3.1 Changes of rat body weights
Loperamide is known as an antagonist that activates the µ-opioid receptor. Activation of µ-opioid receptors in myenteric muscle induces blockage of intracellular calcium channels, which affects signaling pathways such as membrane hyperpolarization (Bohn and Raehal 2006). As a result, intestinal motility is reduced by inhibiting excitatory neurotransmission (Klein et al. 2013).
Loperamide administration causes decreased fecal water and dietary intake due to decreased intestinal peristalsis in rats, which results in constipation and weight loss of the specimen. The residence time of feces in the intestine becomes longer due to decreased intestinal movement, allowing fecal water more time to be absorbed into the body. However, microbial EPS has been shown to have significant water-holding capacity due to its hydrated polymer network mediating function (Zannini 2015). We, therefore, theorized that loperamide-induced clinical symptoms would be alleviated by EPS containing VP30-EPS or pEPS ingestion. The initial body weight, final body weight, body weight gain, food intake, and water intake for each rat group are shown in Table 3.
Table 3 Effects of VP30-EPS or pEPS on body weight, food intake, and water intake in normal and loperamide-treated SD rat groups
Group
|
Initial body weight
(g/rat)
|
Final body weight
(g/rat)
|
Body weight gain
(g/rat)
|
Food intake
(g/day)
|
Water intake
(g/day)
|
NC
|
180.40±8.56a
|
194.60±12.34a
|
12.20±4.09a
|
13.52±2.32a
|
26.80±8.77a
|
LP
|
178.60±3.91a
|
181.72±4.52ab
|
3.12±3.31b
|
11.21±1.54c
|
20.98±9.86a
|
LP+VP30-EPS
|
172.67±5.13a
|
178.67±11.02b
|
6.00±6.08b
|
12.58±1.42b
|
26.30±12.72a
|
LP+pEPS
|
180.50±7.19a
|
184.50±6.86ab
|
4.00±0.82b
|
11.30±1.46c
|
21.82±7.64a
|
a–c Mean values with different letters are significantly different (p < 0.05) according to Duncan’s multiple range test.
Compared to the loperamide-free normal group, all loperamide-administered groups showed significantly decreased food intake, which resulted in lower final body weights and body weight gains compared to the normal group (p<0.05). However, groups treated with VP30-EPS or pEPS showed increased body weight gain and water intake compared to the group treated with only loperamide. Shimotoyodomo et al. (Shimotoyodomo et al. 2000) reported that loperamide administration induced abdominal distension and decreased food intake in experimental animals. In addition, continuous administration of loperamide decreased large intestine peristalsis, thereby lowering fecal movement rates (Cepinskas et al. 1993). Therefore, the observed decrease in dietary intake due to continuous administration of loperamide is explained.
3.2. Effect of VP30-EPS or pEPS on the fecal weights, fecal water contents and number of feces of experimental animals
After administration of VP30-EPS or pEPS to the test groups for three days, changes in fecal weight, fecal water content and number of feces were evaluated. Loperamide lowers the moisture content of feces and causes constipation. Recently, the aquaporin family of cell membrane water channel proteins (AQPs) has been identified as playing an important role in the cellular water transport system (Adeoye et al. 2022). AQP3, AQP4, and AQP8 have been identified as the most important. Intestinal water transport is achieved by the activity of AQP3, and it is reported that this protein enhances water absorption by intestinal epithelial cells. In particular, it has been reported that in constipation caused by loperamide, the AQP3 level is reduced due to decreased AQP3-driven mRNA and protein expression (Yi et al. 2019). Therefore, the fecal water content and number of feces in the experimental groups can be used as biomarkers to compare and evaluate the degree of constipation relief for the experimental groups. Administration of loperamide significantly reduced the wet feces weight of rats (2.36 ± 0.22g/rat, p<0.05) (Table 4).
Table 4 Fecal parameters following oral administration with VP30-EPS or pEPS in loperamide-treated SD rats
Group
|
Wet fecal weight
(g/rat)
|
Dry fecal weight
(g/rat)
|
Fecal water content
(%)
|
NC
|
3.19 ± 0.44a
|
2.49 ± 0.40a
|
22.05 ± 3.15b
|
LP
|
2.36 ± 0.22b
|
2.12 ± 0.20a
|
9.95 ± 3.97c
|
LP+VP30-EPS
|
3.31 ± 0.42a
|
2.39 ± 0.37a
|
27.97 ± 4.47a
|
LP+pEPS
|
3.19 ± 0.34a
|
2.17 ± 0.31a
|
32.00 ± 5.86a
|
a–d Mean values with different letters are significantly different (p < 0.05) according to Duncan’s multiple range test.
