronic diseases by improving the composition of the gut microbiota.
In this study, the decrease in the F/B ratio induced by intake of a nutritionally balanced diet was due, in some cases, to a significant increase in the abundance of the genus
In the B-diet group, the abundance of the phylum Firmicutes, associated with obesity, type 2 diabetes, and IBD decreased, which we attributed to the significant reduction in the abundances of Clostridiaceae (
In this study, intake of
In the current study, a nutritionally balanced diet and probiotic supplements had a synergetic effect on significantly increasing the abundance of
In this study, we conducted an analysis of gut microbiota according to dietary intake in healthy adults. Although the gut microbiota was not in a state of dysbiosis, replacing one meal per day for 2 weeks induced positive changes in the composition of the gut microbiota. However, since we did not measure hematological indicators, only limited physiological changes induced by alterations to the composition of the gut microbiota were observed. Therefore, to confirm the effect of a nutritionally balanced diet and probiotic supplementation on the human body, follow-up studies are needed to analyze the metabolites and physiological indicators together.
The objective of this study was to evaluate the effects of a nutritionally balanced diet consisting of whole grains and vegetables alone or in combination with probiotic supplements on the gut microbiota of healthy adults. Replacing one meal per day with a nutritionally balanced diet improved the intestinal environment by significantly lowering the F/B ratio, decreasing major gut microbiota, such as those of the families Clostridiaceae and Lachnospiraceae and the genera
AUTHOR DISCLOSURE STATEMENT
Y.J.O., K.N., Y.K., S.Y.L., J.I.K, and S.Y.L. are employees of Pulmuone Co., Ltd.. H.S.K. and K.T.H. declare no conflict of interest.
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Article
Original
Prev Nutr Food Sci 2021; 26(2): 121-131
Published online June 30, 2021 https://doi.org/10.3746/pnf.2021.26.2.121
Copyright © The Korean Society of Food Science and Nutrition.
Effect of a Nutritionally Balanced Diet Comprising Whole Grains and Vegetables Alone or in Combination with Probiotic Supplementation on the Gut Microbiota
Young Joo Oh1,2 , Kisun Nam1
, Yanghee Kim1, Seo Yeon Lee1, Hyung Sook Kim3, Jung Il Kang1, Sang Yun Lee1, Keum Taek Hwang2
1Pulmuone Co., Ltd., Seoul 06367, Korea
2Department of Food and Nutrition, and Research Institute of Human Ecology, Seoul National University, Seoul 08826, Korea
3Department of Food and Nutrition, The University of Suwon, Gyeonggi 18323, Korea
Correspondence to:Kisun Nam, Tel: +82-2-2186-8596, E-mail: ksnama@pulmuone.com
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Dysbiosis is a microbial imbalance, which often causes diseases and can be triggered by diet. Here, we determined the effect of a nutritionally balanced diet rich in vegetables and whole grains alone and/or in combination with probiotics on the gut microbiota of healthy adults. We conducted a parallel-group randomized trial enrolling 63 healthy participants who were administered either a balanced diet (B-diet group), a probiotic capsule containing Lactobacillus plantarum PMO 08 (probiotics group), or a balanced diet plus probiotic capsule (synbiotics group) once daily for 2 weeks. The gut microbiota of each participant was analyzed via 16S ribosomal RNA MiSeq-based sequencing. Gastrointestinal symptoms and defecation habits were evaluated using questionnaires. The B-diet group showed significantly reduced Firmicutes-to- Bacteroidetes ratio (P<0.05) and abundances of the genera Blautia (P<0.01), Dorea (P<0.05), and Lachnoclostridium (P<0.05). Furthermore, the abundance of Bacteroides increased (P<0.05) compared to baseline levels. In the synbiotics group, Lactobacillus abundance increased significantly (P<0.05) and defecation difficulty decreased (P<0.05), confirming a synergistic effect of combined intake. All groups showed a significant reduction in the abundance of Clostridiaceae (P<0.001) and alleviation of bloating symptoms (P<0.05). Moreover, the relative abundance of Faecalibacterium significantly increased in the probiotics group (P<0.05). Therefore, the individual or combined intake of a nutritionally balanced diet and L. plantarum PMO 08 beneficially modifies the gut microbiota with the potential to alleviate gastrointestinal symptoms and improve defecation habits.
