Bifidobacterium longum subsp. longum relieves loperamide hydrochloride-induced constipation in mice by enhancing bile acid dissociation

Abstract

Bifidobacterium species are known for their efficacy in alleviating constipation. This study aimed to compare the constipation-relieving effects of different Bifidobacterium species (Bifidobacterium longum subsp. longum, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, and Bifidobacterium adolescentis) and to explore the underlying mechanisms from both the bacterial and host perspectives. We evaluated six Bifidobacterium species for their physiological properties, including growth rate, oligosaccharide utilization, osmotic pressure resistance, cell adhesion, and bile acid dissociation capability. Mice with severe constipation induced by loperamide hydrochloride were treated with these bacteria at a density of 10⁹ CFU per mL for 17 days. Gastrointestinal indices such as fecal water content, time to first black stool defecation, and small intestine propulsion rate were measured to assess constipation relief. Microbiome and metabolome (bile acid and tryptophan) analyses were conducted to elucidate the differences in constipation relief among the species. Our results demonstrated that Bifidobacterium longum subsp. longum exhibited superior physiological traits, including rapid growth, extensive oligosaccharide utilization, and high bile salt dissociation capacity. Notably, only Bifidobacterium longum subsp. longum significantly ameliorated constipation symptoms in the mouse model. Furthermore, this strain markedly restored bile acid and short-chain fatty acid levels in the intestines of constipated mice and altered the composition of the intestinal microbiota. These findings suggest that the enhanced efficacy of Bifidobacterium longum subsp. longum in relieving constipation is associated with its ability to modulate intestinal physiology and microbiota structure and metabolism.

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Introduction

Constipation is a prevalent gastrointestinal disorder affecting approximately 14% of the global population.³ It imposes significant economic burdens on healthcare systems and adversely impacts patients’ physical and mental well-being.^1,2 Current treatments, primarily pharmacological, often have limitations such as single-mode efficacy, severe side effects, and withdrawal symptoms.^4,5 As lifestyle pressures and dietary changes increase the incidence of constipation, there is growing interest in finding effective, safe, and acceptable treatments.

Bifidobacterium species are prominent probiotics in the human gut and are widely utilized in food and pharmaceutical applications.⁶ Species such as Bifidobacterium longum subsp. longum (B. longum), Bifidobacterium longum subsp. infantis (B. infantis), Bifidobacterium breve (B. breve), Bifidobacterium animalis (B. animalis), Bifidobacterium bifidum (B. bifidum), and Bifidobacterium adolescentis (B. adolescentis) are approved for food use under Chinese regulations.⁷ Extensive research has highlighted their benefits, including immune enhancement,⁸ depression alleviation,⁹ tumor suppression,¹⁰ and colitis treatment.¹¹ Increasingly, animal and clinical studies have demonstrated the positive effects of Bifidobacterium on constipation relief by restoring intestinal homeostasis, increasing bowel movements, and promoting colonic motility.¹¹

Bifidobacterium can modulate the intestinal environment and influence metabolic processes to effectively alleviate constipation symptoms. This modulation can occur through various mechanisms, including the strain's superior physiological properties,¹² enhancement of physical and immune barriers,¹³ regulation of intestinal microecology,¹⁴ and metabolism of bile acids and fatty acids.¹⁵ Additionally, Bifidobacterium can inhibit the growth of certain pathogenic bacteria.^16,17 The ability to utilize a broad spectrum of carbon sources varies among different strains, species, and genera,¹⁸ with a greater capacity for carbon source utilization enhancing Bifidobacterium colonization in the gut. Factors such as natural abundance, gastrointestinal tolerance, nutritional availability for proliferation, suitable surface structures for adhesion to the intestinal epithelium or mucus layer, and the production of small metabolic molecules that mediate resistance to competitor colonization¹⁹ all impact gut colonization. B. longum has bile salt tolerance,^20–22 and the resistance to bile salt stress consists of two main mechanisms: excretion of bile salts out of the cell and hydrolysis of bile salts.²³ In addition to this, genomic analysis of B. longum showed that it has a diverse range of carbohydrate-active enzyme genes, which gives B. longum a significant competitive advantage in utilizing host intestinal glycans. Across human cohorts, abfA-cluster abundance can predict functional constipation, and transplantation of abfA cluster-enriched human microbiota to functional constipation-induced germ-free mice improved gut motility.²⁴ Most studies indicate a decrease in Bifidobacterium abundance in constipated patients, and supplementation with Bifidobacterium or prebiotics can increase intestinal Bifidobacterium levels and significantly improve constipation symptoms.²⁵ Previous research in our laboratory has shown that different Bifidobacterium strains exhibit species-specific differences in constipation relief.²⁶ These differences may be attributed to the physiological advantages of the strains themselves or host metabolic factors, which remain largely unexplored.

To investigate this, we selected five strains from each of six different Bifidobacterium species. By studying the physiological properties of these strains and their ability to alleviate constipation, we aimed to preliminarily elucidate the mechanisms underlying Bifidobacterium-mediated constipation relief.

Methods

Bacterial treatment

Six species and 30 strains of Bifidobacterium (5 strains each of B. longum, B. bifidum, B. breve, B. animalis, B. infantis, and B. adolescentis) were obtained from the Strain Bank of the Food Biotechnology Center of Jiangnan University (Jiangsu, China). Specific strain-related information is detailed in Table 1. The strains were activated in modified MRS medium for two generations and then incubated anaerobically at 37 °C. After centrifugation at 8000g for 15 min at 4 °C, the cell pellet was collected, washed twice with sterile saline, resuspended, and the bacterial concentration was determined using the gradient dilution method. The strains were stored at −80 °C until use. On the day of gavage, they were thawed and resuspended in sterile saline to a bacterial density of 10⁹ CFU per mL, homogenized, and kept on ice until administration.

