Table 3.
Human intervention studies using fermented foods containing the probiotic Lacticaseibacillus casei Shirota (LcS).
| Food matrix | Probiotic | Administered daily amount and duration of treatment | Food intake evaluation | Study design | Subjects main characteristics | Effects on gut microbiota | Gut microbiota analysis methods and microbial groups analyzed | Colonization evaluation | Persistence evaluation | Methods of analysis for colonization | References |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Fermented milk | Lacticaseibacillus casei Shirota (LcS) | 80 g containing 3 × 1010 CFU for 4 weeks | No | Cross-over trial, with 4 weeks washout (n = 35) | Malaysian children, 16 normal weight and 19 overweight (17 males and 18 females); age range 7–10 years | Increased Bacteroides ovatus among the normal weight cohort. Increased Lachnospira and Ruminococcus in the overweight participants | 16S rRNA based metagenomics: V3-V4 variable regions of 16S rRNA gene (All bacteria) | No | No | – | (31) |
| Fermented milk (Control: acidified milk) | LcS | 130 g containing 1.3 × 1011 CFU for 3 weeks | No | Double-blind placebo-controlled trial (n = 20, 10 treated and 10 placebo) with 1 week post-intervention washout | English healthy adults; age range 23–70 years | Increased lactobacilli, while no effect on total bacteria, Bacteroides spp., Eubacterium rectale–C. histolyticum subgroup, Atopobium rimae–Collinsella–Eggerthella lenta subgroup, and C. perfringens/histolyticum subgroup or E. coli was observed. A stable and relatively high population of LcS (between 6.7 and 7.1 Log10 CFU/g feces) was maintained in volunteers during the intervention period. LcS persisted in six volunteers until day 28 at 105 CFU/g feces | Fluorescent in situ hybridization (FISH) with molecular probes targeting total bacteria, bifidobacteria, Bacteroides, Clostridia (Clostridium perfringens/ histolyticum sub-group), Eubacterium rectale-C. histolyticum sub-group, Atopobium rimae-Collinsella-Eggerrthella lenta sub-group, Lactobacillus/Enterococcus spp. and E. coli |
Yes | Yes | LcS was enumerated using Lactitol-LBS Vancomycin (LLV) agar. The identity of putative LcS isolates was confirmed by PFGE | (32) |
| Fermented milk (Control: milk drink without bacteria, adjusted to match the LcS fermented milk) | LcS (YIT9029 strain) | 80 g containing 4 × 1010 CFU for 2 months | No | Before/ After trial, with 2 weeks pre-intervention washout (n = 10) |
Japanese elderly frail subjects (3 males and 7 females); age range 73–93 years | No significant increase of total number bacteria, significant increase of bifidobacteria, total lactobacilli and L.a casei subgroup, significant decrease of Enterobacteriaceae and Pseudomonas. LcS detected in all subjects during ingestion (bacterial count of fecal samples: 102-108 CFU/g) | qRT-PCR using group or species-specific primers (Clostridium leptum; C. coccoides; Bacteroides fragilis; Bifidobacterium; Atopobium cluster; Prevotella; C. perifringens; C. difficile; L. acidophilus; L.b brevis; L.a casei; L. fermentum; L. fructivorans; L. plantarum; L. reuteri; L. ruminis; L. sakei; Enterobacteriaceae; Enterococcus; Staphylococcus; Pseudomonas) | Yes | No | qPCR using LcS strain-specific primers | (33) |
| Fermented milk | LcS (YIT9029 strain) | 80 g containing 4 × 1010 CFU for 6 months | No | Randomized placebo-controlled double-blind trial, with 3 weeks pre-intervention washout (n = 72, 36 treated and 36 placebo; n = 20, 10 treated and 10 placebo) | Japanese elderly subjects (19 males and 53 females); age range 72–93 years; staff members (5 males and 15 females); age range (26–49 years) | After 1, 3, and 6 months of ingestion, significant increase of Bifidobacterium in the LcS-fermented milk group. Cell number of C. difficile, C. perfringens, Enterobacteriaceae lower than placebo. LcS recovered from feces of all subjects at 108 CFU/g during the ingestion period | qRT-PCR (Bifidobacterium, Lactobacillus, C. difficile, C. perfringens, Enterobacteriaceae, Staphylococcus, Pseudomonas); YIF-SCAN | Yes | No | qPCR using LcS strain-specific primers | (34) |
