Abstract
A dose-escalation study was conducted to find the effective dose of Lactococcus lactis subsp. cremoris FC for improving defecation in healthy subjects. Twenty-seven subjects were recruited and consecutively ingested a placebo and two dose levels of L. cremoris FC (dose level 1, 1 × 107 cfu; dose level 2, 2 × 107 cfu) capsules daily for two weeks. Frequency of defecation (times/week) was significantly increased by dose level 2, and stool volume (units/week) was significantly increased by dose level 1. This dose-escalation study elucidated that intake of at least 1 × 107 cfu L. cremoris FC improves defecation.
Keywords: Lactococcus lactis, probiotics, defecation, dose-escalation study
Probiotics are live microbial food supplements that have beneficial effects for the host by improving its microbial balance. The most widely used and studied probiotics are lactobacilli and bifidobacteria lactic acid bacteria (LAB), which have been isolated from human and animal intestinal tracts. However, relatively less is known about the probiotic activity of lactococci, as they have not been traditionally considered natural inhabitants of the human gastrointestinal tract [1]. However, several studies have shown the possibility of lactococci residing in the human or animal gastrointestinal tract, and Lactococcus lactis has been isolated from the human intestinal tract [2].
Lactococcus lactis subsp. cremoris (L. cremoris) FC is a LAB that was originally isolated from a fermented milk of the Caucasus mountain region [3]. Fermented milk made with L. cremoris FC has a viscous texture due to the production of exopolysaccharide as a metabolite. We previously reported the probiotic effects of fermented milk made with L. cremoris FC on defecation and intestinal microflora in healthy adults based on a double-blind placebo-controlled study [4, 5], and we detected L. cremoris FC in the feces of subjects up to 2 weeks after administration, suggesting that the bacteria can reach the intestines in a viable form [6]. Moreover, we previously reported that hard capsules containing L. cremoris FC affected defecation and intestinal microflora in healthy adults based on a double-blind placebo-controlled study [7]. This previous study was based on trials in which the effective dose was confirmed beforehand. Here, we describe a dose-escalation study performed prior to the previous study to find the effective dose of hard capsules.
The dietary supplements used in this study were hard capsules containing L. cremoris FC (dose level 1, 1 × 107 colony-forming units [cfu]; dose level 2, 2 × 107 cfu) and a placebo. The nutritional components of the capsules are shown in Table 1. Each daily dose comprised two capsules. The number of bacterial cells was measured at the start and end of testing. The dose level 1 capsules contained 1.0 ± 0.1 × 107 cfu (n=4) bacterial cells in two capsules at the start of the test and 9.4 ± 0.1 × 106 cfu (n=4) of bacterial cells in two capsules at the end of the test. The dose level 2 capsules contained 2.2 ± 0.2 × 107 cfu (n=4) of bacterial cells in two capsules at the start of the test and 2.0 ± 0.1 × 107 cfu (n=4) of bacterial cells in two capsules at the end of the test.
Table 1. Nutritional components of the test capsules.
| Placebo capsule | Dose level 1 test capsule | Dose level 2 test capsule | |
|---|---|---|---|
| Energy (kcal) | 1.8 | 1.8 | 1.4 |
| Protein (g) | 0.12 | 0.14 | 0.16 |
| Fat (g) | 0.01 | 0.01 | 0.02 |
| Carbohydrates (g) | 0.18 | 0.20 | 0.20 |
| Sodium (mg) | 2.0 | 2.0 | 2.0 |
| Calcium (mg) | 70 | 70 | 140 |
| L. cremoris FC (colony-forming units) | - | 1 × 107 | 2 × 107 |
Daily dose: two capsules.
This study was carried out in accordance with the Helsinki Declaration, and informed consent was obtained from all subjects. The study was conducted by KSO Corporation (Tokyo, Japan) at Cestlavie Shimbashi Clinic (Tokyo, Japan). Twenty-seven women (30–60 years old), each of whom reported a frequency of defecation of 3–5 times a week, were recruited for the study. The exclusion criteria were as follows: (i) regular ingestion of antiflatulents and laxatives; (ii) regular ingestion of drugs for treatment of chronic gastroenterological diseases; (iii) ingestion of antibiotics within 2 weeks before the study; (iv) not able to stop ingestion of other potentially interfering health foods (LAB, bifidobacteria, oligosaccharides, dietary fibers, sugar alcohol-rich foods, and others) during the study; (v) food allergy; (vi) history of gastroenterological surgery that would affect digestion and absorption; (vii) severe disease requiring emergency treatment, or severe complication; (viii) participation in another clinical trial or plan to do so after providing informed consent to participate in this study; (ix) pregnancy or nursing a baby, or plan to become pregnant during this study; (x) judgment of unsuitability by the principal doctor.
