Lactobacillus plantarum TWK10 relieves loperamide-induced constipation in rats fed a high-fat diet via modulating enteric neurotransmitters, short-chain fatty acids and gut microbiota

Abstract

Obesity and constipation can alter the intestinal microbiota composition, affecting intestinal barrier integrity, immune function, and metabolic processes. Numerous studies have suggested that Lactobacillus and Bifidobacterium could increase short-chain fatty acid (SCFA) production, thus improving the intestinal microbiota composition, mitigating obesity, and protecting the gastrointestinal tract. Therefore, in this study, we aimed to investigate the impact of Lactobacillus plantarum TWK10 (TWK10) on gut dysbiosis, obesity, and constipation induced by a high-fat diet and loperamide. Over a 5-week experimental period, rats were fed a high-fat diet and subsequently induced with gut dysbiosis and constipation using loperamide. Concurrently, rats were administered different doses of TWK10 or TWK10-fermented soy milk. Following administration of TWK10 or its fermented soy milk, the expression of adipocyte transcription factors such as PPARγ, C/EBPα, and C/EBPβ proteins and adipocyte size were significantly downregulated (p < 0.05). Regarding intestinal motility, compared to the high-fat diet-induced obesity and loperamide-induced constipation group (L), rats receiving TWK10 or its fermented soy milk exhibited regulation of gastrointestinal hormone levels such as gastrin (GT), somatostatin (Sst), calcitonin gene-related peptide (CGRP), and acetylcholinesterase (Ache) in serum. Additionally, there was a notable increase in the intestinal transit ratio, particularly in the 1X TWK10 group, in which it increased by 10.29% (p < 0.05). Furthermore, the consumption of TWK10 or its fermented soy milk significantly increased the number of goblet cells, as well as the thickness of the muscle and mucosal layers in the colon (p < 0.05). Analysis of SCFA content in fecal samples revealed a significant increase in SCFA concentrations, particularly acetic acid, following administration of both TWK10 and its fermented soy milk. Finally, TWK10 was found to modulate the composition of the intestinal microbiota and increase microbial diversity. In conclusion, TWK10 and its fermented soy milk effectively alleviated loperamide-induced constipation in rats fed a high-fat diet. These findings suggest that TWK10 and its fermented soy milk may potentially be functional foods for promoting intestinal health.

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1. Introduction

Obesity is a serious public health problem caused by biological, developmental, genetic, environmental, and behavioral factors.¹ Obesity is strongly associated with changes in the motility of the gastrointestinal tract, including the esophagus, stomach, small intestine, and colon, which can cause digestive disorders such as constipation.^2,3 Intestinal constipation is a disorder of the gastrointestinal tract defined as unsatisfactory bowel movements characterized by difficulty in defecation, low frequency of bowel movements, painful bowel movements, hard stools, or feeling of incomplete bowel movement.⁴ Environmental and behavioral factors appear to play important roles in the etiology of constipation. For example, high-fat meals induce a strong postprandial colonic response in healthy individuals and stimulate retrograde phasic contractions, which may prolong colon transit time.⁵ Mukai et al. showed that high-fat diet (HFD)-fed mice had reduced fecal pellet numbers and prolonged colon transit time, and that the colonic malondialdehyde level was higher in the HFD group than in the normal diet group. Overall, HFD causes constipation via a reduction in colonic mucus and the oxidative stress-dependent mechanism.^6,7 HFD is directly associated with intestinal microbiota dysbiosis, which can cause metabolic disorders and increase appetite, leading to obesity. Moreover, impaired intestinal redox homeostasis and integrity further induces metabolic inflammation.⁸ Loperamide is a prevalent medication for treating diarrhea in humans and animals that acts on μ-opioid receptors in the large intestine, decreasing intestinal peristaltic activity and increasing the absorption of fluids. Loperamide is also used to study bowel dysfunction and constipation in animal models, including rats, mice, and zebrafish, generating a relevant model of irritable bowel syndrome or opioid-induced bowel dysfunction disorder.⁹

Generally, a healthy gut microbiota is highly diverse and produces several active substances, including short-chain fatty acids (SCFAs), vitamins, and anti-inflammatory and antioxidant products. In contrast, low gut microbiota diversity is associated with diseases such as obesity, indicating that changes in the composition of the gut microbiota affect nutrient absorption and energy metabolism.⁸ SCFAs are an important class of gut microbiota bioproducts produced mainly from the fermentation of non-digestible carbohydrates. SCFAs have fewer than six carbon atoms and mainly include acetate, propionate, butyrate, and pentanoate. Acetate, propionate, and butyrate account for 90% of SCFAs produced by the gut microbiota. Recently, the role of SCFAs in energy and lipid metabolism has attracted attention as a potential regulator of metabolic syndrome.¹⁰ Notably, clinical studies have shown that SCFAs may ameliorate obesity.¹¹ Therefore, it has been hypothesized that SCFAs play a key role in the prevention and treatment of metabolic syndromes.