Table 1 List of bacterial strains

Strain	Containing strains	Source
B. longum (C)	CCFM1114 (C1)	Human, Zhangye, Gansu
	FHuBZX30M2 (C2)	Human, Zhongxiang, Hubei
	FJSWXJ3M1 (C3)	Human, Wuxi, Jiangsu
	FSCREG6M53 (C4)	Human, Norgay, Tibet
	FSDLZ50M2 (C5)	Human, Laizhou, Shandong
B. bifidum (L)	JSNT16 2 (L1)	Human, Nantong, Jiangsu
	AHWH24M3 (L2)	Human, Wuhu, Anhui
	FXJCJ32M2 (L3)	Human, Changji, Xinjiang
	JSWX20M6 (L4)	Human, Wuxi, Jiangsu
	M2 03 F02 M6 3 2 (L5)	Human, Norgay, Tibet
B. animalis (A)	BJHD3M6 CDS (A1)	Infants, Haidian, Beijing
	S10 (A2)	Infants, Ji'nan, Shandong
	SC-YA-1-M1 CDS (A3)	Human, Ya'an, Sichuan
	HuNan2016 222 T71 CDS (A4)	Human, Boai County, Henan
	FNMGHHHT2M2 CDS (A5)	Infants, Hulunbuir, Inner Mongolia
B. brevis (D)	FAHWH11M1 (D1)	Human, Wuhu, Anhui
	FGZ18I1M6 (D2)	Human, Guangzhou, Guangdong
	FHuNCS3M4 (D3)	Human, Changsha, Hunan
	FNMGHLBE9M6 (D4)	Human, Hulunbuir, Inner Mongolia
	FXJCJ32M7 (D5)	Human, Changji, Xinjiang
B. infantis (Y)	FGZ17I1M1 (Y1)	Human, Guangzhou, Guangdong
	CCFM1192 (Y2)	Human, Changsha, Hunan
	CCFM1210 (Y3)	Human, Yangzhou, Jiangsu
	FZJJH13M4 (Y4)	Human, Jinhua, Zhejiang
	HeNJZ8M1 (Y5)	Human, Jiaozuo, Henan
B. adolescentis (Q)	CCFM1108 (Q1)	Human, Zhangye, Gansu
	FXJCJ15M4 (Q2)	Human, Changji, Xinjiang
	FJSSZ3M10 (Q3)	Human, Suzhou, Jiangsu
	FQHXN72M5 (Q4)	Human, Xining, Qinghai
	FHNFQ41M3 (Q5)	Human, Fengqiu, Henan

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Physiological characteristics of strains

Growth ability. The activated third-generation bacterial solution was added to a sterile 96-well plate with 300 μL per well, with three parallel control wells set up. The 96-well plates were placed in an anaerobic incubator within an enzyme labeling apparatus, and OD₆₀₀ was measured at 30-minute intervals starting from inoculation (0 h) for 18–24 hours. The growth curve was plotted using incubation time as the horizontal coordinate and absorbance as the vertical coordinate. Based on the growth curves, two time points during the logarithmic phase were selected, and the generation time during this phase was calculated using eqn (1):

In the equation, G is the generation time (min) during the logarithmic growth period; t₁ and t₂ are the selected time points (min); OD₁ and OD₂ are the corresponding OD₆₀₀ values.

Utilization ability of oligosaccharides. The determination of the oligosaccharide utilization capacity of Bifidobacterium was adapted from the method by Tseteslava²⁷ with appropriate modifications. In this study, glucose in the mMRS medium was replaced with 0.5% (m/v) of various oligosaccharides (fructooligosaccharide (FOS), xylo-oligosaccharide (XOS), galactooligosaccharides (GOS), lactosucrose (LS), lactulose (Lac), and isomaltooligosaccharide (IOM)) (Shandong Qilu Biotech, Liaocheng, China). Additionally, 0.1% L-cysteine hydrochloride was added, and 1.50% (m/v) bromocresol violet solution was used as an acid–base indicator to create the oligosaccharide-mMRS medium. After three generations of activation, the bacterial solution was spotted onto plates containing oligosaccharide-mMRS solid medium and incubated anaerobically at 37 °C for 24–48 h. The formation of yellow colonies indicated the strain's ability to utilize the oligosaccharides. mMRS medium with and without glucose served as positive and negative controls, respectively. The generation time (G) was then calculated as described in the ‘Growth ability’ section.

Osmotic pressure resistance. To assess osmotic pressure resistance, NaCl was added to mMRS liquid medium to achieve initial osmolalities of 350 (control), 750, 850, 950, 1050, 1150, 1250, 1350, 1450, 1550, 1750, and 1950 mOsm per kg. The pH was adjusted to 7.0 using HCl or NaOH. Osmolality was increased by approximately 100 mOsm per kg with the addition of 3 g NaCl per liter of mMRS. Subsequent procedures followed those outlined in the ‘Growth ability’ section.

Cell adhesion ability. HT-29 cells in the logarithmic growth phase were used for cell adhesion assays. A volume of 1 mL of cultured HT-29 cytosol was placed in a 6-well plate and incubated for 12 h to form a dense monolayer. After rinsing three times with PBS, DMEM medium was added, and the bacterial concentration was adjusted to 10⁷ CFU per mL. The bacterial suspension was then added to an equal volume of HT-29 cell culture medium and incubated at 37 °C for 3 h. Following incubation, cells were rinsed three times with PBS to remove non-adherent bacteria, fixed with methanol for 30 min, and Gram stained. The number of Bifidobacterium adhering to HT-29 cells was observed microscopically. Cultures without Bifidobacterium served as negative controls, while LGG (Lactobacillus rhamnosus GG) was used as a positive control.

Bile acid dissociation ability. The bile salt hydrolase activity of probiotics was assessed by observing the formation of milky white precipitation rings in bile salt-containing MRS media. The mMRS solid medium was prepared with the addition of TDC at a concentration of 2.5 g L⁻¹, 1.5 g L⁻¹ sodium thioglycolate, and 0.35 g L⁻¹ CaCl₂. A 6 mm diameter circular qualitative filter paper was placed on the solid medium, and 10 μL of bacterial solution with an adjusted OD value was pipetted onto the filter paper surface. The plates were incubated anaerobically and observed after 12 and 24 h to record the presence and size of precipitation circles produced by different strains. The larger precipitation circle formed indicates the greater ability of the strain to dissociate bile salts.