However, when LP was administered to rats along with VP30-EPS or pEPS, the wet fecal weights were not different from that of the NC group (3.31 ± 0.42, 3.19 ± 0.34 and 3.19 ± 0.44 g/rat, respectively). These results showed that intestinal transit ratio of the normal, LP+VP30-EPS and LP+pEPS groups were significantly higher than that of the negative control group (LP group). There was no significant difference in dry fecal weight levels of all groups. The level of fecal water content of the normal, LP, LP+VP30-EPS and LP+pEPS groups were 22.05 ± 3.15%, 9.95 ± 3.97%, 27.97 ± 4.47%, and 32.00 ± 5.86%, respectively. In spite of LP treatment, it was observed that fecal water contents were significantly increased when VP30-EPS or pEPS was administered (p<0.05). Fecal pellet numbers after induced constipation in the LP, LP+VP30-EPS and LP+pEPS groups was significantly different from that of the normal control group (Fig. 1). The three-day averages of fecal pellet number of the LP+VP30-EPS and LP+pEPS groups were significantly higher than that of the LP group (p<0.05), although the fecal pellet numbers on Day 0 in all groups were similar (Fig. 1 A). The pattern of third day data (the last day of experiment) for all groups was consistent with the pattern of average feces numbers (Fig. 1 A and B). These results suggest that pEPS has a positive effect on stool frequency.
Zannini et al. reported that EPS produced by lactic acid bacteria attaches to intestinal mucosa to form an EPS biofilm, helping the adhesion and growth of lactic acid bacteria (Zannini 2015). In addition, it is reported that EPS produced by lactic acid bacteria exhibits strong hydrophilicity and has a high water-holding capacity that enables the survival of microorganisms in a dry intestinal environment (Zannini et al. 2015). We reported in our previous study that the pEPS exhibited significant hydrophilicity with strong water-holding capacity.
3.3. Effect of VP30-EPS or pEPS on the intestinal transit ratio
It is also known that the interstitial cells of Cajal (ICC), which regulate intestinal motility, are not differentiated, developed, or maintained by loperamide, and this phenomenon results in decreased intestinal motility, leading to constipation (Hao et al. 2019). Contraction and relaxation of intestinal smooth muscle cells are reduced by loperamide. The known gene markers related to the contraction and relaxation of intestinal smooth muscle cells are mAchR M2 and M3. Two types of markers are decreased by loperamide, and it is known that contraction and relaxation of intestinal smooth muscle cells affect the decrease. If intestinal peristalsis is reduced due to the administration of loperamide, the intestinal movement distance of feces is reduced, which leads to the accumulation of feces in the intestine.
We expected that supplying rats with VP30-EPS and/or pEPS, which have high water-holding capacity, would increase the moisture content of the rat's feces and reduce the transit time through the large intestine by softening the stool, thereby relieving constipation. For the calculation of intestinal transit ratio, the length of small and large intestines were measured. There was no significant difference in the volume of feces in the intestine or intestinal length in all groups (Table 5, Fig. 2).
Table 5 Effects of VP30-EPS or pEPS on transit ratio and intestine length in loperamide-treated SD rats
|
Transit ratio (%)
|
Small intestine length
(cm)
|
Large intestine length
(cm)
|
NC
|
73.80±4.71a
|
54.55±6.05b
|
10.75±0.53a
|
LP
|
62.10±5.13b
|
59.69±4.41b
|
10.80±1.70a
|
LP+VP30-EPS
|
70.18±4.19a
|
55.84±2.31ab
|
11.60±0.70a
|
LP+pEPS
|
68.52±2.62a
|
59.00±5.18a
|
12.34±0.35a
|
a, b Mean values with different letters are significantly different (p < 0.05) according to Duncan’s multiple range test.