This study was performed using a single-blind dose-escalation design (Fig. 1). The subjects were not informed about the content of the test food. The experimental period included a pre-observation period (2 weeks), placebo intake period (2 weeks), dose level 1 intake period (2 weeks), and dose level 2 intake period (2 weeks). The subjects were administered two capsules daily during each intake period. All subjects were asked to record all other food intake such as meals, banned foods, supplements, health foods, drinks, alcohol, and others.
Fig. 1.
Schedule of the study.
During the study, the subjects recorded the number of times they defecated, stool volume, stool appearance, stool color and smell, and the feeling of defecation completeness daily. The stool volume was expressed based on the number of medium size chicken eggs by visual estimation. Stool appearance was recorded and defined in accordance with a six-point scale as 1, small lumps; 2, hard; 3, banana-like; 4, doughy; 5, pulpy; and 6, watery. Stool color was assessed with a three-point scale as 1, yellow-brown (DIC-240); 2, brown (DIC-308); and 3, blackish dark brown (DIC-311), according to a color sample book (Color Guide, 19th edition, Dainippon Ink and Chemical, Inc., Tokyo, Japan). Stool smell was evaluated with a five-point scale of 1, no smell at all; 2, weak smell; 3, normal; 4, smelly; and 5, very smelly. Sense of defecation completion was recorded with a three-point scale of 1, completely defecated; 2, normal; and 3, incompletely defecated.
For statistical analysis of defecation frequency, stool volume, and stool quality, the Wilcoxon signed-rank test was used to compare the placebo and test capsules intake periods. Differences in the ratios based on stool appearance were statistically evaluated using the χ2 test. All statistical analyses were performed with the JMP14.1 software (SAS Institute Japan, Tokyo, Japan). P values less than 0.05 were considered significant.
Table 2a shows the changes in defecation frequency and stool volume during the different phases of the study. Defecation days were increased in the dose level 1 and 2 intake period compared with those during the placebo period, although the differences were not significant. The frequency of defecation was significantly increased during the dose level 2 intake periods compared with that in the placebo intake period. Stool volume was significantly increased in the dose level 1 and 2 intake periods compared with that in the placebo intake period. However, of note, stool volume was determined according to the number of medium size chicken eggs based on visual estimation by each subject. Thus, there could have been a difference in the size of the units used to determine stool volumes with this method, and the data could have been collected objectively by measuring the weight of the stool.
Table 2. Changes in defecation frequency, stool volume, and stool quality.
| Pre-observation | Placebo | Dose level 1 | Dose level 2 | ||
|---|---|---|---|---|---|
| (a) Defecation frequency and stool volume | |||||
| Defecation days (days/week) | 3.70 ± 0.56 | 4.33 ± 0.97 | 4.52 ± 1.03 | 4.61 ± 1.15 | |
| Frequency of defecation (times/week) | 4.02 ± 0.66 | 4.76 ± 1.09 | 5.06 ± 1.25 | 5.41 ± 1.80* | |
| Stool volume (units/week) a | 6.92 ± 3.73 | 8.77 ± 6.32 | 9.56 ± 6.22* | 10.52 ± 6.90** | |
| (b) Stool quality | |||||
| Color | 2.11 ± 0.33 | 2.02 ± 0.34 | 1.98 ± 0.29 | 2.03 ± 0.23 | |
| Smell | 3.28 ± 0.80 | 3.15 ± 0.71 | 3.06 ± 0.70 | 3.10 ± 0.50 | |
| Sense of defecation completeness | 2.21 ± 0.32 | 1.98 ± 0.37 | 1.98 ± 0.43 | 1.95 ± 0.34 | |
Values are means ± SD.
a Units show the stool volume as the number of medium size chicken eggs by visual estimation.