Animals and experimental design

Eighty five-week-old specific pathogen-free (SPF) grade BALB/c male mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). The methods and procedures used in this animal experiment were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Jiangnan University (SYXK 2021-0056), reviewed and approved by the Animal Ethics Committee of Experimental Animals of Jiangnan University (qualified no. jn. no 20221030b1350515 [463]), and complied with the European Union Guide for Laboratory Animals (Directive 2010/63/EU). All mice were provided ad libitum access to feed and water, housed in IVC cage boxes, and fed standard mouse chow under controlled environmental conditions: temperature 25 ± 2 °C, relative humidity 50 ± 5%, with a 12-hour light/dark cycle. Animal experiments commenced after one week of acclimatization.

The mice were randomly divided into ten groups (eight mice per group): a blank control group (control), a model group (model), two positive drug groups (Lin and Pin), and six probiotic intervention groups. The probiotic intervention group was a mixture of 5 strains of the same species with the same viable counts of bacteria resulting in a final bacterial concentration of 10⁹ CFU per mL. Specific details shown in Table 2. From days 8–24, the blank control group received sterile saline gavage, while other groups were administered 20 mg per (kg bw) loperamide hydrochloride (Xi'an Janssen Pharmaceutical Company, Ltd, Xi'an, China). One hour later, the model group received sterile saline gavage, positive drug groups received resuspended Linaclotide (AstraZeneca Pharmaceutical Co., Ltd, Wuxi, China) or Pinaverium Bromide (Abbott Laboratories Ltd, Bangkok, Thailand) solutions in sterile saline, and probiotic groups received resuspended Bifidobacterium suspension at a concentration of 10⁹ CFU per mL in sterile saline. The gavage dose was maintained at 0.2 mL per administration. The detailed animal experimental process is illustrated in Fig. 1.

Fig. 1 The animal experiment schedule.

Table 2 List of animal testing groups

Group	Gavage substance	Gavage concentration	Gavage volume
Control	Normal saline	—	0.2 mL
Model	Loperamide hydrochloride	20 mg per (kg bw)	0.2 mL
Lin	Linaclotide	0.1 mg per (kg bw)	0.2 mL
Pin	Pinaverium bromide	30 mg per (kg bw)	0.2 mL
C	C1 + C2 + C3 + C4 + C5	10^9 CFU per mL	0.2 mL
L	L1 + L2 + L3 + L4 + L5	10^9 CFU per mL	0.2 mL
A	A1 + A2 + A3 + A4 + A5	10^9 CFU per mL	0.2 mL
D	D1 + D2 + D3 + D4 + D5	10^9 CFU per mL	0.2 mL
Y	Y1 + Y2 + Y3 + Y4 + Y5	10^9 CFU per mL	0.2 mL
Q	Q1 + Q2 + Q3 + Q4 + Q5	10^9 CFU per mL	0.2 mL

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Collection of animal tissues and serum

Fecal samples were collected weekly and stored at −80 °C. At the experiment's conclusion, mice were fasted overnight and anesthetized with intraperitoneal ketamine (100 mg per kg body weight). Blood was collected via retro-orbital bleeding, and mice were euthanized by cervical dislocation. Serum was obtained by centrifuging blood samples at 3000g for 15 minutes and stored at −80 °C. The entire gastrointestinal tract was excised for imaging, and remaining tissues were flash-frozen in liquid nitrogen for storage at −80 °C.

Gastrointestinal function assessment

Faeal water content. For fecal water content experiments, mouse feces were collected once a week. Individual mice were placed in clean cages lined with filter paper post-gavage. Fresh feces were collected in sterile 0.5 mL microcentrifuge tubes, weighed wet, lyophilized, and reweighed. Fecal water content was calculated using the following equation:

Time of first black stool defecation. On day 24, mice were gavaged with 0.2 mL of a loperamide hydrochloride and activated charcoal solution, prepared as described by Zhang et al.²⁸ The time from gavage to the first black stool appearance was recorded. Treatment efficacy was assessed by comparing transit times between treatment and model groups.

Small intestine propulsion rate. Following a 12-hour fast on day 25, all groups except the control received 0.2 mL of loperamide hydrochloride and activated charcoal mixture via gavage. The control group received saline and activated charcoal. After 30 minutes, mice were euthanized, and the small intestine (pylorus to cecum) was excised.²⁹ The small intestine propulsion rate was calculated as:

Nontargeted metabolomics

Lyophilized fecal samples (20 mg) were homogenized in 1 mL of pre-cooled extraction solution (methanol : acetonitrile : water, 2 : 2 : 1 v/v/v). After protein precipitation at −20 °C for 1 hour, samples were centrifuged at 12 000g for 15 minutes at 4 °C. The supernatant was vacuum-dried, reconstituted in 200 μL acetonitrile : water (1 : 1 v/v), and filtered through a 0.22 μm membrane. Metabolite detection employed a Dionex UltiMate 3000 UHPLC system coupled with a Q Exactive high-resolution mass spectrometer (Thermo Fisher Scientific). Chromatographic separation was performed on a Waters ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm) at 35 °C. Mobile phases consisted of 0.1% formic acid in water (A) and acetonitrile (B) for positive ion mode, and 5 mM ammonium acetate (A) and acetonitrile (B) for negative ion mode. The flow rate was 0.3 mL min⁻¹ with a gradient elution program. Sample injection volume was 2 μL, with autosampler temperature maintained at 4 °C.