Keywords: Firmicutes-to-Bacteroidetes ratio, gut health, gut microbiota, Lactobacillus plantarum PMO 08, nutritionally balanced diet
INTRODUCTION
The intestinal ecosystem is closely related to human health. Intestinal dysbiosis is associated with various diseases, such as inflammatory bowel disease (IBD), obesity, non-alcoholic liver disease, and high blood pressure (Singh et al., 2017). The composition of the gut microbiota is dependent on internal factors, such as sex, race, and age, and is affected by external environmental factors, such as antibiotics, drugs, stress, and diet. Diet is a source of nutrients for microorganisms and is considered the most important factor influencing the gut microbiota (Shin et al., 2019). Arumugam et al. (2011) introduced the following three enterotypes of the human gut microbiome according to dietary habits: the
Modern society is exposed to the “Western-style diet”, which is characterized by excessive intake of energy-rich foods, such as fat, cholesterol, and animal protein. This type of diet causes inflammatory conditions, and sustained inflammatory reactions can lead to various diseases, such as obesity, metabolic syndrome, and colon cancer (Cândido et al., 2018). Specifically, chronic inflammation is caused by lipopolysaccharides, a component of the cell wall of Gram-negative bacteria that is closely related to dietary factors, such as animal fat intake (Schoeler and Caesar, 2019). Therefore, the composition of the gut microbiota has been well studied based on investigations of dietary control for preventing chronic diseases (Chae, 2016; Barrett et al., 2018; Garcia-Mantrana et al., 2018; Losasso et al., 2018). Compared to the regular diet, the Mediterranean diet, which comprises olive oil, whole grains, nuts, vegetables, and fruits, among other foods, increases the levels of
Based on recent findings of probiotic function, studies are being conducted on the potential of probiotics for the treatment and prevention of chronic diseases (Shokryazdan et al., 2017). Probiotics are defined as living microorganisms that have a beneficial effect on the host when consumed in adequate amounts. Many of their beneficial effects, such as normalization of intestinal microbiota, competitive exclusion of pathogens, regulation of the intestinal transit, and modulation of the immune response, have been reported (FAO/WHO, 2001; Lee et al., 2018). This study included
This study aimed to evaluate the effect of a nutritionally balanced diet consisting of whole grains and vegetables alone or in combination with probiotics on the composition of gut microbiota in healthy adults. Participants were provided with one nutritionally balanced meal per day in the form of a lunch meal box for 2 weeks, and the abundance of major intestinal microorganisms was compared before and after the intervention. Furthermore,
MATERIALS AND METHODS
Subjects and experiment design
This study enrolled healthy adult volunteers aged 19∼65 years from October 8, 2019 to October 22, 2019. All participants provided written informed consent. Participants were randomly assigned to one of three groups and administered either a nutritionally balanced meal (B-diet group), a probiotic capsule (probiotics group), or a nutritionally balanced meal with a probiotic capsule (symbiotic group) once per day for 2 weeks following a 2-week washout period. Feces were collected before and after intake of the experimental diet, and all participants completed questionnaires related to defecation and dietary habits. Anthropometric biomarkers such as the body weight (kg), body fat percentage (%), and body mass index (BMI; m/kg2) were measured at the beginning and end of the intervention periods using a Body Composition Analyzer (InBody 230; InBody Co., Ltd., Seoul, Korea). The overall clinical study design is shown in Fig. 1. This study was conducted with approval of the Institutional Review Board of Suwon University (approval number: 1910-045-01).
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Figure 1. Schematic outline of the study protocol for the B-diet, probiotics, and synbiotics groups.