Soymilk, a soybean product, is a popular non-dairy milk alternative because of its improved functional and nutritional properties and potential health benefits. Recently, fermentation has gained attention as a novel processing approach to improve the nutrient bioavailability, oxidation resistance, and flavor attributes of soymilk products.¹² Fermented soymilk has attracted attention in food research because of its antioxidant, anti-inflammatory, anti-obesity, and immunomodulatory effects, as well as its ability to promote intestinal peristalsis.¹³ Fermented soymilk prepared using special lactic acid bacteria (LAB), such as Lactobacillus paracasei subsp. paracasei NTU 101, shows anti-obesity effects by reducing adipogenesis, which may be attributed to the conversion of isoflavones into daidzein and genistein.¹⁴ Additionally, L. plantarum promoted lipid metabolism, increased SCFA content in the colon, and reversed HFD-induced intestinal flora disorder.¹⁵ Additionally, L. acidophilus LA-5 and Bifidobacterium longum BB-46 altered the gut microbiota of individuals with obesity and improved the fat profile.¹⁶ Moreover, L. plantarum CQPC02 partially reversed loperamide-induced constipation in mice by increasing motilin, gastrin, endothelin, and acetylcholinesterase levels and decreasing oxidative stress. Furthermore, L. plantarum CQPC02 increased Bacteroides and Akkermansia abundance and decreased Firmicutes abundance and the Firmicutes/Bacteroides ratio in the feces of mice with constipation.¹⁷

Currently, two types of gut dysbiosis induction methods have been used in in vivo studies: (1) HFD to induce dietary-based gut dysbiosis and (2) loperamide to slow gastrointestinal motility, induce constipation, and cause gut dysbiosis.¹⁸ However, the effects of LAB-fermented soymilk on loperamide- and HFD-induced obesity, constipation, and gut dysbiosis remain unknown. Therefore, in this study, we aimed to investigate the effects and potential molecular mechanisms of Lactobacillus plantarum TWK10 (TWK10) on HFD- and loperamide-induced gut dysbiosis, obesity, and constipation in Sprague–Dawley (SD) rats. We examined adipocyte accumulation, gastrointestinal hormone levels, fecal SCFA content, and microbiota diversity to elucidate the molecular mechanisms of TWK10.

2. Materials and methods

2.1. Preparation of fermented soymilk

L. plantarum TWK10 was isolated from Taiwanese fermented cabbage and cultured in Lactobacilli de Man, Rogosa and Sharpe (MRS) broth for 24 h at 37 °C. It was deposited in the depository at the Bioresource Collection and Research Center, Food Industry Research and Development Institute (HsinChu, Taiwan), and was given the accession number BCRC 910734. The TWK10 powder was obtained from Synbio Tech Inc. (Kaohsiung, Taiwan). No sugar-added soymilk (Chuan Gui Bio-Organic Co., Taoyuan, Taiwan) and TWK10 were used as intervention materials. The soymilk was heated in a water bath at 90 °C for 1 h and cooled immediately to 37 °C.¹⁸ The TWK10 strain was cultured in MRS medium. The reinoculation solution of MRS broth with 1% LAB was inoculated into 1 L soymilk. Subsequently, the mixture was incubated at 37 °C for 48 h under anaerobic conditions. After fermentation, the soymilk was freeze-dried (FD24, Kingmech Co., Taipei, Taiwan) to obtain dry powders. The dry soymilk powder was ground and stored at −20 °C.

2.2. Animal experimental design

The experiment was approved by the Animal Care and Research Ethics Committee of the Fu Jen Catholic University (IACUC approved number A10931). Eighty 6-week-old male Sprague–Dawley rats were purchased from BioLASCO Taiwan Co., Ltd. Animals were individually housed in stainless steel screen-bottomed cages at 25 °C, with a relative humidity of 60% and under a 12 h light/dark cycle. Rats were allowed free access to standard laboratory chow (Ralston Purina, St Louis, MO, USA) and water. After one week of acclimation, a five-week feeding trial was conducted. The rats were randomly divided into 2 basic feeding regime groups: one group was given the laboratory diet, with a physiological fuel value consisting of 15.56% of calories derived from fat. The other feeding groups were given a HFD that had a physiological fuel value of 5.30 kcal g⁻¹, providing 54.36% of calories from fat due to the high content of lard. Table 1 lists the experimental group diet ingredients. Consumption of HFD was assessed each day over a 5-week period. Diets were modified versions of the D12266B formulation from Research Diets, Inc. used previously to induce obesity in rats. Fig. 1 depicts the overall experimental scheme. The rats were randomly divided into 10 groups (8 rats each) as follows:

1. Normal control (NC) group: the rats were fed a laboratory diet throughout the course of the experiment.

2. Loperamide control (LC) group: the rats were fed a laboratory diet for 2 weeks, followed by injection with loperamide (2 mg per kg bw twice a day). The animals were maintained on a laboratory diet for the rest of the experimental period.

3. Loperamide (L) group: the rats were fed a HFD for 2 weeks, followed by injection with loperamide (2 mg per kg bw twice a day). The animals were maintained on a HFD for the rest of the experimental period (HFD/L).

4. Positive control (PC) group: HFD/L rats that received bisacodyl, 3.30 mg per kg bw per day for 3 weeks.

5. TWK10-1 (T1) dose group: HFD/L rats that received TWK10 powder, 7.60 × 10⁸ CFU per kg bw per day for 3 weeks.

6. TWK10-2 (T2) dose group: HFD/L rats that received TWK10 powder, 1.52 × 10⁹ CFU per kg bw per day for 3 weeks.

7. TWK10-4 (T3) dose group: HFD/L rats that received TWK10 powder, 3.04 × 10⁹ CFU per kg bw per day for 3 weeks.

8. TWK10 fermented soymilk-1 (F1) dose group: HFD/L rats that received TWK10-fermented soymilk powder, 0.70 g per kg bw per day for 3 weeks.

9. TWK10 fermented soymilk-2 (F2) dose group: HFD/L rats that received TWK10-fermented soymilk powder, 1.40 g per kg bw per day for 3 weeks.

10. TWK10 fermented soymilk-4 (F4) dose group: HFD/L rats that received TWK10-fermented soymilk powder, 2.80 g per kg bw per day for 3 weeks.