Short-chain fatty acid (SCFA) analysis

The analysis of short-chain fatty acids (SCFAs) in fecal samples was conducted following established protocols.²⁸ Approximately 30 mg of freeze-dried mouse feces was weighed and suspended in 0.5 mL of saturated sodium chloride for 30 minutes. The samples were homogenized using a tissue homogenizer, followed by the addition of 20 μL of 10% sulfuric acid and shaking for 30 seconds. Subsequently, 1 mL of ether was added to each sample, and the mixtures were centrifuged at 4 °C, 12 000g for 15 minutes. The upper ether phase was collected, mixed with 0.3 g of sodium sulfate, and centrifuged again under the same conditions. The supernatant was transferred to a gas chromatography-mass spectrometry (GC-MS) vial for analysis. Standard curves of various SCFAs (Sinopharm Chemical Reagent Corporation, Shanghai, China) were generated using the external standard method, and the concentrations of SCFAs (μmol g⁻¹) in the samples were calculated from these standard curves. The chemical reagents used in the experiments were chromatographically pure.

Determination of bile acid (BA) contents in feces

Approximately 30 mg of lyophilized cecal content was weighed, and 1 mL of methanol was added for homogenization. The mixture was centrifuged at 12 000g for 15 minutes at 4 °C. The supernatant was concentrated and resuspended in methanol, then centrifuged again under the same conditions. The supernatant was transferred to an injection vial for analysis. Samples were analyzed using a DIONEX UltiMate 3000 HPLC system equipped with a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) and an ACQUITY UPLC® HSS T3 column (1.8 μm, 2.1 × 100 mm). The autosampler temperature was set to 15 °C, and 2 μL of sample was injected at a flow rate of 0.30 mL min⁻¹ with a column temperature of 35 °C for gradient elution. The mobile phase consisted of 1 mM ammonium acetate aqueous solution (A) and 1 mM ammonium acetate methanol solution (B). The gradient elution program was: 0 min, 20% B; 0–6 min, 20% B; 6–25 min, 100% B; 25–29 min, 100% B; 29–29.1 min, 100%–20% B; and 29–35 min, 20% B. The mass spectrometer operated in negative electrospray ionization (ESI) mode with a spray voltage of −2.50 kV, sheath gas flow rate of 30 arb, and auxiliary gas flow rate of 10 arb. The capillary temperature was set to 325 °C, with full scans performed at a resolution of 70 000 over a scanning range of 100 to 750. The chemical reagents used in the experiments were chromatographically pure.

Determination of tryptophan contents in feces

Approximately 30 mg of lyophilized colon contents was weighed and mixed with 900 μL of MeOH/H₂O (1 : 1). The mixture was centrifuged at 15 000g for 10 minutes at 4 °C. The supernatant was concentrated and resuspended in MeOH/H₂O (1 : 9), followed by another centrifugation under the same conditions. The supernatant was then transferred to an injection vial for analysis. Samples were analyzed using a DIONEX UltiMate 3000 HPLC system equipped with a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) and an ACQUITY UPLC BEH C18 column (1.7 μm, 100 mm × 2.10 mm). The injection volume was set to 2 μL. The solvent system comprised of 0.1% (v/v) formic acid (A) and acetonitrile (B) at a flow rate of 0.3 mL min⁻¹. The gradient program was: 5% B (0–3 min), 30% B (3–9 min), 100% B (9–15 min), 100% B (15–16.5 min), and 5% B (16.5–20 min). The mass spectrometer operated in positive electrospray ionization (ESI+) mode with a spray voltage of +3500 V. Nitrogen served as the sheath gas at a flow rate of 35 units and auxiliary gas at a flow rate of 15 units. The capillary temperature was set to 320 °C. The chemical reagents used in the experiments were chromatographically pure.

16S rDNA sequencing and bioinformatics analysis

DNA was extracted from mouse feces using the Fecal FastDNA spin kit (MP Biomedical, catalogue no. 6570200) according to the manufacturer's instructions. The extracted DNA served as the template for polymerase chain reaction amplification of bacterial V3–V4 fragments using forward primer 341F (5′-CCTAYGGGRBGCASCAG-3′) and reverse primer 806R (5′-GGACTACNNGGGTATCTAAT-3′). The microbial genomic DNA was electrophoresed on a 1.5% agarose gel and stained with nucleic acid dye. PCR products were purified using the DNA Gel/PCR purification miniprep kit (BW-DC3511-01, BIOMIGA, USA). DNA concentrations were determined by NanoDrop spectrophotometry, and samples were mixed at equal concentrations to create DNA libraries. Amplicons were sequenced on the MiSeq platform (Illumina, San Diego, CA, USA) using the MiSeq kit. Sequencing data were analyzed using QIIME II software with reference to Zhang.²³

Statistical analysis

The statistical analyses and graphical representations of the experimental data were expressed as “mean ± standard deviation” and performed using GraphPad Prism 8.0 (GraphPad Inc., San Diego, CA, USA) and IBM SPSS Statistics 22.0 (IBM Corp., Armonk, NY, USA). One-way ANOVA was employed to analyze differences between groups, followed by Fisher's least significant difference (LSD) test to compare the experimental and model groups. Statistical significance was set at P < 0.05.

Results

B. longum exhibits superior physiological traits in growth, oligosaccharide utilization, and bile salt hydrolysis

The growth and proliferation of Bifidobacterium in the intestine are influenced by several factors, including growth rate, oligosaccharide utilization, osmotic pressure resistance, cell adhesion ability, and bile salt hydrolysis capacity. Generation time, defined as the duration for a single microorganism to divide and reproduce one generation, is a critical indicator of growth rate; shorter generation times indicate faster growth. Fig. 2A evaluates the growth capacity of 30 strains across six Bifidobacterium species. The results demonstrate that B. longum has the shortest average generation time with minimal inter-strain variation, followed by B. animalis. In contrast, the other four species exhibit longer average generation times and greater inter-strain variability. Functional oligosaccharides are difficult to absorb gastrointestinally but can be metabolized by gut bacteria, selectively promoting the growth of beneficial bacteria and enhancing intestinal motility and metabolic functions. Qualitative assessments of 30 Bifidobacterium strains revealed that all B. longum strains could utilize all oligosaccharides except Lac (Fig. 2B), whereas other strains showed significant variability in oligosaccharide utilization.

