Original Paper Received: July 15, 2015 Accepted: September 13, 2015 Published online: October 24, 2015
Ann Nutr Metab 2015;67:257–266 DOI: 10.1159/000441066
Intestinal Microbiota Profiles of Healthy Pre-School and School-Age Children and Effects of Probiotic Supplementation Chongxin Wang a Satoru Nagata a, b Takashi Asahara c Norikatsu Yuki c Kazunori Matsuda c Hirokazu Tsuji c Takuya Takahashi a, c Koji Nomoto a, c Yuichiro Yamashiro a
a
Probiotics Research Laboratory, Juntendo University Graduate School of Medicine, b Department of Pediatrics, Tokyo Women’s Medical University, and c Yakult Central Institute, Tokyo, Japan
Abstract Objectives: This study aims to establish the baseline profile of intestinal microbiota in pre-school and school-age Japanese children and to investigate the effects of a probiotic on the microbiota. Methods: We analyzed the intestinal microbiota and investigated the effects (before, during and after the ingestion period) on intestinal microbiota and the environment of 6 months of daily ingestion of a probiotic (Lactobacillus casei strain Shirota (LcS)-fermented milk). Results: We performed an open trial in 23 children (14 boys, 9 girls; age 7.7 ± 2.4 years (mean ± SD); BMI 19.6 ± 4.6). The composition of intestinal microbiota of healthy pre-school and school-age children resembled that of adults. During probiotic supplementation, the population levels of Bifidobacterium and total Lactobacillus increased significantly, while those of Enterobacteriaceae, Staphylococcus and Clostridium perfringens decreased significantly. A significant in-
© 2015 S. Karger AG, Basel 0250–6807/15/0674–0257$39.50/0 E-Mail [email protected] www.karger.com/anm
crease in fecal concentrations of organic acids and also a decrease in fecal pH were observed during the ingestion period. However, the patterns of fecal microbiota and intestinal environment were found to revert to the baseline levels (i.e. before ingestion) within 6 months following the cessation of probiotic intake. Conclusion: Regular intake of an LcScontaining probiotic product may modify the gut microbiota composition and intestinal environment in pre-school and school-age children while maintaining the homeostasis of the microbiota. © 2015 S. Karger AG, Basel
Introduction
The intestinal microbiota of a typical adult can weigh 1 kg or more. This ‘tissue’ comprises about 1014 microorganisms, meaning that the number of these microbes is almost 10 times greater than the total number of human body cells [1]. These gut microbes are believed to contribute significantly not only to host nutrition and metabolism but also to the developmental regulation of intestinal angiogenesis, protection from pathogens and developProf. Yuichiro Yamashiro Probiotics Research Laboratory, Juntendo University Graduate School of Medicine 3rd Floor, Hongo-Asakaze Bldg, 2-9-8 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan) E-Mail yamasiro @ juntendo.ac.jp
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 4/25/2016 11:16:44 PM
Key Words Fecal microbiota · Gut microbiota · Lactobacillus casei strain Shirota · Organic acids · Pre-school and school-age children · Probiotics
Preingestion
During ingestion
Post-ingestion
3 weeks
1 2 3 4 5 6 month months months months months months
6 months
LcS-fermented milk
Fig. 1. A schematic of the study design, Fecal sample
ment of immune functions [1]. The intestinal microbiome undergoes dynamic changes during different stages of host growth and development, with the most dramatic compositional changes believed to occur during infancy and childhood [2]. Probiotics are viable bacteria that exhibit a diverse range of beneficial effects on host health, due primarily to the impact of the bacteria on the improvement of gut microbial balance and the intestinal environment. Thus, orally administered probiotics are expected to be resistant to gastric acid, bile and proteolytic enzymes, permitting these bacteria to reach