Fig. 1 The schedule of the animal experiments.

Table 1 The groups and sample dosages of the experimental animals

Group	Sample dosage	Constipation induced (loperamide – 2 mg per kg bw twice a day)	High fat diet	Normal diet
Control (NC)	Sterile water	—	—	+
Loperamide-control (LC)	Sterile water	+	—	+
Loperamide (L)	Sterile water	+	+	—
Positive control (PC)	Bisacodyl – 3.30 mg per kg bw per day	+	+	—
TWK10-1X (T1)	TWK10 powder – 7.60 × 10^8 CFU per kg bw per day	+	+	—
TWK10-2X (T2)	TWK10 powder – 1.52 × 10^9 CFU per kg bw per day	+	+	—
TWK10-4X (T4)	TWK10 powder – 3.04 × 10^9 CFU per kg bw per day	+	+	—
Fermented soy milk-1X (F1)	TWK10-fermented soy milk powder – 0.70 g per kg bw per day	+	+	—
Fermented soy milk-2X (F2)	TWK10-fermented soy milk powder – 1.40 g per kg bw per day	+	+	—
Fermented soy milk-4X (F4)	TWK10-fermented soy milk powder – 2.80 g per kg bw per day	+	+	—

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The TWK10 powder and TWK10 fermented soymilk powder were diluted with sterile normal saline and were orally administered with 1 mL of a diluted solution at different doses, and then the rats in the NC or LC groups were orally administered with 1 mL of sterile normal saline. The rats were first fed a HFD, and then 2 weeks later, loperamide was injected and they were also fed a HFD throughout the experiment (weeks 2–5), which mimics unbalanced dietary habits and gut dysmotility in humans. At the end of the experimental period, the rats were fasted overnight and sacrificed by CO₂ asphyxiation. Blood samples were centrifuged at 3000g for 10 min at 4 °C and stored at −80 °C until analysis. The epididymal fat pad, cecum and feces were immediately stored at −80 °C until analysis.

2.3. Estimation of feed intake, body weight gain, and feed efficiency ratio

The daily feed intake of each rat was recorded and calculated based on the average daily intake during the experimental period (5 weeks). Body weight gain was determined by the difference between the initial and the end body weight during the experimental period. The feed efficiency ratio was estimated using the following equation: grams of body weight gain per grams of total food intake × 100%.

2.4. Determination of body fat

There are two major compartments of abdominal fat. One is the subcutaneous fat pad, which is present between the skin and the abdominal wall – the fat around the groin and lumbar region. The other is the visceral fat pad, which surrounds the abdominal organs. Mesenteric, epididymal, and perinephric fat present the sum of total visceral fat mass.¹⁹ The percentage of total body fat in each rat was calculated using the following formula: grams of the subcutaneous and visceral fat mass per final body weight × 100%.

2.5. Estimation of fecal weight

The rat fecal pellets were collected from individual cages once daily on the experimental days 0, 28, and 35. The fecal pellets were counted and then dried in an oven at 65 °C for 16–24 hours, and then weighed. The fecal water content was calculated according to the following equation: fecal water content (%) = {[wet weight (g) − dry weight (g)]/wet weight (g)} × 100%.²⁰

2.6. Measurement of the intestinal transit time

A charcoal meal test is widely used for the measurement of intestinal transit time in rodents. On week 5, all experimental rats were fasted for 12 h and then fed the sample. After 10 min, 1 mL of a suspension containing 10% active charcoal in 5% carboxymethyl cellulose was orally administered as a marker; 30 min later, the animals were sacrificed to measure the length of the intestine and the distance traveled by the charcoal. The intestinal transit time was calculated according to the following equation: intestinal transit ratio (%) = the distance traveled by the activated carbon (cm)/the length of the intestine (cm) × 100%.²¹

2.7. Measurement of SCFAs in feces

The concentrations of SCFAs (acetic, propionic, and butyric acids) in feces were analyzed by gas chromatography. On experimental day 35, fecal pellets from each rat were collected into a centrifuge tube after extraction and weighed. 1 mL of 1 N HCl was added to 0.5 g of feces in a centrifuge tube. Subsequently, 1 mL of diethyl ether was added and vortexed, and the diethyl ether was collected from the centrifuge tube by centrifugation (4000g for 5 min, 4 °C). 150 μL of diethyl ether extraction solution was applied to a gas chromatograph (Shimadzu GC-2010 Plus, Shimadzu Scientific Instruments, Co., Ltd, Kyoto, Japan) equipped with a flame ionization detector and Zebron ZB-WAXPLUS GC column (30 m × 0.32 mm × 1.0 μm). The operation conditions were as follows: the temperature of the column oven was set at 120 °C for 1 min, and at 200 °C; thereafter, the temperature of the injection port and detector was set at 250 °C, and the flow rate of the He gas was 1 mL min⁻¹. The concentrations of the SCFAs were calculated using a standard curve drawn with standard acetic, propionic, and butyric acids. A 400 mmol L⁻¹ 2-methylvaleric acid solution was used as the internal standard.^22,23 All standard solutions were stored in glass vials for a maximum of 6 months at 4 °C.