Fig. 2 Evaluation of physiological properties of different Bifidobacterium. (A) Growth generation time. (B) Heatmap of oligosaccharide utilization by Bifidobacterium. (C) Generation time with various oligosaccharides as sole carbon sources. (D) Osmotic pressure resistance. (E) HT-29 cell adhesion capacity. (F) Bile salt hydrolysis capacity.

Further analysis of oligosaccharide utilization revealed that B. longum had the shortest average generation time when grown on GOS, LS, Lac, and XOS as sole carbon sources. Conversely, B. bifidum exhibited shorter generation times on FOS and IOM, with a small difference from B. longum (Fig. 2C). This indicates that B. longum can access a diverse range of nutrients and grow rapidly. Osmotic stress tolerance is crucial for the stable and prolonged beneficial effects of probiotics. As shown in Fig. 2D, B. animalis displayed the highest mean osmotic stress tolerance, while other species showed no significant differences.

Cell adhesion is essential for bacterial colonization and probiotic efficacy, with HT-29 cells serving as an in vitro model for assessing intestinal epithelial adhesion. Fig. 2E demonstrates that B. bifidum had the highest HT-29 cell adhesion capacity, whereas B. infantis had the lowest. Qualitative experiments assessed bile salt hydrolysis capacity among Bifidobacterium strains. The heatmap in Fig. 2F indicates that all B. longum and B. adolescentis strains could hydrolyze bile salts, with B. longum exhibiting superior hydrolysis capabilities.

In summary, B. longum demonstrates rapid growth, broad-spectrum oligosaccharide utilization, and robust bile salt hydrolysis, conferring significant physiological advantages. B. bifidum excels in cell adhesion and oligosaccharide utilization, while B. animalis shows superior osmotic stress tolerance, and B. adolescentis excels in bile salt hydrolysis. These findings suggest a potential link between the physiological properties of different Bifidobacterium species and their ability to alleviate constipation. Consequently, we evaluated the efficacy of these strains in relieving constipation and explored the role of their physiological properties in ameliorating constipation symptoms.

In a severe constipation model, only B. longum among the six species demonstrated significant constipation relief

Based on the aforementioned analysis of the physiological properties of B. longum, the following was to investigate its effects on host metabolism and gut microbiota in alleviating constipation. A severe constipation model was established using loperamide hydrochloride (administered at twice the normal dose). This induction resulted in a significant reduction in fecal water content (P < 0.01) (Fig. 3A) and a notable increase in both the time to first black stool defecation and the small intestine propulsion rate (P < 0.001) (Fig. 3B and C). These results confirm the successful establishment of a severe constipation model using loperamide hydrochloride.

Fig. 3 Effects of different Bifidobacterium species on gastrointestinal indicators in constipated mice. (A) Fecal water content. (B)Time to first black stool defecation. (C) Small intestine propulsion rate. Data are presented as means ± standard deviations. One-way ANOVA followed by Fisher's LSD test for control and strain groups; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the model group.

Upon simultaneous oral administration of six different Bifidobacterium species, only B. longum significantly increased fecal water content in constipated mice compared to the model group (P < 0.01) (Fig. 3A). The time to first black stool defecation and the small intestine propulsion rate are indicative of gastrointestinal transit capacity. Post-intervention, four Bifidobacterium species (B. longum, B. animalis, B. infantis, and B. adolescentis) significantly reduced the time to first black stool defecation in constipated mice compared to the model group (P < 0.05) (Fig. 3B), with B. longum showing the shortest defecation time. Moreover, both B. longum and B. bifidum significantly enhanced the small intestine propulsion rate (P < 0.05) (Fig. 3C). According to the 2022 edition of the Technical Code for Health Food Evaluation, only B. longum effectively alleviated loperamide hydrochloride-induced constipation under severe conditions.

B. longum significantly modulated fecal metabolite levels in constipated mice

The investigation into intestinal metabolites in constipated mice aimed to preliminarily determine whether B. longum's constipation-relieving effects are linked to host intestinal metabolites. Fecal untargeted metabolomics was characterized using LC-MS, yielding 134 metabolite abundances in positive ion mode and 137 in negative ion mode. A combined analysis revealed 179 significant metabolites, with raw data normalized for further analysis. Permutation tests and PCA analysis between the blank control and model groups demonstrated that Q² < 0, with both Q² and R² coordinates on the left side being lower than those on the right side (Fig. S1A†). Fig. S1B and C† show that fecal metabolites of the blank control and model groups were completely separated, as were those of the Bifidobacterium intervention groups from the control group, with samples from the same group clustering together. This indicates that loperamide hydrochloride-induced constipation significantly affects fecal metabolites in mice, and different Bifidobacterium interventions result in statistically significant changes in fecal metabolites, with minimal intra-group variation.

To identify potential differential metabolites in the feces following Bifidobacterium intervention, we performed Fold Change (FC) and P-value calculations on the data. Fig. 4A–B presents volcano plots of differential metabolites across various treatment groups and specifically after B. longum intervention. Comparing the metabolic profiles of each treatment group with the model group (P < 0.05, FC > 2 or FC < 0.5), it was observed that the differential metabolites in the NC group were predominantly amino acids, tryptophan, and fatty acids. group C was enriched mainly with amino acids, nucleotides, and tryptophan; group L was primarily concentrated in amino acids; group A in tryptophan and nucleotides; group D in organic acids and proteins; group Y in amino acids and antibiotic metabolites; and group Q in amino acids, biotin, and organic acids. Specifically analyzing the differential metabolites post-B. longum treatment (Fig. 4B), we identified 21 significantly different metabolites (P < 0.05, FC > 2 or FC < 0.5), with 16 metabolites significantly upregulated and 5 significantly downregulated. These metabolites were mainly amino acids, bile acids, tryptophan, and fatty acids. KEGG enrichment analysis of these differential metabolites (Fig. 4C) revealed enrichment in pathways related to tryptophan biosynthesis and phenylalanine metabolism following B. longum intervention. Consequently, our focus shifted to bile acids, tryptophan, and fatty acid metabolism for further targeted metabolite analysis.