the intestinal tract in a viable form. Having reached the intestinal tract, these microorganisms are thought to modify the intestinal microbiota and subsequently contribute to the reduction of various disease risks [3, 4]. The probiotic approach is aimed primarily at repairing the perturbations of the gut microbiota composition (dysbiosis) and to restoring the resilience of the microbiota [5]. Over the last 2 decades, there has been a growing interest in the manipulation of intestinal microbiota with probiotics for the prevention and treatment of certain diseases. However, reports on the analysis of intestinal microbiota in pre-school and school-age children and the effects of probiotic supplementation on the microbiota in these children are sparse. In this context, the present study was designed to establish the baseline profile of intestinal microbiota in pre-school and schoolage children and, in addition, to investigate the effects of probiotic supplementation on the microbiota. Specifically, we investigated the effects of daily intake of probiotic Lactobacillus casei strain Shirota (LcS)-fermented milk on the fecal microbiota and intestinal environment of healthy pre-school and school-age Japanese children before, during and after 6 months of probiotic ingestion and after another 6 months following the termination of ingestion (the post-ingestion period). 258
Ann Nutr Metab 2015;67:257–266 DOI: 10.1159/000441066
Subjects and Methods Ethical Approval Information The trial was approved by the Clinical Research Ethics Committee of Juntendo University Hospital and was performed in compliance with the principles of the Helsinki Declaration (adopted in 1964; amended in 1975, 1983, 1989, 1996 and 2000), whereby a thorough explanation was given to the subjects’ parents or guardians prior to the trial regarding the purpose and methods of the study in order to obtain a written informed consent from the parents or guardians. Test Diet The test diet consisted of a commercially available fermented milk product (Yakult 400; Yakult Honsha Co., Ltd., Tokyo, Japan) containing LcS (YIT 9029) at 4 × 1010 cells per 80-ml bottle. LcSfermented milk was made from glucose/fructose/liquid sugar, skimmed milk and flavor ingredients. The test diet was offered as an 80-ml bottle containing 1.0 g protein, 0.1 g fat, 14.4 g carbohydrate and 15 mg sodium and providing 62 kcal. LcS was present in the product at a concentration of about 4 × 1010 viable LcS cells per bottle. The test diet was provided by Yakult Honsha Co., Ltd. Study Schedule and Ingestion of the Test Diet The trial was conducted as an open study, evaluating the parameters at defined time points during pre-ingestion, ingestion and post-ingestion periods (fig. 1). The subjects consented to dietary limitations for a minimum of 3 weeks prior to the start of the study; these limitations precluded the children from ingestion (during this interval) of any product that might contain LcS and other lactic acid bacteria or probiotics. Following this interval, the subjects were instructed to ingest 1 bottle of LcS-fermented milk per day for 6 months during the period from January to July, 2012. Examination Methods Fecal Bacteriologic Examination Fecal sampling: fresh fecal samples were obtained from the children on day 1 (i.e. before ingestion) on months 1, 3 and 6 of the ingestion period and at 6 months after the termination of ingestion (fig. 1). At each time point, for each child, approximately 1-gram samples of feces were collected separately into a sterile fecal collection tube (Sarstedt AG & Co., Numbrecht, Germany) containing 2 ml RNAlater (Ambion Inc., Austin, Tex., USA; for the
Wang/Nagata/Asahara/Yuki/Matsuda/ Tsuji/Takahashi/Nomoto/Yamashiro
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 4/25/2016 11:16:44 PM
showing the duration of the study, schedule of probiotic ingestion and the time points of sample collection (as indicated by gray arrows).