2.8. Western blot analysis

Protein expression levels of PPARγ, C/EBPα, and C/EBPβ in the epididymal fat sample were determined by western blotting. The epididymal fat was immediately placed in liquid nitrogen and then stored in a refrigerator at −80 °C. 0.5 g of epididymal fat protein was extracted using 500 μL of tissue protein lysate. Protein concentration was determined using a BCA protein quantification kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. The protein (40 μg) was subjected to 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for 3 h, and then transferred to polyvinylidene difluoride (PVDF) membranes for 1.5 h at 100 volts at 4 °C; then, the membranes were blocked with 3% bovine serum albumin in Tris-buffered Tween 20 for 1 h and then incubated overnight with a primary antibody at 4 °C. The following day, PVDF membranes were immersed in a secondary antibody (1 : 25 000) for 1 h. Finally, protein bands were visualized using an ECL working solution, and band intensity was quantified using a Gel-Pro analyzer (V4.0, Media Cybernetic Inc., Rockville, MD, USA).³ In this study, primary antibodies were used as follows: anti-actin (1 : 5000) (Chemicon International Inc., Temecula, CA, USA), anti-PPARγ (1 : 1000) (Cayman Chemical Co., Ann Arbor, MI, USA), and anti-C/EBPα and anti-C/EBPβ (1 : 1000) (Cell Signaling Technology Inc., Boston, MA, USA).

2.9. Measurement of gastrin (GT), somatostatin (Sst), calcitonin gene related peptide (CGRP), and acetylcholinesterase (Ache)

The concentrations of GT, Sst, CGRP, and Ache were quantified using an ELISA kit (Taiclone Biotech Corp., Taipei, Taiwan) in accordance with the manufacturer's protocols.

2.10. 16S rDNA sequencing of the gut microbiota

The method was modified according to a previous study.²⁴ To extract microbial genomic DNA from the intestines of rats subjected to different treatments, fresh ceca were collected from rats in a sterilized tube containing CO₂, immediately frozen upon collection, and stored in a refrigerator at −80 °C until analysis. The total genomic DNA of microorganisms contained in the ceca was extracted using a QIAamp Fast DNA Stool Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions, and the changes in the intestinal microorganisms were analyzed using a 16S rRNA gene pyrosequencing method.²⁴ DNA quality was assessed using 260/280 nm absorption ratios >1.8.

2.11. Statistical analysis

Each experimental group consisted of eight animals. Data are presented as means ± standard deviation. Significant differences between groups were determined using one-way analysis of variance (ANOVA), followed by Duncan's tests using PASW Statistics software (version 18.0; SPSS, IBM Co., Armonk, NY, USA). Principal component analysis (PCA) and analysis of similarities (ANOSIM) of the metabolite signatures were performed using the vegan package. Correlation between different variables was determined using Pearson's test or its nonparametric Spearman equivalent. Other data were visualized and analyzed using Prism 6.01 software (GraphPad Software, USA). Statistical significance was set at p < 0.05, and all comparisons were performed relative to the NC group, with significant differences indicated as *p < 0.05 and **p < 0.01.

3. Results

3.1. Effect of TWK10 and TWK10-fermented soymilk on body weight changes, feed efficiency, fat pad weight, and adipocyte size

Previous studies have shown that HFD intake is associated with fat pad mass and adipocyte size. We examined the changes in body weight, feed efficiency, fat pad weight, and adipocyte size to investigate the anti-obesity effects of TWK10 and TWK10 fermented soymilk. No significant difference (p > 0.05) was observed in the initial average body weights among the groups; however, the rats in the L group (522.55 ± 36.93 g) had a significantly higher (p < 0.01) body weight than those in the NC group (453.09 ± 37.38 g) after 5 weeks on a HFD (Fig. 2A). However, TWK10 or TWK10-fermented soymilk intake at various doses suppressed HFD-induced weight gain after 5 weeks compared with that in the L group (Fig. 2B). Additionally, we calculated the feed efficiency (Fig. 2C) and found that the feed efficiency of the L group was almost twice that of the NC group. However, feed efficiency decreased by approximately 15.4 and 10.9% in the PC and F1 groups (p < 0.05), respectively, compared with that in the L group. Moreover, subcutaneous fat weight, total fat mass, and visceral fat weight increased significantly in the L group (Fig. 2D–F). However, TWK10-fermented soymilk intake significantly suppressed HFD-induced increase in visceral fat (Fig. 2F). Compared with that in the L group, total fat weight decreased significantly by approximately 26.9, 22.8, and 24.0% in the T1, F2, and F4 groups, respectively (Fig. 2D). Expectedly, adipocyte size increased in the L group compared with that in the NC group (Fig. 2G and H), indicating adipocyte hypertrophy. However, TWK10 or TWK10-fermented soymilk intake suppressed HFD-induced increase in adipocyte size. Notably, adipocyte size decreased significantly (p < 0.01) by 24.0% in the T1 group compared with that in the L group. Collectively, these results indicate that TWK10 or TWK10-fermented soymilk intake suppresses adipocyte accumulation in rats.

Fig. 2 The effects of dietary Lactobacillus plantarum TWK10 or TWK10-fermented soy milk on weekly body weight (A), weight gain (B), feed efficiency (C), total fat weight (D), subcutaneous fat weight (E), visceral fat weight (F), histological structures of adipose tissue (G), and adipocyte size relative to the L group (H) of loperamide-induced constipation in SD rats fed a high-fat diet. *p < 0.05 when compared with the NC group, **p < 0.01 when compared with the NC group; ^#p < 0.05 when compared with the L group, ^##p < 0.01 when compared with the L group. NC: normal control group; LC: loperamide-induced constipation control group with no treatment and no high fat diet; L: loperamide-induced constipation group with no treatment; PC: loperamide-induced constipation positive control group with administration of bisacodyl at 3.3 mg per kg bw; T1: loperamide-induced constipation group with administration of L. plantarum TWK10 at a dose of 7.60 × 10⁸ CFU per kg bw; T2: loperamide-induced constipation group with administration of L. plantarum TWK10 at a dose of 1.52 × 10⁹ CFU per kg bw; T4: loperamide-induced constipation group with administration of L. plantarum TWK10 at a dose of 3.04 × 10⁹ CFU per kg bw; F1: loperamide-induced constipation group with administration of L. plantarum TWK10 fermented soy milk powder at a dose of 0.7 g per kg bw; F2: loperamide-induced constipation group with administration of L. plantarum TWK10 fermented soy milk powder at a dose of 1.4 g per kg bw; F4: loperamide-induced constipation group with administration of L. plantarum TWK10 fermented soy milk powder at a dose of 2.8 g per kg bw.