Experimental diet
The nutritionally balanced diet used in the study was a modified version of the 211 diet, a balanced meal plan developed by Pulmuone (Seoul, Korea). The 211 diet emphasizes selection of protein foods with low saturated fats, vegetables, and whole grains to prevent excessive carbohydrate consumption (Ha et al., 2020). Nutritionally balanced meals are rich in whole grains (e.g., oats, barley, quinoa, lentil, and chickpeas), vegetables (e.g., broccoli, carrot, cabbage, tomato, and mushroom), nuts and beans, and have a low content of red meat. The nutritionally balanced diet was high in fiber and low in saturated fats, and was provided in commercialized lunch boxes produced by the Pulmuone Green Juice Co., Ltd. (Chungbuk, Korea). On weekdays, a refrigerated lunch box containing a nutritionally balanced diet was delivered each day, and two frozen lunch boxes were provided on Friday for the weekend. Table 1 shows the nutritional content of each product and a list of the food ingredients. The probiotic capsule (400 mg) containing 10 billion colony forming unit (CFU) of
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Table 1 . Nutrient content of the balanced diet1).
Weekday (Mon∼Fri) Weekend (Sat∼Sun) Energy (kcal) 561 592 Carbohydrate (g) 88 (63%)2) 84 (57%) Protein (g) 23 (16%) 28 (19%) Fat (g) 13 (21%) 16 (24%) Dietary fiber (g) 14 4 1)Consists of the following: whole grains (oats, sorghum, adlay, quinoa, and brown rice), beans (kidney beans, black beans, lentils, lima beans, chickpeas, pea, and corn), vegetables (egg-plant, potato, sweet potato, red pepper, sweet pumpkin, carrot, wild parsley, romaine lettuce, garlic, Japanese apricot, white radish, broccoli, black olive, king oyster mushroom, celery, asparagus, cabbage, lettuce, button mushroom, onion, lotus root, cucumber, red onion, red cabbage, cheongyang chili, red pepper, green pepper, cauliflower, tomato, paprika, zucchini, and fragrant mushroom)..
2)Percent of calories from each nutrient..
Assessment of defecation habits
Defecation habits were evaluated using the Bristol Stool Scale (Lewis and Heaton, 1997) and constipation diagnosis questionnaires (Longstreth et al., 2006), which include stool frequency and regularity, gut transit time, feeling of incomplete evacuation, difficulty in defecation (the act of pushing and squeezing), and bloating, assessed on a four-point scale. Average scores before and after the intervention period were compared.
Analysis of gut microbiota composition
Fecal samples before and after the intervention period were collected using a stool collection kit (Noble Biosciences, Inc., Gyeonggi, Korea) provided by Green Cross Genome Corporation (Gyeonggi, Korea). Collection kits containing fecal samples collected by the subjects were collected and sent to Green Cross Genome Corporation for analysis.
DNA extraction, amplification, and sequencing
DNA extraction from feces was performed using the Chemagic DNA Stool Kit (PerkinElmer, Inc., Waltham, MA, USA). A NEXTFLEX 16S V4 Amplicon-Seq kit (Bioo Scientific Corp., Austin, TX, USA) was used to create a library of the extracted DNA, which was evaluated for size and quality using the 2200 TapeStation (Agilent Technologies, Inc., Santa Clara, CA, USA). Sequencing was performed using a MiSeq Reagent Kit v2 (2×150 bp) and the MiSeq Sequencing System (Illumina, Inc., San Diego, CA, USA).
Bioinformatics analysis
Pre-processing and amplicon sequence variant clustering of the results file from the Illumina MiSeq Platform were performed using QIIME 2 (https://dev.qiime2.org). For microbial identification, BLCA software (https://github.om/qunfengdong/BLCA) and the NCBI RefSeq database (http://www.ncbi.nlm.nih.gov/refseq) were used. The final processed data were calculated as the number of reads per layer, and each calculated number of reads was converted into relative abundance for analysis.
Statistical analysis
All results were analyzed using SPSS, version 21.0 software (SPSS Inc., Chicago, IL, USA). Results were expressed as the mean±standard deviation (SD) or standard error of mean (SEM). Defecation habits and the relative abundances of gut microbiota before and after the intervention period were verified by paired Student’s
RESULTS
General characteristics of participants
Table 2 provides information regarding the study participants. A total of 65 individuals were recruited, of which 63 were included in the analysis. The participants had an average age of 36.8±8.0 years, average weight of 65.0±2.6 kg, average BMI of 23.7±0.8 mg/kg2, and average body fat percentage of 30.2±1.4%. There were no significant differences between groups.