| Fermented milk | LcS | 80 g containing 4 × 1010 CFU for 16 weeks | No | Randomized controlled trial (n = 68, 34 treated and 34 control) | Japanese type 2 diabetes patients with stable glycemic control (49 males and 19 females); age range 30–79 years | Total Lactobacillus and L.a casei, L. gasseri, L. reuteri subgroups significantly increased. At the end of the study the fecal counts of the Clostridium coccoides group and C. leptum subgroup significantly higher in the intervention group | RT-qPCR; qPCR; YIF-SCAN | No | No | – | (35) |
| Fermented milk | LcS | 80 g containing 1 × 1010 CFU for 2 weeks | During the study, daily food intake, concomitant medications, and bowel movement were recorded | Before/After trial, with 2 weeks pre-intervention washout and 2 weeks post-intervention washout (n = 21) | Chinese healthy young adults (14 females and 7 males); age range 18–25 years | No effect on gut microbiota composition. LcS detected in all the subjects during the ingestion period (about 107 CFU/g feces); LcS was detected in only 5 subjects at the end of the 14 days post-intervention washout | 16S rRNA based metagenomics: V3–V4 variable regions of 16S rRNA gene (All bacteria) | Yes | Yes | ELISA strain-specific | (36) |
| Fermented milk Yakult | LcS | 100 g containing 1 × 1010 CFU for 4 weeks | No | Before/After trial, with 2 weeks pre-intervention washout and 2 weeks post-intervention washout (n = 62) | Chinese healthy adults (3 groups with different constipation levels); (54 females and 8 males); age range 18–45 years | LcS intervention increased the Pseudobutyrivibrio and Roseburia abundances in HS (hard stool) and decreased the Pseudobutyrivibrio abundance in SS (Soft stool) | 16S rRNA based metagenomics: V3–V4 variable regions of 16S rRNA gene (All bacteria) | No | No | – | (37) |
| Fermented milk | LcS | 65 g containing 6.5 × 109 CFU for 6 weeks | No | Controlled trial (n = 18, 6 treated and 12 control) | Dutch healthy children (6 males and 12 females); age range 12–18 years | No effect on microbiota composition | Intergenic Spacer Profiling (IS-pro) (All bacteria) | No | No | – | (38) |
| Fermented milk (Control: acidified milk) | LcS (YIT9029 strain) | 65 g containing 1.5 × 1010 CFU for 6 months | No | Randomized, double-blind, placebo-controlled parallel trial (n = 68, 35 treatment and 33 placebo) | Japanese elderly subjects (57 females and 11 males); mean age 86.3 ± 7.8 years | Significant increase of Bifidobacterium. Enterobacteriaceae numbers at the end of ingestion period in both groups were significantly lower than those before the ingestion period, but when the changes between two periods were compared, there were no significant differences between the two groups. No differences in Clostridium levels | RT-qPCR for Bifidobacterium, Enterobacteriaceae, Clostridium perfringens | No | No | – | (39) |
| Fermented milk Yakult (Control: acidified milk) | LcS (YIT9029 strain) | 100 g containing 1 × 1011 CFU for 8 weeks | Daily consumption self-recorded in a diary to check the compliance rate of consumption. Consumption of other fermented milk, yogurt, lactic acid bacteria beverages and probiotic/prebiotic products had to be avoided | Randomized, double-blind, placebo-controlled parallel trial, with 2 weeks pre-intervention washout and 2 weeks post-intervention washout (n = 48, 23 treated and 24 control) | Japanese healthy young adults (25 males and 22 females); average age 22.8 ± 0.3 years | Significantly higher numbers of LcS species and significant lower Bacteroidaceae in supplemented subjects. LcS detected during the ingestion period but not during post-supplementation period | 16S rRNA based metagenomics: V1–V2 variable regions of 16S rRNA gene (All bacteria) | Yes | Yes | Cultivation on Lactitol-LBS Vancomycin (LLV); colony identification through PCR using LcS specific set of primers | (40) |