3.2. Effect of TWK10 and TWK10-fermented soymilk on fecal parameters and gastrointestinal motility

In addition to obesity, loperamide injection and HFD intake induced constipation and gut dysmotility. Therefore, we examined the experimental rats’ constipation indicators, including fecal number, fecal water content, and the intestinal transit ratio. Compared with that in the NC group, the fecal number and water content decreased (p < 0.01) by 33.6% and 68.3%, respectively, in the L group (Fig. 3A–C). In contrast, the fecal water content increased significantly (p < 0.05) by 16.9% and 25.7% in the PC and TWK10 (T4) groups, respectively, compared with the L group (Fig. 3C). The intestinal transit ratio was measured before euthanasia using activated carbon (Fig. 3D and E). Compared with the NC group (73.9%), there was a significant decrease (p < 0.05) in the intestinal transit ratio in the L group (65.8%). Notably, TWK10 intake significantly increased (p < 0.05) the intestinal transit ratio to 72.6% in the T1 group, compared with the L group. Moreover, TWK10-fermented soymilk intake also increased the intestinal transit ratio in the F1 (70.2%) and F2 (68.4%) groups, compared with the L group. These results suggest that TWK10 intake increases fecal water content and promotes intestinal motility in rats.

Fig. 3 The effects of dietary Lactobacillus plantarum TWK10 or TWK10-fermented soy milk on fecal pellets (A), fecal number (B), fecal water content (C), intestinal charcoal transit (D), and intestinal transit ratio (E) of SD rats fed a high-fat diet of the loperamide-induced constipation group. Data are presented as means ± SD (n = 8). Student's t-test was used to compare two groups of data. *p < 0.05 when compared with the NC group, **p < 0.01 when compared with the NC group; ^#p < 0.05 when compared with the L group, ^##p < 0.01 when compared with the L group. The abbreviations are the same as in Fig. 2.

3.3. Effect of TWK10 and TWK10-fermented soymilk on the serum levels of gastrointestinal hormones

To further investigate the mechanisms of TWK10, we examined the serum levels of gastrointestinal hormones. There was an increase in the serum levels of GT, Sst, and CGRP and a decrease in the serum level of Ache in the L group (Fig. 4A–D) compared with those in the NC group. However, TWK10 intake (T4 group) significantly reduced (p < 0.05) serum Sst and CGRP levels by approximately 33.3 and 22.9%, respectively, and increased serum Ache levels by 33.8% compared with those in the L group. Additionally, TWK10-fermented soymilk intake significantly decreased (p < 0.01) serum CGRP levels by 23.6–31.6% compared with those in the L group (Fig. 4C). These results indicate that TWK10 or TWK10-fermented soymilk intake ameliorates loperamide- and HFD-induced hormonal disorders in the gastrointestinal tract of rats.

Fig. 4 The effect of dietary Lactobacillus plantarum TWK10 or TWK10-fermented soy milk on serum gastrointestinal hormones-GT (A), Sst (B), CGRP (C), Ache (D) and the fecal short chain fatty acids – total SCFAs (E), acetic acid (F), propionic acid (G), and butyric acid (H) of SD rats fed a high-fat diet of the loperamide-induced constipation group. Data are presented as means ± SD (n = 8). Student's t-test was used to compare two groups of data. *p < 0.05 when compared with the NC group, **p < 0.01 when compared with the NC group; ^#p < 0.05 when compared with the L group, ^##p < 0.01 when compared with the L group. GT, gastrin; Sst, somatostatin; CGRP, calcitonin gene related peptide; Ache, acetylcholinesterase. The abbreviations are the same as in Fig. 2.

3.4. Effect of TWK10 and TWK10-fermented soymilk on the organic acid concentrations in cecal contents

Loperamide treatment and HFD intake significantly decreased the cecal concentrations of total SCFAs and acetic and propionic acids (Fig. 4E–G). However, TWK10 fermented soymilk intake (T4 group) markedly increased (p < 0.05) the cecal concentrations of all SCFAs compared with those in the L group (Fig. 4E). Importantly, butyric acid was not detected in rats in all groups (n = 1–8; Fig. 4H). TWK10-fermented soymilk intake (F4 group) significantly increased (p < 0.05) the concentrations of total SCFAs, acetic acid, and propionic acid by 3.1-, 3.8-, and 2.4-fold, respectively (Fig. 4E–G) compared with those in the L group. These results indicate that TWK10 fermented soymilk promotes SCFA production in the cecum.

3.5. Effect of TWK10 and TWK10-fermented soymilk on colon histology

Hematoxylin and eosin and Alcian blue staining were performed to investigate the effect of TWK10 treatment on the histology of the colon (Fig. 5). The thicknesses of the muscle and mucosa decreased significantly (p < 0.01) by approximately 14.7% and 20.4% in the LC and L groups, respectively, compared with those in the NC group. Notably, TWK10-fermented soymilk intake markedly increased the thicknesses of the muscle and mucosa by 11.3–24.4% and 18.20–23.4%, respectively, compared with those in the L group (Fig. 5B and C). The area ratio of goblet cells was 26.1% lower in the L group than that in the NC group, and TWK10 and TWK10-fermented soymilk intake significantly increased (p < 0.01) the area ratio of goblet cells (Fig. 5F).