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Table 2 . General characteristics of study participants.
B-diet group Probiotics group Synbiotics group P -valueSex [n (%)] Male 8 (38.1) 7 (33.3) 7 (33.3) Female 13 (61.9) 14 (66.7) 14 (33.3) Age (yrs) 36.9±7.9 36.7±7.60 36.7±8.4 0.995 Body weight (kg) 64.8±2.60 65.1±2.56 65.3±2.78 0.992 BMI (kg/m2) 23.9±0.84 23.4±0.64 23.6±0.78 0.898 Body fat (%) 30.6±1.22 30.5±1.41 29.7±1.56 0.881 Values are expressed as the mean±SD (n=21)..
B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotic capsule containing
Lactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08..BMI, body mass index..
Microbial composition at baseline
The main phyla analyzed in the feces of all subjects participating in the experiment are shown in Table 3. The gut microbiome of the participants was composed of the phyla Bacteroidetes (49.53±3.84%), Firmicutes (41.87±0.37%), Proteobacteria (3.58±0.65%), Actinobacteria (1.03±0.38%), Verrucomicrobia (1.35±0.98%), and Fusobacteria (1.21±0.97%), with the top six most abundant phyla accounting for 98.5% of the microbiota. The ratio of Firmicutes-to-Bacteroidetes (F/B) ratio was 1.15±0.23, and there were no significant differences between groups.
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Table 3 . Relative abundances of baseline gut microbiota communities at the phylum level in all participants.
All subjects (n=63) Test groupNS B-diet group Probiotics group Synbiotics group Bacteroidetes 49.53±3.84 44.42±4.23 55.33±3.25 48.84±4.05 Firmicutes 41.87±3.37 45.17±3.51 37.45±2.85 42.98±3.75 Proteobacteria 3.58±0.65 4.01±0.68 4.14±0.97 2.60±0.32 Actinobacteria 1.03±0.38 0.87±0.20 0.89±0.28 1.32±0.65 Fusobacteria 1.21±0.97 2.67±2.30 0.32±0.25 0.63±0.35 Verrucomicrobia 1.35±0.98 1.47±0.98 0.44±0.32 2.13±1.64 Others 1.36±0.50 0.99±0.44 1.43±0.49 1.66±0.55 F/B ratio 1.15±0.23 1.34±0.23 0.80±0.12 1.31±0.29 Values are expressed as the mean±SEM (n=21)..
F/B ratio, Firmicutes-to-Bacteroidetes ratio. B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotic capsule containing
Lactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08. NS, no significant difference between groups..
Changes in gut microbiota induced by dietary intake
Changes in the main phyla according to dietary intake are shown in Fig. 2. The B-diet group increased the abundance of Bacteroidetes by 113% (
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Figure 2. Relative abundance of bacteria constituting the gut microbiome at the phylum level. T0, baseline value; T2, after 2 weeks of intervention. B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotics capsule containing
Lactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08. *P <0.05 compared with baseline (T0), calculated using pairedt -tests.
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Figure 3. Effect of each diet on changes in the Firmicutes-to-Bacteroidetes (F/B) ratio. T0, baseline value; T2, after 2 weeks of intervention. Values are expressed as the mean±SEM. B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotics capsule containing
Lactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08. *P <0.05 compared with baseline (T0), calculated using pairedt -tests.
Changes in the gut microbiota according to dietary intake at the family level are shown in Fig. 4. The B-diet group showed a significant increase in the abundance of Bacteroidaceae (
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Figure 4. Effect of each diet on relative bacterial abundances at the family level. T0, baseline value; T2, after 2 weeks of consumption. B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotics capsule containing
Lactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08. *P < 0.05, **P <0.01, and ***P <0.001 compared with baseline (T0), calculated using pairedt -tests.
Changes in the gut microbiota according to dietary intake at the genus level are shown in Fig. 5 and Fig. 6. The B-diet group showed an increase in the abundance of
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Figure 5. Effect of each diet on relative abundances of bacteria in the major microbiota at the genus level. Relative abundances of the top five genera are shown. T0, baseline value; T2, after 2 weeks of consumption. Values are expressed as the mean±SEM. B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotics capsule containing
Lactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08. *P <0.05 and **P <0.01 compared with values at baseline (T0), calculated by pairedt -tests.