Fig. 4 The effects of different Bifidobacterium species on nontargeted metabolomics in constipated mice. (A) Volcano plot of typical differential metabolites across different Bifidobacterium species. (B) Volcano plot of typical differential metabolites for B. longum. (C) Functional enrichment analysis of differential metabolites for B. longum.

B. longum modulates bile acid levels in the intestines of constipated mice but does not significantly affect tryptophan levels

Bile acid levels are causally related to colonic transit capacity, while dysregulation of tryptophan levels also impacts colonic motility. Targeted metabolomic analysis of bile acid content in cecal contents and tryptophan content in colonic contents of constipated mice was conducted using LC-MS, as shown in Fig. 5. Loperamide hydrochloride-induced constipation significantly reduced total bile acid levels in cecal contents (P < 0.001) and altered specific bile acid levels (Fig. 5A). Post-B. longum treatment, total bile acid levels were significantly upregulated, notably increasing CDCA, T-β-MCA, and GHDCA levels (P < 0.05). Conversely, all six Bifidobacterium species significantly reduced UDCA levels in constipated mice (P < 0.01). This aligns with our earlier findings that all B. longum strains possess bile salt hydrolysis capability, which was more pronounced than other Bifidobacterium species.

Fig. 5 The effects of different Bifidobacterium species on bile acid and tryptophan content in the intestines of constipated mice. (A) Bile acids. (B) Tryptophan.

Measurement of tryptophan levels in colonic contents revealed that 5-HT levels were significantly upregulated in the model group compared to the control group (P < 0.001). However, after Bifidobacterium intervention, all six species significantly downregulated 5-HT and XA levels in constipated mice (P < 0.05), with B. bifidum also significantly reducing IPA levels (P < 0.05). In summary, B. longum effectively restored certain bile acid levels in constipated mice, thereby enhancing gastrointestinal motility to alleviate constipation, but there were no major differences in tryptophan metabolites between B. longum and other Bifidobacterium.

B. longum significantly upregulates SCFA levels in constipated mice

Short-chain fatty acids (SCFAs) are pivotal in maintaining intestinal health, influencing intestinal transit capacity. In this study, constipated mice induced by loperamide hydrochloride exhibited significantly lower levels of six SCFAs (acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid) (P < 0.0001) (Fig. 6). This finding suggests a potential link between constipation and abnormal SCFA levels, corroborating our previous results that B. longum can utilize various oligosaccharides and proliferate rapidly. Post-intervention with Bifidobacterium, B. longum significantly increased the fecal content of acetic acid and propionic acid in constipated mice compared to the model group (P < 0.01) (Fig. 6A and B). Additionally, B. bifidum notably elevated propionic acid levels (P < 0.05) (Fig. 6B). Other Bifidobacterium interventions did not yield statistically significant changes in SCFA levels. In summary, B. longum effectively increased acetic and propionic acid levels in the feces of constipated mice, thereby alleviating constipation.

Fig. 6 The effects of different Bifidobacterium on SCFA content in the feces of constipated mice. (A) Acetic acid. (B) Propionic acid. (C) Butyric acid. (D) Valeric acid. (E) Isobutyric acid. (F) Isovaleric acid.

B. longum influences the composition of the intestinal flora in constipated mice, significantly increasing the relative abundance of Parabacteroides and Parasutterella

Alterations in intestinal flora are believed to contribute to constipation, with flora diversity indicative of its structure and composition. Analyzing the intestinal flora diversity in constipated mice, α-diversity results are shown in Fig. 7A and B, and β-diversity results in Fig. 7C. The α-diversity Chao1 and Fisher indices were significantly reduced in the model group compared to the blank control group (P < 0.01), suggesting that constipation is associated with altered intestinal flora. Both B. longum and B. bifidum significantly restored the α-diversity Chao1 and Fisher indices in the intervention group compared to the model group (P < 0.05), except for B. animalis and B. adolescentis, which only increased the α-diversity Chao1 index (P < 0.05). β-Diversity analysis via PCoA indicated significant changes in fecal flora between the model and blank groups (P < 0.05), suggesting loperamide hydrochloride-induced constipation alters host intestinal flora structure. All six Bifidobacterium species significantly modified the β-diversity of intestinal flora post-intervention (P < 0.05). Fig. 7D reveals that intervention groups were primarily enriched at the microbial phylum level of p_Firmicutes, p_Bacteroidetes, and p_Verrucomicrobia.

Fig. 7 The effects of different Bifidobacterium on the diversity and structure of the gut microbiota of constipated mice at the phylum level. (A) Chao1 index of α-diversity. (B) Fisher index of α-diversity. (C) A Bray–Curtis analysis based on the relative abundance of operational taxonomic units (OTUs) was performed to evaluate β-diversity. (D) Phyla of the gut microbiota. (E) LEfSe cladogram analysis. (F) Distribution histogram based on LDA of genus level. (G) The relative abundance of Parabacteroides. (H) The relative abundance of Parasutterella. (I) The relative abundance of Ruminiclostridium 5. (J) The relative abundance of Prevotellaceae UCG-001.

Differential gut microbial characteristics post-Bifidobacterium intervention were clarified using LEfSe analysis (Fig. 7E). Different interventions resulted in distinct intestinal flora changes, with unique characteristic flora for each group. At the genus level (Fig. 7F), the characteristic flora post-loperamide hydrochloride intervention included gutmetagenome, while the blank control group's characteristic flora were CandidatusSaccharimonas, CandidatusGastranaerophilalesbacteriumZag_111, Enterorhabdus, Bilophila, Adlercreutzia, and Anaerotruncus. Post-B. longum intervention, the relative abundance of Parabacteroides, Escherichia_Shigella, and Parasutterella significantly increased (Fig. 7G and H). Additionally, B. longum significantly upregulated the relative abundance of Ruminiclostridium 5 and Prevotellaceae UCG-001 (Fig. 7I and J). These findings suggest that B. longum can enrich specific flora and alter the intestinal flora structure and composition in constipated mice, thereby ameliorating constipation symptoms.