analysis of fecal microbiota), an RNA stabilization solution and into empty tubes (for organic acids analysis). The tubes were stored at 4 ° C (for the analysis of fecal microbiota) or at –20 ° C (for organic acids analysis) for about 36 h. The fecal samples were maintained in a cooling box with refrigerants and were immediately sent to the Yakult Central Institute where these samples were preserved in a –20 ° C freezer block until further processing. Primary treatment of fecal samples for fecal microbiota analysis: fecal samples were weighed and then suspended in 9 volumes of RNAlater (Ambion). After incubating for 10 min at room temperature, the fecal homogenate was added to 1 ml of sterilized phosphate buffer saline (pH 7.2), and then centrifuged at 5,000 g for 10 min. The supernatant was discarded, and the pellet was stored at –80 ° C pending use for the extraction of nucleic acids. Isolation of RNA and DNA: RNA [6, 7] and DNA [8] were extracted using the methods described in the respective references. The resulting nucleic acid fractions each were suspended in 1 ml of nuclease-free water (Ambion). Determination of bacterial count by reverse transcriptionquantitative polymerase chain reaction (RT-qPCR): a standard curve was generated using RT-qPCR data (using the quantification cycle, that is, the cycle number when threshold fluorescence was reached) and the corresponding cell count, which was determined microscopically with 4, 6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, Calif., USA) staining [9] for the dilution series of the standard strains described elsewhere [6, 7]. To determine the density of bacteria present in a given sample, each extracted RNA sample was subjected to 10-fold serial dilutions in nuclease-free water; dilutions were subjected to RT-qPCR; the quantification cycle values in the linear range of the assay were applied to a standard curve generated in the same experiment to obtain the bacterial cell count in each nucleic acid sample, and the results were converted to the number of bacteria per sample. The procedure was performed in triplicate for a given RNA sample. The specificity of the RT-qPCR assay using groupor species-specific primers was determined as described previously [6, 7]. The methods used for the detection of Clostridium difficile [10] and those for methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant coagulase-negative Staphylococcus (MRCNS), methicillin-sensitive S. aureus (MSSA) and methicillin-sensitive coagulase negative Staphylococcus (MSCNS) [11] have been described previously. The method for quantitative analysis of LcS has been described previously [8]. The lower detection limit of LcS was 5.7 log10 cells/g feces.
Measurements of the Fecal Organic Acid Concentrations and pH A portion of the homogenized feces was isolated, weighed, mixed with 0.15 M perchloric acid in a 4-fold volume and reacted at 4 ° C for 12 h. Next, the mixture was centrifuged at 4 ° C at 20,400 g for 10 min, and the supernatant was filtered through a 0.45-μm membrane filter (Millipore Japan, Tokyo) and sterilized. The concentrations of organic acids in the resulting samples were measured using a waters high-performance liquid chromatography system (Waters 432 Conductive Detector; Waters, USA) and a Shodex RSpack KC-811 column (Showa Denko, Tokyo) [12]. We calculated the concentrations of organic acids in the experimental specimens based on a standard curve. The standard curve was generated using a mixed solution standard consisting of 1–20 mM succinic acid, lactic acid, formic acid, acetic acid, propionic acid, iso
Statistical Analysis The results of pre, during and post ingestion were compared using the non-parametric Wilcoxon signed-rank test (fecal microbiota and fecal organic acids) and the parametric paired t test (fecal pH). The fecal bacteria detection rate was analyzed using the Fisher exact probability test. We used the IBM SPSS Statistics Desktop version 22.0 software (IBM Japan Ltd., Tokyo, Japan). In all tests, p < 0.05 was regarded as significant.
Effects of Probiotic on Healthy Children
Results
The Baseline Patterns of Intestinal Microbiota in Pre-School and School-Age Children Twenty-three healthy children (14 boys and 9 girls, age (mean ± SD) 7.7 ± 2.4 (range 4–12) years; BMI 19.6 ± 4.6), were recruited on the basis of no recent (i.e. during the 2 weeks preceding the commencement of the trial) history of long-term (≥1 week) antibiotic therapy or chronic inflammatory or viral illness, no current drug therapy, no known allergies and no participation in other clinical trials. The composition and profile of intestinal microbiota of healthy pre-school and school-age children (table 1) were found to be similar to those reported previously in healthy adults [7]. The Baseline Patterns of Fecal Organic Acids in Healthy Pre-School and School-Age Children The concentrations of fecal organic acids and fecal pH of healthy pre-school and school-age children (table 2) were found to be relatively stable and comparable to those of healthy adults reported previously [13]. Effects of Ingesting LcS-Fermented Milk on the Fecal Microbiota There was no significant difference in terms of the total number of bacteria in the fecal samples throughout the study period. The level of Bifidobacterium increased significantly at months 3 and 6 of the ingestion period when compared to the pre-ingestion levels (p < 0.05 and p < 0.01, respectively). The level of total Lactobacillus increased significantly at months 1, 3 and 6 of the ingestion period when compared to the pre-ingestion levels (p < 0.01). However, at 6 months post ingestion, the values of both Bifidobacterium and total Lactobacillus were found to return to their baseline levels and did not differ significantly from the pre-ingestion values. Ann Nutr Metab 2015;67:257–266 DOI: 10.1159/000441066
259
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 4/25/2016 11:16:44 PM
butyric acid, butyric acid, isovaleric acid and valeric acid. The fecal pH of each experimental specimen was measured by directly inserting the glass electrode of a D-51 pH meter (Horiba Seisakusho Co., Ltd., Tokyo) into the homogenized feces.