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Figure 6. Effect of each diet on relative abundances of bacteria in the microbiota at the genus level. Genera with significant differences in even one of the groups are shown. (A) Relative abundances of bacterial taxa accounting for more than 1%. (B) Relative abundances of bacterial taxa accounting for less than 1%. T0, baseline value; T2, after 2 weeks of consumption. Values are expressed as the mean±SEM. B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotics capsule containing
Lactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08. *P <0.05 and **P <0.01 compared with values at baseline (T0), calculated by pairedt -tests.
Relative abundances of Lactobacillus and Bifidobacterium
The relative abundances of the genus
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Figure 7. Effect of each diet on (A)
Lactobacillus and (B)Bifidobacterium composition of the microbiota. T0, baseline value; T2, after 2 weeks of consumption. Values are expressed as the mean±SEM. B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotics capsule containingLactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08. *P <0.05 compared with values at baseline (T0), calculated by pairedt -tests.
Changes in defecation habits induced by dietary intake
Defecation habits of the subjects are shown in Table 4. Before the dietary intervention, the average Bristol score of the subjects was 3.9±1.20 points, and 55.6% of the subjects scored 4 points with a normal fecal shape. An evaluation of each item (including frequency, regularity, transit time, the difficulty of defecation, incomplete evacuation, and bloating) showed that there were no significant differences between the groups at baseline (T0). The significance of dietary intake was evaluated using paired
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Table 4 . Defecation habits and fecal symptoms of study participants.
Items Periods B-diet group P -valueProbiotics group P -valueSynbiotics group P -valueBristol score T0 4.24±0.23 0.590 4.00±0.26 0.760 3.48±0.28 0.162 T2 4.10±0.23 4.10±0.27 3.86±0.21 Stool frequency T0 3.14±0.20 0.649 3.10±0.19 1.000 3.19±0.19 0.329 T2 3.05±0.20 3.10±0.22 3.33±0.19 Stool regularity T0 2.05±0.13 0.666 2.19±0.15 0.493 2.14±0.13 0.055 T2 2.00±0.14 2.10±0.15 1.86±0.14 Gut transit time T0 1.57±0.18 0.493 1.48±0.11 1.000 1.81±0.19 0.379 T2 1.48±0.15 1.48±0.15 1.67±0.21 Difficult of defecation T0 1.81±0.18 0.540 1.86±0.16 0.104 2.05±0.16 0.030 T2 1.90±0.14 1.67±0.16 1.76±0.15 Incomplete evacuation T0 2.29±0.18 0.025 1.86±0.16 0.056 2.29±0.12 0.004 T2 1.86±0.14 1.62±0.16 1.81±0.16 Bloating T0 2.71±0.18 0.009 2.43±0.19 0.042 2.71±0.18 0.038 T2 2.10±0.18 2.05±0.21 2.24±0.15 Values are expressed as the mean±SD (n=21)..
Significant differences were measured using paired
t -tests..The seven types of Bristol stool scale are as follows: type 1, separate hard lumps; type 2, lumpy and sausage-like; type 3, sausage-shape with cracks on the surface; type 4, like a smooth, soft sausage or snake; type 5, soft blobs with clear-cut edges; type 6, mushy consistency with ragged edges; type 7, liquid consistency with no solid pieces..
Constipated, including type 1 and type 2 on the Bristol stool scale; normal, including type 3, type 4, and type 5 on the Bristol stool scale; inflammation, including type 6 and type 7 on the Bristol stool scale..
B-diet group, whole grains, and vegetable-enriched diet group; probiotics group,
Lactobacillus plantarum PMO 08-administered group; synbiotics group, whole grains, and vegetable-enriched diet group combined withL. plantarum PMO 08..
Dietary compliance
Through surveying intake of the diet provided during the experiment and any side effects, all participants were found to have complied with the provided diets and probiotic supplements during the study period, and no participants experienced any side effects (data not shown).