| Fermented milk Yakult | LcS | 100 g containing 1 × 1010 CFU for 14 days | Subjects filled-in a diary on a daily basis on food intake. They were asked to avoid fermented dairy products and eat their habitual diet | Before/After trial, with 14 days pre-intervention washout baseline and 14 days post-intervention washout (n = 25) | Chinese healthy adults with BMI 18.5–29.9; (9 males and 16 females); age range 20–40 years | Eleven species had positive correlation with LcS, including Anaerostipes sp., Bifidobacterium adolescentis, Bacteroides uniformis and eight uncultured bacterial strains. Fourteen species had negative correlation with LcS, including Roseburia hominis, R. intestinalis, Clostridium sp., Bacteroides, Lachnospiraceae and Blautia wexlerae, and eight uncultured bacterial strains. LcS was able to survive in the gut but not to persist after supplementation | DGGE (universal bacteria) and sequencing of selected excised bands | Yes | Yes | Culture method combined with ELISA. | (41) |
| Fermented milk Yakult | LcS (YIT9029 strain) | 80 g containing 4 × 1010 CFU for 6 months | Not evaluated, but subjects were asked to abstain from ingestion of any product that might contain LcS, other lactic bacteria or probiotics for 3 weeks before the treatment | Before/After trial, with 3 weeks pre-intervention washout and 6 months follow-up (n = 23) | Japanese healthy children (14 males and 9 females); age range 4–12 years | During probiotic supplementation, the population levels of Bifidobacterium and total Lactobacillus increased significantly, while those of Enterobacteriaceae, Staphylococcus and Clostridium perfringens decreased significantly. The patterns of fecal microbiota and intestinal environment were found to revert to the baseline levels within 6 months following the cessation of probiotic intake. LcS was detected during the ingestion period but not at 6 months post supplementation | RT-qPCR for Total bacteria; Clostridium coccoides group; C. leptum subgroup; Bacteroides fragilis group; Bifidobacterium, Atopobium cluster; Prevotella; C. perfringens; C. difficile; Lactobacillus; L. gasseri subgroup; L.b brevis; L.a 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 | Yes | Yes | qPCR using LcS strain-specific primers | (42) |
| Fermented milk (Control: milk drink without bacteria, adjusted to match the LcS fermented milk) | LcS | 80 g containing 4 × 1010 CFU for 4 weeks | Subjects filled in a diary of food consumption. They were asked not to consume any other fermented food or oligosaccharide containing food during the survey period | Randomized, double-blind, placebo-controlled trial, with 2 weeks pre-intervention washout and 2 weeks follow up (n = 118, 67 treated and 67 control) | Japanese gastrectomized subjects (42 men and 25 women in each group); mean age 63 ± 11 years | Decreased fecal Staphylococcus level. LcS did not colonize the gut | RT-qPCR for total bacteria, Clostridium coccoides group; C. leptum subgroup; Bacteroides fragilis group; Bifidobacterium; Atopobium cluster; Prevotella, C. perfringens; total Lactobacillus, Enterobacteriaceae, Enterococcus, Staphylococcus and LcS | Yes | Yes | qPCR using LcS strain-specific primers | (43) |
a
Recently renamed Lacticaseibacillus.
b
Recently renamed Levilactobacillus.