The Wilcoxon signed-rank test revealed significant differences compared with placebo intake: *p<0.05; **p<0.01.
Figure 2 shows the changes in ratios based on stool appearance. There were no significant differences among capsule intake periods and the placebo intake period. However, there were significant changes in stool appearance assessed in the placebo (p<0.05), dose level 1 (p<0.01), and dose level 2 (p<0.001) intake periods compared with changes observed in the pre-intake period. Specifically, the proportion of “banana-like” stool increased, while the proportions of “small lumps” and “hard” stool decreased during capsule intake. There were few subjects who produced diarrhea stool, described as pulpy and watery, during the study (0.7% in the placebo period, 1.7% in dose level 1 period, and 1.6% in dose level 2 period). Intake of L. cremoris FC had no effect on stool color, stool smell, and sense of defecation completeness (Table 2b). Thus, the intake of test capsules tended to increase the proportion of soft stool and reduced that of hard stool, although there were no significant differences compared with the placebo intake period. We previously described that fermented milk containing 2 × 1010–2 × 1011 cfu L. cremoris FC significantly improved stool appearance [4, 5]. Therefore, differences in the dose or form of the test food could explain the lack of a significant effect obtained in the study, and thus further studies are needed to explore the reasons.
Fig. 2.
Changes in ratios of stool appearance.
The χ2 test revealed significant differences compared with the pre-observation period: *p<0.05; **p<0.01; ***p<0.001.
This dose-escalation study showed that intake of at least 1 × 107 cfu L. cremoris FC sufficiently improves defecation. These findings for the effective dose of L. cremoris FC were applied to a double-blind placebo-controlled study [7] in which a 2-week intake of capsules containing 1 × 107 cfu L. cremoris FC significantly increased defecation days per week, defecation times per week, and stool volumes. Moreover, the numbers of total bacteria and Lactobacillus in feces were significantly increased during the test diet intake period. In general, an increase in the number or percentage of lactobacilli or bifidobacteria is considered to increase the contents of organic acids, as metabolic products of the bacteria, which reduces the stool pH and suppresses the proliferation of harmful bacteria such as putrefactive bacteria [8, 9]. In one of our previous studies, intake of fermented milk containing L. cremoris FC increased the percentage of Lactobacillus and reduced that of Clostridium perfringens [5]. Therefore, it is likely that intake of the dietary supplement in the present study also changed the intestinal microbiota, contributing to the improvement of defecation frequency and stool conditions. We are currently conducting a clinical study using the same diet (ethics committee approval number: KE/FK/1001EC/2018, Gadjah Mada University, Indonesia), in which we will evaluate the effect on intestinal microflora and subsequent effects such as stool and organic acid production.
It is important to note the limitations of this study. We conducted a dose-escalation study to find the effective dose of an L. cremoris FC supplement. The study was performed using two bacterial doses, 1 × 107 cfu and 2 × 107 cfu, and it showed that at least 1 × 107 cfu bacteria improved defecation. To elucidate the minimum effective dose of L. cremoris FC that has an effect on defecation, further study using a lower dose of L. cremoris FC is needed. In addition, the difference between the two doses was small (ratio of dose level 1 to dose level 2, 1:2), and bacterial numbers were mostly indicated as log numbers; thus, the indicated doses could be just within the error range. In further studies, we should test using supplements with bigger dose differences.
Moreover, our study subjects included only healthy individuals and did not consider individuals with gastrointestinal issues. We previously reported that L. cremoris FC alleviates symptoms of colitis via suppression of inflammatory cytokines in mice induced by dextran sulfate sodium [10]. Further, the diversity of microflora in inflammatory bowel diseases is reduced due to loss of normal anaerobic bacteria belonging to Bacteroides, Eubacterium, and Lactobacillus species [11]. Therefore, further studies are required to determine whether the probiotic and anti-inflammatory effects of L. cremoris FC might make it beneficial for patients with inflammatory bowel disease.
Overall, the results of the present study demonstrate that intake of a supplement containing at least 1 × 107 cfu L. cremoris FC improves defecation frequency and stool volume. Thus, L. cremoris FC could be considered a probiotic that is beneficial for defecation in healthy adults.
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