Table 1. The patterns of intestinal microbiota of healthy pre-school and primary school-age Japanese children Organisms
Total bacteria Obligate anaerobes Clostridium coccoides group C. leptum subgroup Bacteroides fragilis group Bifidobacterium Atopobium cluster Prevotella C. perfringens C. difficile Facultative anaerobes Total Lactobacillus L. gasseri subgroup L. brevis L. casei subgroup L. fermentum L. fructivorans L. plantarum subgroup L. reuteri subgroup L. ruminis subgroup L. sakei subgroup Enterobacteriaceae Enterococcus Staphylococcus MRSA MRCNS MSSA MSCNS Aerobes Pseudomonas
4–5 years (n = 6)
6–7 years (n = 5)
8–9 years (n = 7)
10–12 years (n = 5)
log10 cells/g fecesa
prevalence, %
log10 cells/g fecesa
prevalence, %
log10 cells/g fecesa
prevalence, %
log10 cells/g fecesa
prevalence, %
10.5±0.3
100
10.7±0.3
100
10.5±0.1
100
10.4±0.3
100
9.6±0.3 10.0±0.3 9.7±0.4 9.8±0.6 8.8±0.8 8.2 4.7±0.8 3.4
100 100 100 100 100 17 67 17
9.6±0.2 10.0±0.4 10.2±0.4 9.7±0.6 9.1±0.7 10.1 5.3±1.6
Ann Nutr Metab 2015;67:257–266 DOI: 10.1159/000441066
Intestinal Microbiota Profiles of Healthy Pre-School and School-Age Children and Effects of Probiotic Supplementation Chongxin Wang a Satoru Nagata a, b Takashi Asahara c Norikatsu Yuki c Kazunori Matsuda c Hirokazu Tsuji c Takuya Takahashi a, c Koji Nomoto a, c Yuichiro Yamashiro a
a
Probiotics Research Laboratory, Juntendo University Graduate School of Medicine, b Department of Pediatrics, Tokyo Women’s Medical University, and c Yakult Central Institute, Tokyo, Japan
Abstract Objectives: This study aims to establish the baseline profile of intestinal microbiota in pre-school and school-age Japanese children and to investigate the effects of a probiotic on the microbiota. Methods: We analyzed the intestinal microbiota and investigated the effects (before, during and after the ingestion period) on intestinal microbiota and the environment of 6 months of daily ingestion of a probiotic (Lactobacillus casei strain Shirota (LcS)-fermented milk). Results: We performed an open trial in 23 children (14 boys, 9 girls; age 7.7 ± 2.4 years (mean ± SD); BMI 19.6 ± 4.6). The composition of intestinal microbiota of healthy pre-school and school-age children resembled that of adults. During probiotic supplementation, the population levels of Bifidobacterium and total Lactobacillus increased significantly, while those of Enterobacteriaceae, Staphylococcus and Clostridium perfringens decreased significantly. A significant in-
© 2015 S. Karger AG, Basel 0250–6807/15/0674–0257$39.50/0 E-Mail [email protected] www.karger.com/anm
crease in fecal concentrations of organic acids and also a decrease in fecal pH were observed during the ingestion period. However, the patterns of fecal microbiota and intestinal environment were found to revert to the baseline levels (i.e. before ingestion) within 6 months following the cessation of probiotic intake. Conclusion: Regular intake of an LcScontaining probiotic product may modify the gut microbiota composition and intestinal environment in pre-school and school-age children while maintaining the homeostasis of the microbiota. © 2015 S. Karger AG, Basel
Introduction
The intestinal microbiota of a typical adult can weigh 1 kg or more. This ‘tissue’ comprises about 1014 microorganisms, meaning that the number of these microbes is almost 10 times greater than the total number of human body cells [1]. These gut microbes are believed to contribute significantly not only to host nutrition and metabolism but also to the developmental regulation of intestinal angiogenesis, protection from pathogens and developProf. Yuichiro Yamashiro Probiotics Research Laboratory, Juntendo University Graduate School of Medicine 3rd Floor, Hongo-Asakaze Bldg, 2-9-8 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan) E-Mail yamasiro @ juntendo.ac.jp
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 4/25/2016 11:16:44 PM
Key Words Fecal microbiota · Gut microbiota · Lactobacillus casei strain Shirota · Organic acids · Pre-school and school-age children · Probiotics
Preingestion
During ingestion
Post-ingestion
3 weeks
1 2 3 4 5 6 month months months months months months
6 months
LcS-fermented milk
Fig. 1. A schematic of the study design, Fecal sample