Relief of constipation by B. longum correlates with the strain's bile salt dissociation ability and modulation of intestinal flora and metabolism

Further correlation analysis was conducted to explore the relationships between the physiological properties of Bifidobacterium, indicators of constipation, microbial and metabolite levels. The results are illustrated in Fig. 8. Establishing correlations between the physiological properties of Bifidobacterium and constipation indicators (Fig. 8A) revealed that the oligosaccharide utilization capacity and bile acid hydrolysis capacity of Bifidobacterium were positively correlated with fecal water content. Conversely, growth generation time, osmotic pressure tolerance capacity, and cell adhesion capacity were negatively correlated with fecal water content. Additionally, growth generation time and cell adhesion capacity showed a positive correlation with the time to pass the first black stool, while oligosaccharide utilization capacity, bile acid hydrolysis capacity, and osmotic pressure tolerance capacity were negatively correlated with this parameter. Notably, bile acid hydrolysis capacity was significantly negatively correlated with the time to first black stool defecation (P < 0.05). Furthermore, growth generation time, cell adhesion capacity, and bile acid hydrolysis capacity were positively correlated with small intestinal propulsion rate, whereas oligosaccharide utilization capacity and osmotic pressure tolerance were negatively correlated with this rate.

Fig. 8 Correlation analysis of intestinal microbiota and physicochemical indexes in mice. (A) Pearson correlation analysis of the correlation between the physiological characteristics and intestinal bacterial abundance. (B) Pearson correlation analysis of the correlation between the intestinal bacterial abundance and physiological characteristics. (C) Pearson correlation analysis of the correlation between the intestinal bacterial abundance and constipation-related biomarkers.

A Pearson correlation analysis of the physiological characteristics of Bifidobacterium with the relative abundance of microorganisms at the genus level (Fig. 8B) demonstrated that the growth generation time of Bifidobacterium was significantly and positively correlated with the relative abundance of Lachnospiraceae FCS020 group, Akkermansia, and GCA-900066575 (P < 0.05). Conversely, oligosaccharide utilization was significantly negatively correlated with the relative abundance of Lachnospiraceae FCS020 group and GCA-900066575 (P < 0.05). The bile salt dissociation ability of Bifidobacterium was significantly positively correlated with the relative abundance of Marvinbryantia and significantly negatively correlated with Muribaculum and Ruminiclostridium 1 (P < 0.05). The relative abundance of microorganisms at the genus level was also correlated with constipation-related gastrointestinal indicators and differential metabolite levels (Fig. 8C). For instance, the relative abundance of Catabacter, Enterorhabdus, Escherichia-Shigella, Parabacteroides, and Ruminiclostridium 5 was significantly positively correlated with fecal water content (P < 0.05), while Lactobacillus and Blautia showed a significant negative correlation (P < 0.05). Additionally, the relative abundance of Ruminococcus 2 was positively correlated with the time to first black stool defecation (P < 0.05), whereas several other genera including Adlercreutzia, Ruminococcaceae NK4A214 group, and [Eubacterium] nodatum group were negatively correlated (P < 0.05). The analysis further revealed significant correlations between various genera and metabolite levels. For example, the relative abundance of Anaerotruncus, Marvinbryantia, and others was positively correlated with acetic acid content in feces (P < 0.05), while Candidatus soleaferrea and Staphylococcus showed negative correlations (P < 0.05). The relative abundance of Romboutsia was negatively correlated with propionic acid content (P < 0.05). Additionally, certain genera such as Ruminiclostridium 5, Adlercreutzia, and others were positively correlated with total bile acid levels, while others like Blautia showed negative correlations (P < 0.05).

The findings suggest a significant positive correlation between the relative abundance of characteristic intestinal flora such as Parabacteroides, Escherichia_Shigella, and Parasutterella, and various physiological indicators including fecal water content, small intestinal propulsion rate, butyric acid content, and CDCA level after intervention with B. longum. This indicates that the bile salt dissociation ability of Bifidobacterium is linked to constipation relief, highlighting that B. longum can ameliorate intestinal flora disorders induced by loperamide hydrochloride, thereby alleviating constipation.

Discussion

Constipation is a multifaceted condition,³⁰ implicating various digestive organs and host metabolic cycles. Beyond the roles of digestive organs and neural networks in the transition from food intake to fecal excretion, the host's intestinal microbiota significantly influences this process.³¹ Moreover, the physiological properties and probiotic functions of specific strains are crucial in mitigating constipation. Bifidobacterium, a commonly utilized probiotic, exhibits considerable variability in its efficacy across different species.⁷ This study aims to elucidate the differential impacts of various Bifidobacterium species on constipation relief and to preliminarily explore the underlying mechanisms.

Previous studies have demonstrated that Bifidobacterium significantly reduces whole-bowel transit time and alleviates symptoms such as abdominal pain, nausea, and irregular bowel movements.³² Ishizuka. et al.³³ reported that B. lactis GCL2505 notably promoted the proliferation of intestinal Bifidobacterium, increased bowel movement frequency, and enhanced fecal weight. Consistent with these findings, our results indicate that different Bifidobacterium species exhibit varying efficacies in alleviating severe constipation induced by loperamide hydrochloride in mice. Notably, only B. longum effectively improved fecal dryness and hardness while enhancing gastrointestinal motility, thereby relieving constipation. Similarly, Wang et al.³⁴ found that a single-strain study involving three different Bifidobacterium species revealed that B. longum promoted intestinal peristalsis and optimized water and electrolyte metabolism, leading to constipation relief. A recent clinical trial further corroborated these findings, demonstrating that a two-week supplementation with B. longum CLA8013 significantly increased bowel movement frequency and improved stool consistency, straining, and pain during defecation in constipated patients.³⁵ These observations suggest that B. longum holds a distinct advantage in ameliorating constipation-related symptoms, potentially attributable to its unique genetic characteristics, probiotic properties, and metabolic capacity.