DISCUSSION
This study was conducted to evaluate the effect of a nutritionally balanced diet enriched in whole grains and vegetables alone or in combination with probiotics on the composition of the gut microbiota. Nutritionally balanced diets were provided to participants in lunch boxes. Meals provided on weekdays contained 561 kcal, with a ratio (%) of carbohydrate to protein to fat of 63:16:21 and 14 g of dietary fiber. Meals provided on weekends contained 592 kcal, with a ratio (%) of carbohydrates to protein to fat of 57:19:24 with 4 g of dietary fiber. We evaluated the synergistic effect of a nutritionally balanced diet in combination with a probiotic supplement containing
Firmicutes and Bacteroidetes account for more than 90% of the gut microbiota, and a high F/B ratio is associated with IBD, type 2 diabetes, and obesity (Magne et al., 2020). The F/B ratio is an important indicator of body weight control as it is higher in patients with obesity and decreases with reduced caloric intake (Turnbaugh et al., 2006). Ley et al. (2006) first identified a causal relationship between the microbiome and obesity, which was confirmed by inducing obesity in lean mice via transplanting the feces of obese mice, showing that the ability to extract energy from food could be affected by intestinal microorganisms. Therefore, the nutritionally balanced diet used in the current study is expected to help prevent chronic diseases by improving the composition of the gut microbiota.
In this study, the decrease in the F/B ratio induced by intake of a nutritionally balanced diet was due, in some cases, to a significant increase in the abundance of the genus
In the B-diet group, the abundance of the phylum Firmicutes, associated with obesity, type 2 diabetes, and IBD decreased, which we attributed to the significant reduction in the abundances of Clostridiaceae (
In this study, intake of
In the current study, a nutritionally balanced diet and probiotic supplements had a synergetic effect on significantly increasing the abundance of
In this study, we conducted an analysis of gut microbiota according to dietary intake in healthy adults. Although the gut microbiota was not in a state of dysbiosis, replacing one meal per day for 2 weeks induced positive changes in the composition of the gut microbiota. However, since we did not measure hematological indicators, only limited physiological changes induced by alterations to the composition of the gut microbiota were observed. Therefore, to confirm the effect of a nutritionally balanced diet and probiotic supplementation on the human body, follow-up studies are needed to analyze the metabolites and physiological indicators together.
The objective of this study was to evaluate the effects of a nutritionally balanced diet consisting of whole grains and vegetables alone or in combination with probiotic supplements on the gut microbiota of healthy adults. Replacing one meal per day with a nutritionally balanced diet improved the intestinal environment by significantly lowering the F/B ratio, decreasing major gut microbiota, such as those of the families Clostridiaceae and Lachnospiraceae and the genera
AUTHOR DISCLOSURE STATEMENT
Y.J.O., K.N., Y.K., S.Y.L., J.I.K, and S.Y.L. are employees of Pulmuone Co., Ltd.. H.S.K. and K.T.H. declare no conflict of interest.
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Table 1 . Nutrient content of the balanced diet1)
Weekday (Mon∼Fri) Weekend (Sat∼Sun) Energy (kcal) 561 592 Carbohydrate (g) 88 (63%)2) 84 (57%) Protein (g) 23 (16%) 28 (19%) Fat (g) 13 (21%) 16 (24%) Dietary fiber (g) 14 4 1)Consists of the following: whole grains (oats, sorghum, adlay, quinoa, and brown rice), beans (kidney beans, black beans, lentils, lima beans, chickpeas, pea, and corn), vegetables (egg-plant, potato, sweet potato, red pepper, sweet pumpkin, carrot, wild parsley, romaine lettuce, garlic, Japanese apricot, white radish, broccoli, black olive, king oyster mushroom, celery, asparagus, cabbage, lettuce, button mushroom, onion, lotus root, cucumber, red onion, red cabbage, cheongyang chili, red pepper, green pepper, cauliflower, tomato, paprika, zucchini, and fragrant mushroom).
2)Percent of calories from each nutrient.