In LP-treated rats, the transit ratio significantly increased when the rats were treated with VP30-EPS or pEPS. VP30-EPS or pEPS consumption increased the transit ratio to the level of the control group. Various research groups have reported a positive correlation between milk fermented by lactic acid bacteria and fecal transit ratios. Ge et al. reported that L. acidophilus and B. bifidum release neuro-messengers that promote intestinal motility (Ge et al. 2017). Milk fermented with B. lactis and B. animalis reduced constipation symptoms and colonic transit time in humans and animals, respectively (Agrawal et al. 2008; Bouvier et al. 2001). Marteau et al. also reported that regular intake of milk fermented with Bifidobacterium spp. has a positive effect on large intestine functionality (Marteau et al. 2002). NC and LP denote normal control and loperamide respectively. Various groups have reported that live active or inactivated lactic acid bacteria, or lactic acid bacterial EPS or microbial fermented foods can enhance in vivo host intestinal peristalsis (Table 6).
Table 6 Constipation improvement efficacy reporting case of probiotics or produced EPS
No
|
Species
|
Model
(in vitro/in vivo/clinical study)
|
Dosing level
(Stimulation reagent, experimental day)
|
Efficacy
|
Ref
|
1
|
B. bifidum G9-1
|
In vivo (Sprague-Dawley rat)
|
1.0 x 1010 CFU
(Loperamide, 4 days)
|
1. Fecal pellet number: 40 pellets/day (30 pellets/day of control)
2. Fecal water content: 60 % (50% of control)
|
Makzaki et al. 2021
|
2
|
Mixture of B. adolescentis CCFM626, CCFM667, CCFM669
|
In vivo (BALB/c mice)
|
1.0 x 1010 CFU
(Loperamide, 17 days)
|
1. Fecal weight: 1 g (0.5 g of control)
2. Fecal moisture: 60% (about 40% of control)
3. Intestinal transit ratio: 100% (30% of control)
|
Wang et al. 2017
|
3
|
L. plantarum NCU116
|
In vivo (Kunming mice)
|
1.0 x 109 CFU
(Loperamide, 15 days)
|
1. Fecal pellet number: 15.22 pellets (8.78 pellets of control)
2. Fecal pellet weight: 0.41 g (0.21 g of control)
3. Fecal moisture: 49.86% (34.65% of control)
4. Intestinal transit ratio: 73.83% (50.40% of control)
|
Li et al. 2015
|
4
|
L. plantarum CQPC02-fermented soybean milk
|
In vivo (Kunming mice)
|
2 mL
(Loperamide, 17 days)
|
1. Fecal weight: 0.83 g (0.45 g of control)
2. Fecal pellet number: 40 pellets (22 pellets of control)
3. Fecal moisture: 45% (20% of control)
4. Intestinal transit ratio: 80.5% (25.2% of control)
|
Yi et al. 2019
|
5
|
Mixture of L. rhamnosus CCFM1068, FFJND15-L2, FHeNJZ7-1, FTJDJ11-1, FZJHZ11-7
|
In vivo (BALB/c mice)
|
5 x 109 CFU
(Loperamide, 29 days)
|
1. Fecal moisture: 60% (40% of control)
2. Intestinal transit ratio: 60% (40% of control)
|
Wang et al. 2020
|
6
|
Heat-killed L. plantarum nF1
|
In vivo (Sprague-Dawley rat)
|
1.6 x 1011 CFU
(Loperamide, 5 weeks)
|
1. Fecal moisture: 90% (75% of control)
2. Intestinal transit ratio: 95% (55% of control)
|
Park et al. 2021
|
7
|
L. paracasei NTU101
|
In vivo (Sprague-Dawley rat)
|
2.3 x 1010 CFU
(Loperamide, 20 days)
|
1. Fecal pellet number: 67.43 pellets (44.83 pellets of control)
2. Fecal weight: 14.05 g (10.63 g of control)
3. Fecal moisture: 47.54% (36.50% of control)
4. Intestinal transit ratio: 62.57% (47.43% of control)
|
Chen et al. 2020
|
8
|
L. plantarum LRCC5193
|
In vivo (Sprague-Dawley rat)
|
2.5 x 1010 CFU
(Loperamide, 2 weeks)
|
1. Fecal weight: 0.4 g (0.25 g of control)
2. Fecal moisture: 40% (25% of control)
3. Intestinal transit ratio: 60% (35% of control)
|
Eor et al. 2019
|
9
|
Mixture of B. subtilis CBD-2, KMKW4
|
In vivo (BALB/c mice)
|
1.0 x 107 CFU
(Loperamide, 2 weeks)
|
1. Fecal pellet number: 15 pellets (10 pellets of control)
2. Fecal weight: 0.15 g (0.1 g of control)