ment of immune functions [1]. The intestinal microbiome undergoes dynamic changes during different stages of host growth and development, with the most dramatic compositional changes believed to occur during infancy and childhood [2]. Probiotics are viable bacteria that exhibit a diverse range of beneficial effects on host health, due primarily to the impact of the bacteria on the improvement of gut microbial balance and the intestinal environment. Thus, orally administered probiotics are expected to be resistant to gastric acid, bile and proteolytic enzymes, permitting these bacteria to reach the intestinal tract in a viable form. Having reached the intestinal tract, these microorganisms are thought to modify the intestinal microbiota and subsequently contribute to the reduction of various disease risks [3, 4]. The probiotic approach is aimed primarily at repairing the perturbations of the gut microbiota composition (dysbiosis) and to restoring the resilience of the microbiota [5]. Over the last 2 decades, there has been a growing interest in the manipulation of intestinal microbiota with probiotics for the prevention and treatment of certain diseases. However, reports on the analysis of intestinal microbiota in pre-school and school-age children and the effects of probiotic supplementation on the microbiota in these children are sparse. In this context, the present study was designed to establish the baseline profile of intestinal microbiota in pre-school and schoolage children and, in addition, to investigate the effects of probiotic supplementation on the microbiota. Specifically, we investigated the effects of daily intake of probiotic Lactobacillus casei strain Shirota (LcS)-fermented milk on the fecal microbiota and intestinal environment of healthy pre-school and school-age Japanese children before, during and after 6 months of probiotic ingestion and after another 6 months following the termination of ingestion (the post-ingestion period). 258
Ann Nutr Metab 2015;67:257–266 DOI: 10.1159/000441066
Subjects and Methods Ethical Approval Information The trial was approved by the Clinical Research Ethics Committee of Juntendo University Hospital and was performed in compliance with the principles of the Helsinki Declaration (adopted in 1964; amended in 1975, 1983, 1989, 1996 and 2000), whereby a thorough explanation was given to the subjects’ parents or guardians prior to the trial regarding the purpose and methods of the study in order to obtain a written informed consent from the parents or guardians. Test Diet The test diet consisted of a commercially available fermented milk product (Yakult 400; Yakult Honsha Co., Ltd., Tokyo, Japan) containing LcS (YIT 9029) at 4 × 1010 cells per 80-ml bottle. LcSfermented milk was made from glucose/fructose/liquid sugar, skimmed milk and flavor ingredients. The test diet was offered as an 80-ml bottle containing 1.0 g protein, 0.1 g fat, 14.4 g carbohydrate and 15 mg sodium and providing 62 kcal. LcS was present in the product at a concentration of about 4 × 1010 viable LcS cells per bottle. The test diet was provided by Yakult Honsha Co., Ltd. Study Schedule and Ingestion of the Test Diet The trial was conducted as an open study, evaluating the parameters at defined time points during pre-ingestion, ingestion and post-ingestion periods (fig. 1). The subjects consented to dietary limitations for a minimum of 3 weeks prior to the start of the study; these limitations precluded the children from ingestion (during this interval) of any product that might contain LcS and other lactic acid bacteria or probiotics. Following this interval, the subjects were instructed to ingest 1 bottle of LcS-fermented milk per day for 6 months during the period from January to July, 2012. Examination Methods Fecal Bacteriologic Examination Fecal sampling: fresh fecal samples were obtained from the children on day 1 (i.e. before ingestion) on months 1, 3 and 6 of the ingestion period and at 6 months after the termination of ingestion (fig. 1). At each time point, for each child, approximately 1-gram samples of feces were collected separately into a sterile fecal collection tube (Sarstedt AG & Co., Numbrecht, Germany) containing 2 ml RNAlater (Ambion Inc., Austin, Tex., USA; for the
Wang/Nagata/Asahara/Yuki/Matsuda/ Tsuji/Takahashi/Nomoto/Yamashiro
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 4/25/2016 11:16:44 PM
showing the duration of the study, schedule of probiotic ingestion and the time points of sample collection (as indicated by gray arrows).