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Table 2 . General characteristics of study participants
B-diet group Probiotics group Synbiotics group P -valueSex [n (%)] Male 8 (38.1) 7 (33.3) 7 (33.3) Female 13 (61.9) 14 (66.7) 14 (33.3) Age (yrs) 36.9±7.9 36.7±7.60 36.7±8.4 0.995 Body weight (kg) 64.8±2.60 65.1±2.56 65.3±2.78 0.992 BMI (kg/m2) 23.9±0.84 23.4±0.64 23.6±0.78 0.898 Body fat (%) 30.6±1.22 30.5±1.41 29.7±1.56 0.881 Values are expressed as the mean±SD (n=21).
B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotic capsule containing
Lactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08.BMI, body mass index.
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Table 3 . Relative abundances of baseline gut microbiota communities at the phylum level in all participants
All subjects (n=63) Test groupNS B-diet group Probiotics group Synbiotics group Bacteroidetes 49.53±3.84 44.42±4.23 55.33±3.25 48.84±4.05 Firmicutes 41.87±3.37 45.17±3.51 37.45±2.85 42.98±3.75 Proteobacteria 3.58±0.65 4.01±0.68 4.14±0.97 2.60±0.32 Actinobacteria 1.03±0.38 0.87±0.20 0.89±0.28 1.32±0.65 Fusobacteria 1.21±0.97 2.67±2.30 0.32±0.25 0.63±0.35 Verrucomicrobia 1.35±0.98 1.47±0.98 0.44±0.32 2.13±1.64 Others 1.36±0.50 0.99±0.44 1.43±0.49 1.66±0.55 F/B ratio 1.15±0.23 1.34±0.23 0.80±0.12 1.31±0.29 Values are expressed as the mean±SEM (n=21).
F/B ratio, Firmicutes-to-Bacteroidetes ratio. B-diet group, whole grains and vegetable-enriched diet; probiotics group, probiotic capsule containing
Lactobacillus plantarum PMO 08; synbiotics group, whole grains and vegetable-enriched diet combined withL. plantarum PMO 08. NS, no significant difference between groups.
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Table 4 . Defecation habits and fecal symptoms of study participants
Items Periods B-diet group P -valueProbiotics group P -valueSynbiotics group P -valueBristol score T0 4.24±0.23 0.590 4.00±0.26 0.760 3.48±0.28 0.162 T2 4.10±0.23 4.10±0.27 3.86±0.21 Stool frequency T0 3.14±0.20 0.649 3.10±0.19 1.000 3.19±0.19 0.329 T2 3.05±0.20 3.10±0.22 3.33±0.19 Stool regularity T0 2.05±0.13 0.666 2.19±0.15 0.493 2.14±0.13 0.055 T2 2.00±0.14 2.10±0.15 1.86±0.14 Gut transit time T0 1.57±0.18 0.493 1.48±0.11 1.000 1.81±0.19 0.379 T2 1.48±0.15 1.48±0.15 1.67±0.21 Difficult of defecation T0 1.81±0.18 0.540 1.86±0.16 0.104 2.05±0.16 0.030 T2 1.90±0.14 1.67±0.16 1.76±0.15 Incomplete evacuation T0 2.29±0.18 0.025 1.86±0.16 0.056 2.29±0.12 0.004 T2 1.86±0.14 1.62±0.16 1.81±0.16 Bloating T0 2.71±0.18 0.009 2.43±0.19 0.042 2.71±0.18 0.038 T2 2.10±0.18 2.05±0.21 2.24±0.15 Values are expressed as the mean±SD (n=21).
Significant differences were measured using paired
t -tests.The seven types of Bristol stool scale are as follows: type 1, separate hard lumps; type 2, lumpy and sausage-like; type 3, sausage-shape with cracks on the surface; type 4, like a smooth, soft sausage or snake; type 5, soft blobs with clear-cut edges; type 6, mushy consistency with ragged edges; type 7, liquid consistency with no solid pieces.
Constipated, including type 1 and type 2 on the Bristol stool scale; normal, including type 3, type 4, and type 5 on the Bristol stool scale; inflammation, including type 6 and type 7 on the Bristol stool scale.
B-diet group, whole grains, and vegetable-enriched diet group; probiotics group,
Lactobacillus plantarum PMO 08-administered group; synbiotics group, whole grains, and vegetable-enriched diet group combined withL. plantarum PMO 08.
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