3. Fecal moisture: 45% (35% of control)
4. Intestinal transit ratio: 50% (35% of control)
|
Kim et al. 2014
|
10
|
B. subtilis CBD-2-fermented SD-P2A2
|
In vivo (BALB/c mice)
|
1.0 x 104 CFU
(Loperamide, 2 weeks)
|
1. Fecal pellet number: 15 pellets
(10 pellets of control)
2. Fecal weight: 0.15 g (0.10 g of control)
3. Fecal moisture: 55% (45% of control)
4. Intestinal transit ratio: 50% (40% of control)
|
Kim et al. 2016
|
11
|
L. kefiranofaciens
|
In vivo (Sprague-Dawley rat)
|
300 mg/kg of EPS
(low-fiber diet, 57 days)
|
1. Fecal weight: 3 g (1g of control)
2. Fecal moisture: 55% (40% of control)
|
Maeda et al. 2004
|
Clinical study
|
Functional constipation patient
(4 weeks)
|
1. Stool frequency (before/after of administration): 2/5
2. Stool consistency (before/after of administration): 12/6 (hard); 6.5/12.5 (normal); 1/1 (Loose)
|
Turan et al. 2014
|
12
|
L. kefiranofaciens DN1
|
In vivo (BALB/c mice)
|
2.0 x 108 CFU
(2 weeks)
|
1. Fecal weight: 0.194 g (0.150 g of control)
2. Fecal moisture: 45.66% (28.99% of control)
|
Jeong et al. 2017
|
Specifically, Maeda et al. (2004) artificially induced constipation in Sprague-Dawley (SD) rats by treatment with a low-fiber diet and orally administered EPS isolated from L. kefiranofaciens at a dose of 100 or 300 mg/kg for 14 days. They reported that fecal moisture content and weight increased in a dose-dependent manner with bacterial EPS (Maeda et al. 2004). In a follow-up study they treated 20 patients with symptoms of chronic constipation for 4 weeks with purified EPS isolated from L. kefiranofaciens and observed the clinical effects. All 20 patients showed increased excretion frequency and decreased stool retention time (Turan et al. 2014). Jeong's group identified L. kefiranofaciens DN1 with the best EPS production ability among 22 L. kefiranofaciens strains (Jeong et al. 2017), and orally administered L. kefiranofaciens DN1 (2 Í 108 CFU) to Balb/c female mice. They reported that the fecal weight and moisture contents of experimental animals were increased by oral administration of L. kefiranofaciens DN1 (Jeong et al. 2017). The EPS yield of L. kefiranofaciens was 2.5 g/L, a value about 14 times lower than the EPS yield of VP30 (36.47 g/L). Although L. kefiranofaciens EPS has been shown to reduce constipation through various studies, the commercialization potential of L. kefiranofaciens EPS is limited due to low EPS production.
3.4. Blood analysis
The microbiological safety of VP30 was verified in our previous study but in vivo toxicity evaluation studies using VP30-EPS or pEPS extract were not conducted. Therefore, we conducted toxicity evaluations using ALT and AST to determine whether the intake of VP30-EPS or pEPS induces hepatotoxicity. Serum ALT was within the normal range in all group (Table 7).
Table 7 Effects of VP30-EPS or pEPS on ALT, AST in loperamide-treated SD rats
Group
|
ALT
(U/L)
|
AST
(U/L)
|
NC
|
31.00±9.37a
|
108.13±17.67b
|
LP
|
30.58±7.51a
|
130.40±13.83a
|
LP+VP30-EPS
|
25.90±6.15a
|
102.14±9.30b
|
LP+VP30 EPS
|
27.50±5.28a
|
94.14±11.41b
|
a–c Mean values with different letters are significantly different (p < 0.05) according to Duncan’s multiple range test.
Serum AST was statistically increased by LP treatment, but both VP30-EPS and pEPS maintained serum AST levels within the normal range. Based on these results, the intake of VP30-EPS and pEPS is considered harmless to humans. The intake of 107 to 1010 CFU/day of Bifidobacterium or Lactobacillus increased stool weight, stool moisture content, number of stools, and bowel movement distance. Most Bifidobacterium and Lactobacillus are known to produce EPS, but their productivity, chemical structures and molecular sizes differ (Kavitake et al. 2020).