analysis of fecal microbiota), an RNA stabilization solution and into empty tubes (for organic acids analysis). The tubes were stored at 4 ° C (for the analysis of fecal microbiota) or at –20 ° C (for organic acids analysis) for about 36 h. The fecal samples were maintained in a cooling box with refrigerants and were immediately sent to the Yakult Central Institute where these samples were preserved in a –20 ° C freezer block until further processing. Primary treatment of fecal samples for fecal microbiota analysis: fecal samples were weighed and then suspended in 9 volumes of RNAlater (Ambion). After incubating for 10 min at room temperature, the fecal homogenate was added to 1 ml of sterilized phosphate buffer saline (pH 7.2), and then centrifuged at 5,000 g for 10 min. The supernatant was discarded, and the pellet was stored at –80 ° C pending use for the extraction of nucleic acids. Isolation of RNA and DNA: RNA [6, 7] and DNA [8] were extracted using the methods described in the respective references. The resulting nucleic acid fractions each were suspended in 1 ml of nuclease-free water (Ambion). Determination of bacterial count by reverse transcriptionquantitative polymerase chain reaction (RT-qPCR): a standard curve was generated using RT-qPCR data (using the quantification cycle, that is, the cycle number when threshold fluorescence was reached) and the corresponding cell count, which was determined microscopically with 4, 6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, Calif., USA) staining [9] for the dilution series of the standard strains described elsewhere [6, 7]. To determine the density of bacteria present in a given sample, each extracted RNA sample was subjected to 10-fold serial dilutions in nuclease-free water; dilutions were subjected to RT-qPCR; the quantification cycle values in the linear range of the assay were applied to a standard curve generated in the same experiment to obtain the bacterial cell count in each nucleic acid sample, and the results were converted to the number of bacteria per sample. The procedure was performed in triplicate for a given RNA sample. The specificity of the RT-qPCR assay using groupor species-specific primers was determined as described previously [6, 7]. The methods used for the detection of Clostridium difficile [10] and those for methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant coagulase-negative Staphylococcus (MRCNS), methicillin-sensitive S. aureus (MSSA) and methicillin-sensitive coagulase negative Staphylococcus (MSCNS) [11] have been described previously. The method for quantitative analysis of LcS has been described previously [8]. The lower detection limit of LcS was 5.7 log10 cells/g feces.