Fig. 5 The effect of dietary Lactobacillus plantarum TWK10 or TWK10-fermented soy milk on the histopathological findings for the colon (A), muscle thickness (B), mucosa thickness (C), colon tissue with the Alcian blue stain (D), and area ratio of goblet cells (E) of SD rats fed a high-fat diet of the loperamide-induced constipation group. d: decrease of goblet cells and thickness of the mucosa layer. 1 = minimal; 2 = slight; 3 = moderate; 4 = moderate/severe. The normal colon is characterized by abundant goblet cells and a substantial thickness of the mucosal layer. The multifocal decrease of goblet cells and the thickness of the mucosa layer was scored as moderate (3) in the LC group, slight (2) in the L group, normal (0) in the PC group, and slight (2) in the T1, T2, T4, F1, F2 and F4 groups, respectively. H&E stain, magnification ×40. Data are presented as means ± SD (n = 8). Student's t-test was used to compare two groups of data. *p < 0.05 when compared with the NC group, **p < 0.01 when compared with the NC group; ^#p < 0.05 when compared with the L group, ^##p < 0.01 when compared with the L group. The abbreviations are same as in Fig. 2.

3.6. Effect of TWK10 and TWK10-fermented soymilk on PPARγ, C/EBPα, and C/EBPβ protein expressions

Western blotting was performed to elucidate the potential molecular mechanisms of TWK10 and TWK10-fermented soymilk in HFD-fed rats injected with loperamide. TWK10 intake significantly reduced C/EBPα expression (p < 0.01) and downregulated PPARγ and C/EBPβ protein expression compared with the L group (Fig. 6B). Notably, TWK10-fermented soymilk intake (F4 group) significantly decreased (p < 0.01) the protein expression of PPARγ, C/EBPα, and C/EBPβ by approximately 39.0, 65.5, and 21.0%, respectively, compared with the L group (Fig. 6C). Notably, TWK10 and TWK10-fermented soymilk intake suppressed weight gain, adipocyte size, and adipogenesis.

Fig. 6 The effect of dietary Lactobacillus plantarum TWK10 or TWK10-fermented soy milk on PPARγ, C/EBPα and C/EBPβ protein expression in adipose tissue of loperamide-induced constipation in SD rats fed a high-fat diet. (A) Western blot analysis of protein expression. (B) Quantified protein levels in the TWK10 intake group. (C) Quantified protein levels in the TWK10-fermented soy milk intake group. Data are presented as means ± SD (n = 3). Student's t-test was used to compare two groups of data. ^#p < 0.05 when compared with the L group, ^##p < 0.01 when compared with the L group. The abbreviations are the same as in Fig. 2.

3.7. Effect of TWK10 on the gut microbiota in rats

To investigate the effect of TWK10 on the gut microbiota, we examined the species richness and uniformity. TWK10 intake (T4 group) significantly reversed the (p < 0.01) loperamide-induced decrease in the Shannon index in rats fed a HFD (Fig. 7A). Notably, TWK10 intake (3.04 × 10⁹ CFU per kg bw) increased the species richness and uniformity. Additionally, PCA showed that the six groups formed distinct clusters, with a relatively large distance between the NC and L groups (Fig. 7B). After TWK10 intervention, the distance was reduced, and the flora structure gradually recovered to that of the NC group. Collectively, these results indicate that TWK10 reverses HFD- and loperamide-induced alterations in the gut microbiota. An ANOSIM test was performed to determine dissimilarities in the flora structure among the different groups (Fig. 7C). Although there was a marked difference between the L and NC groups, TWK10 treatment significantly reduced this difference. Overall, this result indicates that TWK10 may ameliorate intestinal floral disorder in rats.

Fig. 7 The effect of dietary Lactobacillus plantarum TWK10 on α-diversity (A), principal component analysis (B), similarity (C), and relative abundance of specific bacteria genus of intestinal microbiota compositions Enterocloster (D), Blautia (E), Eubacterium (F), Enterocloster-Pearson (G), Blautia-Pearson and (H), Eubacterium-Pearson (I) of the intestinal microbiota compositions of loperamide-induced constipation in SD rats fed a high-fat diet. The Shannon index shows species richness and evenness. The results are the means for n = 6. Student's t-test was used to compare two groups of data (A); statistical significance was determined by a Pearson correlation analysis (C–I). The abbreviations are same as in Fig. 2.

Changes in the relative abundances of species are shown in Fig. 7D–F. There was a marked decrease in the relative abundances of Enterocloster and Blautia and a significant increase in the relative abundance of Eubacterium in the L group, compared with those in the NC group. However, TWK10 intake reversed the HFD- and loperamide-induced imbalance in the intestinal microbiota of the rats. Furthermore, Pearson's correlation analysis was performed to examine the relationship between the different doses of TWK10 and the abundances of specific genera (Fig. 7G–I). Oral administration of TWK10 was positively correlated with the relative abundances of Enterocloster (R = 0.5959; p = 0.0021) and Blautia (R = 0.5288; p = 0.0079) and negatively correlated with Eubacterium abundance (R = −0.5087; p = 0.01113) in HFD-fed rats with loperamide-induced constipation.