Measurements of the Fecal Organic Acid Concentrations and pH A portion of the homogenized feces was isolated, weighed, mixed with 0.15 M perchloric acid in a 4-fold volume and reacted at 4 ° C for 12 h. Next, the mixture was centrifuged at 4 ° C at 20,400 g for 10 min, and the supernatant was filtered through a 0.45-μm membrane filter (Millipore Japan, Tokyo) and sterilized. The concentrations of organic acids in the resulting samples were measured using a waters high-performance liquid chromatography system (Waters 432 Conductive Detector; Waters, USA) and a Shodex RSpack KC-811 column (Showa Denko, Tokyo) [12]. We calculated the concentrations of organic acids in the experimental specimens based on a standard curve. The standard curve was generated using a mixed solution standard consisting of 1–20 mM succinic acid, lactic acid, formic acid, acetic acid, propionic acid, iso
Statistical Analysis The results of pre, during and post ingestion were compared using the non-parametric Wilcoxon signed-rank test (fecal microbiota and fecal organic acids) and the parametric paired t test (fecal pH). The fecal bacteria detection rate was analyzed using the Fisher exact probability test. We used the IBM SPSS Statistics Desktop version 22.0 software (IBM Japan Ltd., Tokyo, Japan). In all tests, p < 0.05 was regarded as significant.
Effects of Probiotic on Healthy Children
Results
The Baseline Patterns of Intestinal Microbiota in Pre-School and School-Age Children Twenty-three healthy children (14 boys and 9 girls, age (mean ± SD) 7.7 ± 2.4 (range 4–12) years; BMI 19.6 ± 4.6), were recruited on the basis of no recent (i.e. during the 2 weeks preceding the commencement of the trial) history of long-term (≥1 week) antibiotic therapy or chronic inflammatory or viral illness, no current drug therapy, no known allergies and no participation in other clinical trials. The composition and profile of intestinal microbiota of healthy pre-school and school-age children (table 1) were found to be similar to those reported previously in healthy adults [7]. The Baseline Patterns of Fecal Organic Acids in Healthy Pre-School and School-Age Children The concentrations of fecal organic acids and fecal pH of healthy pre-school and school-age children (table 2) were found to be relatively stable and comparable to those of healthy adults reported previously [13]. Effects of Ingesting LcS-Fermented Milk on the Fecal Microbiota There was no significant difference in terms of the total number of bacteria in the fecal samples throughout the study period. The level of Bifidobacterium increased significantly at months 3 and 6 of the ingestion period when compared to the pre-ingestion levels (p < 0.05 and p < 0.01, respectively). The level of total Lactobacillus increased significantly at months 1, 3 and 6 of the ingestion period when compared to the pre-ingestion levels (p < 0.01). However, at 6 months post ingestion, the values of both Bifidobacterium and total Lactobacillus were found to return to their baseline levels and did not differ significantly from the pre-ingestion values. Ann Nutr Metab 2015;67:257–266 DOI: 10.1159/000441066
259
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 4/25/2016 11:16:44 PM
butyric acid, butyric acid, isovaleric acid and valeric acid. The fecal pH of each experimental specimen was measured by directly inserting the glass electrode of a D-51 pH meter (Horiba Seisakusho Co., Ltd., Tokyo) into the homogenized feces.
Table 1. The patterns of intestinal microbiota of healthy pre-school and primary school-age Japanese children Organisms
Total bacteria Obligate anaerobes Clostridium coccoides group C. leptum subgroup Bacteroides fragilis group Bifidobacterium Atopobium cluster Prevotella C. perfringens C. difficile Facultative anaerobes Total Lactobacillus L. gasseri subgroup L. brevis L. casei subgroup L. fermentum L. fructivorans L. plantarum subgroup L. reuteri subgroup L. ruminis subgroup L. sakei subgroup Enterobacteriaceae Enterococcus Staphylococcus MRSA MRCNS MSSA MSCNS Aerobes Pseudomonas
4–5 years (n = 6)
6–7 years (n = 5)
8–9 years (n = 7)
10–12 years (n = 5)
log10 cells/g fecesa
prevalence, %
log10 cells/g fecesa
prevalence, %
log10 cells/g fecesa
prevalence, %
log10 cells/g fecesa
prevalence, %
10.5±0.3
100
10.7±0.3
100
10.5±0.1
100
10.4±0.3
100
9.6±0.3 10.0±0.3 9.7±0.4 9.8±0.6 8.8±0.8 8.2 4.7±0.8 3.4
100 100 100 100 100 17 67 17
9.6±0.2 10.0±0.4 10.2±0.4 9.7±0.6 9.1±0.7 10.1 5.3±1.6