4. Discussion

LAB-fermented soymilk is beneficial for human health because of the increased bioavailability of substances such as aglycone isoflavones and bioactive peptides in the human intestinal tract. Notably, fermented soy milk possesses several health-promoting effects, including anti-obesity, anti-hypertensive, and anti-hyperglycemic effects.²⁵ However, studies on the effects and mechanisms of fermented soy milk on adipocyte accumulation and gastrointestinal hormones in HFD-fed mice with loperamide-induced constipation are limited. To the best of our knowledge, this is the first study to examine the effect of LAB-fermented soy milk in rats with HFD- and loperamide-induced obesity, constipation, and gut dysbiosis. Consistent with previous findings,^14,26,27 LAB-fermented soy milk suppressed body weight gain and decreased fat weight in HFD- and loperamide-induced rats. A high-fat diet can induce fat accumulation in adipocytes, leading to swelling. Moreover, fat accumulation around the abdominal viscera has more severe negative health implications than obesity.²⁸ In this study, HFD-fed rats exhibited apparent obesity, subcutaneous and visceral fat accumulation, and adipocyte enlargement. However, TWK10 and TWK10-fermented soymilk intake suppressed weight gain and adipocyte volume and decreased visceral fat and total fat mass (Fig. 2). Additionally, the expression of genes that regulate adipocyte differentiation, such as PPARγ and C/EBP proteins, is important. Specifically, C/EBPβ is quickly activated at the start of adipocyte differentiation. Multiple regulatory factors work together to enhance the expression of C/EBPα and PPARγ by interacting with C/EBP-responsive elements in the promoter regions of these genes. Notably, C/EBPα and PPARγ play a prominent role in regulating the expression of adipocyte genes necessary for the development of functionally mature adipocytes. C/EBPα is associated with PPARγ and can activate the expression of fat-specific genes to synthesize, extract, and store long-chain fatty acids, as well as to stop the proliferation of cells.^29,30 Fermented soy has been reported to affect parameters associated with metabolism and obesity.³¹ For example, Cheng et al. (2015) reported that the intake of an aqueous extract of soy fermented with L. paracasei subsp.paracasei NTU 101 inhibited the protein expressions of C/EBPβ, PPARγ, and C/EBPα, and affected lipogenesis by reducing heparin-releasable lipoprotein lipase activity.⁴⁸ Additionally, Lee et al. (2013) showed that the intake of soy fermented with L. paracasei subsp. paracasei NTU 101 or L. plantarum NTU 102 significantly inhibited obesity, which was attributed to the inhibition of preadipocyte differentiation.¹⁴ Similarly, TWK10 and TWK10-fermented soymilk intake significantly decreased the protein expressions of PPARγ, C/EBPα, and C/EBPβ (Fig. 6). Among the three adipogenic factors, TWK10 and TWK10-fermented soymilk exhibited the highest inhibitory effect on C/EBPα expression (Fig. 6). PPARγ is adipose tissue-specific and plays an important role in adipogenesis, lipid metabolism, and insulin sensitivity. Therefore, it could be speculated that TWK10 and TWK10-fermented soymilk regulate adipogenesis and lipid storage via PPARγ inhibition. Collectively, these results indicate that TWK10 and TWK10-fermented soymilk intake attenuates HFD-induced body weight gain by inhibiting adipogenesis and adipocyte hypertrophy.

Constipation is more frequent in patients with obesity than in healthy individuals. The intake of some foods, such as high-fat meals, plays an essential role in the etiology of constipation.^2,5 Therefore, we used loperamide to slow gastrointestinal motility and induce gut dysbiosis and constipation in this study. Loperamide is a μ-opioid receptor agonist that alters various gastrointestinal hormones, suppresses intestinal peristalsis, and inhibits the excretion of intestinal fecal pellets, resulting in various intestinal motility disorders.^32–34 Enteric nervous system-related factors secreted by the enteric nerve network in the gastrointestinal tract act as neuromodulators and neurotransmitters, promoting intestinal peristalsis. Acetylcholinesterase is an excitatory neurotransmitter, while somatostatin is an inhibitory peptide neurotransmitter. Gastrin, which is mainly secreted by the gastric sinus, promotes gastrointestinal motility, and its secretion is inhibited by somatostatin. CGRP is involved in gastrointestinal nociception, inflammation, gastric acid secretion, and motility.³⁵ Some dietary interventions, including LAB and fermented foods, exert beneficial effects on constipation. L. plantarum YS-3 reduced the fecal weight, particle number, and water content of constipated mice. Additionally, L. plantarum YS-3 increased the gastrointestinal transit rate and regulated the levels of gastrointestinal hormones, such as acetylcholinesterase and somatostatin.³⁶ Moreover, L. plantarum YS2 treatment increased serum levels of gastrin and acetylcholine and reduced somatostatin levels in mice with constipation.³⁷ Furthermore, L. plantarum CQPC01-fermented soybean milk exerts an anti-constipation effect, which is attributed to its isoflavone content. Notably, the higher the content of specific active substances, the greater the inhibitory effect of soybean milk on constipation.³⁸ Consistent with previous findings, TWK10 intake ameliorated loperamide-induced constipation in rats by preserving fecal water content and increasing the intestinal transit rate (Fig. 3C and E). Additionally, TWK10 and TWK10-fermented soymilk intake reversed loperamide-induced changes in muscle and mucosal thickness (Fig. 5B and C) and serum levels of somatostatin, calcitonin gene-related peptide, and acetylcholinesterase (Fig. 4B–D). Gastrin expression is regulated by diverse signals, including nutrients, hormones, neurotransmitters, pH, and stomach distension.³⁹ HFD consumption affects gastrin secretion in the digestive tract. For example, fatty meal intake significantly increased serum gastrin in healthy male gerbils, while high-fat diet (HFD) intake had the same effect in Mongolian gerbils.^40,41 Similarly, there was a significant increase in serum gastrin levels in HFD-fed rats in the present study (Fig. 4A). However, the unique experimental design of each study makes it difficult to generalize the effects of HFD on gastrin level.

Dysbiosis is an imbalance in the intestinal microflora associated with obesity and constipation. The gut microbiota plays a vital role in regulating intestinal motility, secretion, and absorption via metabolite activities, preventing the growth of pathogenic bacteria and modulating neurotransmitter synthesis.^33,42Enterocloster is a genus of anaerobic fusiform rods that commonly inhabit the human gut.⁴³Blautia is a genus of anaerobic bacteria with probiotic characteristics that occurs widely in the feces and intestines of mammals. Blautia has attracted attention for its role in alleviating inflammatory and metabolic diseases and its antibacterial activity against specific microorganisms.⁴⁴Eubacterium, a genus of obligate anaerobic rods, is a common gastrointestinal commensal species implicated as the cause of intra-abdominal infections and bacteremia.⁴⁵ PCA, ANOSIM, and the Shannon index showed that TWK10 intake reversed loperamide-induced alteration in gut microbiota composition in HFD-fed rats (Fig. 7A–C). Previous studies have shown that Eubacterium may be associated with constipation-related diseases. Moreover, high Eubacterium abundance is associated with remission in patients with ulcerative colitis.⁴⁶L. paracasei intervention significantly increased the relative abundances of Blautia and Lactobacillus and decreased the abundances of Alloprevotella, Helicobacter, Bacteroides, and Enterococcus in mice with HFD-induced obesity.⁴⁷ Importantly, TWK10 intake at different doses was positively correlated with the relative abundances of Enterocloster and Blautia, but was negatively correlated with Eubacterium abundance. Research findings suggest that HFD intake decreases gut microbial richness and biodiversity, as well as SCFA production. Notably, total SCFA concentration was lower in the L group than in the LC group (Fig. 4E). Additionally, TWK10 intake markedly increased the abundance of the SCFA-producing Blautia in the gut of rats and intestinal SCFA levels. Hwang et al. (2017) indicated that HFD depleted genes were involved in succinate synthesis by reducing Lactobacillus.¹⁸ Moreover, HFD decreased the abundance of butyrate-producing bacteria such as Parabacteroides and Bacteroides (in the Bacteroidales order) that possess the buk and ptb genes necessary for butyrate synthesis. Conversely, HFD increased Bacteroidales, especially Bacteroides, which can convert pyruvate into acetyl-CoA. Although acetyl-CoA can be subsequently metabolized to butyrate, depletion of the buk and ptb genes inhibits butyrate synthesis. Thereby, unused acetyl-CoA indirectly increases fat storage by feedback inhibition.¹⁷ Similar to our results, butyric acid was detected in the ceca of rats fed with TWK10-fermented soymilk (Fig. 4H). Moreover, TWK10-fermented soymilk intake significantly suppressed HFD-induced increase in visceral fat (Fig. 2F), total fat weight (Fig. 2D), and adipocyte size (Fig. 2G and H). Furthermore, TWK10 intake significantly ameliorated loperamide-induced constipation in HFD-fed rats by reducing the number of harmful bacteria and increasing the number of beneficial bacteria; the relationship between the key intestinal microbes and TWK10 intervention and lipid metabolism requires further verification.

5. Conclusions

TWK10 and TWK10-fermented soymilk intake ameliorates the condition of HFD- and loperamide-induced rats by increasing the fecal water content and intestinal transit rate and improving colon histopathology. In additionally, TWK10 and TWK10-fermented soymilk intake prevents HFD-induced obesity by reducing weight gain, adipocyte size, and adipogenesis. Notably, TWK10 had a greater inhibitory effect on HFD-induced obesity and loperamide-induced gut dysbiosis and constipation in rats than TWK10 fermented soymilk. Mechanistically, TWK10 ameliorated HFD-induced obesity and loperamide-induced constipation by regulating the expression of adipogenesis-related proteins, gut microbiota diversity, and the levels of SCFAs. Conclusively, these findings indicate that TWK10 possess potential application as a probiotic to prevent constipation and related symptoms. Moreover, it is crucial to reduce dietary fat intake and increase the consumption of probiotics to improve gut microbiota diversity and health.

Author contributions

Liu, TH: conceptualization, methodology, investigation, data curation, and visualization. Chen, GL: methodology, investigation, data curation, and formal analysis. Lin, CH: methodology, investigation, data curation, and formal analysis. Tsai, TY: conceptualization, review and editing, supervision, and project administration. Cheng, MC: methodology, writing – review and editing, and project administration. All authors have reviewed the manuscript and have given their approval for its submission.

Statement of animal experiments

We confirm that all experiments involving rat samples in our study were conducted in compliance with relevant laws and guidelines.

1. Compliance with national laws and guidelines: all animal experiments were performed in accordance with the Animal Protection Act, 2021, and Guidelines for the Care and Use of Laboratory Animals, 2018, issued by the Council of Agriculture Executive Yuan. This ensures that our research met the ethical standards set forth for the treatment and care of laboratory animals.

2. Institutional guidelines: our study followed the institutional guidelines for animal care and use as outlined by the Animal Care and Research Ethics Committee of Fu Jen Catholic University. These guidelines are designed to ensure humane treatment and minimize the suffering during research.

3. Approval from the Institutional Committee: the experiments were approved by the Animal Care and Research Ethics Committee of Fu Jen Catholic University under protocol number A10931. This committee reviewed our research proposal to ensure compliance with ethical standards.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of interest

There are no conflicts to declare.

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Footnote

† These authors contributed equally to this work.

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