To further elucidate the mechanisms by which B. longum alleviates constipation, we conducted a comprehensive analysis encompassing both the physiological properties of the strain and its effects on the host's intestinal flora and metabolism. The intrinsic physiological characteristics of B. longum significantly influence its probiotic efficacy. Generation time, defined as the duration required for a single microorganism to complete one division cycle, is a critical determinant of growth rate. Our findings indicate that B. longum exhibits a superior growth capacity compared to other Bifidobacterium species, with minimal inter-strain variability. Functional oligosaccharides, which are resistant to absorption in the stomach and intestines, are metabolized by intestinal bacteria into SCFAs and gases. These metabolites lower intestinal pH, inhibit pathogenic bacteria, enhance peristalsis, and alleviate constipation.³⁶ Our results demonstrate that B. longum efficiently utilizes a diverse array of oligosaccharides and thrives in various carbon source environments. This rapid growth and metabolic versatility likely underpin its effectiveness in relieving constipation, as a critical mass of Bifidobacterium is necessary for therapeutic benefits. Supporting our findings, Wang et al.³⁷ reported that dietary oligosaccharides increased fecal water content, reduced intestinal transit time, elevated levels of Lactobacillus and Bifidobacterium, and boosted SCFA concentrations in constipated mice. Specifically, B. longum significantly elevated acetic acid levels, corroborating our results. The lower incidence of gastrointestinal disorders in breastfed infants, attributed to the oligosaccharides in breast milk fostering a higher proportion of Bifidobacterium, further underscores the importance of oligosaccharide utilization.³⁸ Additionally, mixtures of GOS and FOS have been shown to selectively stimulate Bifidobacterium growth in infants.³⁹ Bile acids, synthesized in the liver and metabolized by bile salt hydrolase (BSH)-producing bacteria into secondary bile acids, play a crucial role in gut motility. Our qualitative assays revealed that all B. longum strains possess BSH activity, indicating their ability to hydrolyze bile salts. Animal studies have shown that germ-free mice supplemented with BSH-active bacterial consortia exhibit higher levels of secondary bile acids, enhanced colonic propulsion, and reduced colonic transit times.⁴⁰ These findings suggest a strong link between the physiological properties of B. longum—including rapid growth, oligosaccharide utilization, and bile salt hydrolysis—and its efficacy in alleviating constipation.

Bifidobacterium metabolites, such as acetic acid, enhance transepithelial resistance (TEER) and down-regulate tumor necrosis factor (TNF-α) levels, thereby augmenting tight junction protein expression.⁴¹ These metabolites also facilitate the repair of monolayer wounds in colon adenocarcinoma cells (Caco-2) and bolster intestinal epithelial function.⁴² In a study where constipation was induced through a low-fiber diet, Bifidobacterium BBG9-1 increased the relative abundance of butyric acid-producing bacteria in the intestines of constipated rats, consequently elevating butyric acid concentrations and promoting colonic motility.⁴³ Our results indicate that B. longum significantly up-regulated acetic and propionic acid levels in the feces of constipated mice. Numerous studies have demonstrated that acetic acid promotes the release of 5-HT, enhancing the intestinal peristaltic reflex and accelerating the transport rate of intestinal contents.⁴⁴ Additionally, B. longum can effectively inhibit Clostridium difficile infection by producing acetic acid, competing with pathogenic bacteria for ecological niches, and forming an intestinal chemoprotective barrier.⁴⁵ SCFAs are undoubtedly key factors influencing host intestinal motility, and alterations in their levels can significantly impact the development of constipation.

Bile acid metabolism is also intricately linked to constipation. Bile acids activate specific receptors, thereby regulating host metabolism.⁴⁶ These molecules activate receptors in the intestine, liver, and periphery, which in turn regulate immune homeostasis, metabolism, and intestinal motility.⁴⁷ Studies have shown that patients with constipation exhibit altered bile acid metabolism, with lower gut bile acid levels and longer colonic transit times compared to healthy individuals.⁴⁸ Our results revealed that B. longum intervention significantly up-regulated total bile acid levels in the intestines of constipated mice, including significant increases in CDCA, T-β-MAC, and GHDCA levels, while UDCA levels significantly decreased. Thus, elevated bile acid levels correlate with improved colonic transit,⁴⁹ a conclusion supported by our findings.

The metabolic changes induced by Bifidobacterium intervention are closely tied to alterations in the intestinal flora. Parabacteroides, a core group in the human gut,⁵⁰ emerged as a characteristic genus following B. longum supplementation, similar to findings by Yuan et al.⁵¹ This suggests that disruptions in the intestinal flora directly or indirectly affect host intestinal motility. The alleviating effect of B. longum on constipation is thus intricately linked to the gut microbiota and its metabolic activities. Previous reports indicate significant differences between the gut microbiota of constipated patients and healthy populations, with microbial changes potentially affecting intestinal viability and secretory functions through competitive colonization and metabolite concentration alterations.⁵² Early studies have identified Eubacterium as having a distributional profile of potential driver bacteria.⁵³ Our results show that Eubacterium's relative abundance is significantly correlated with gastrointestinal indicators such as time to first black stool defecation. Eubacterium has been associated with SCFA production and bile acid metabolism in studies by Kanauchi et al.⁵⁴ and Han et al.⁵⁵ SCFAs produced by microbial metabolism influence colonic 5-HT and MTL-containing enteroendocrine cells, playing a role in colonic physiological regulation.⁵⁶ This explains the changes in host SCFA and bile acid levels following B. longum intervention, likely connected to alterations in gut flora composition. Additionally, our results show that Enterorhabdus's relative abundance is significantly positively correlated with gastrointestinal indicators such as fecal water content and small intestinal propulsion rate. This finding aligns with previous studies by Hu⁵⁷ and Zhang,²⁸ which reported increased Enterorhabdus abundance following substance/probiotic interventions to ameliorate constipation symptoms. Ruminiclostridium, known to contribute to overall metabolic processes,⁵⁸ showed increased relative abundance following probiotic dietary modifications, correlating positively with secondary bile acid and bound bile acid content,⁵⁹ consistent with our results. Prevotellaaceae UCG-001, a genus within the Prevotellaceae family, is widely recognized for promoting SCFA production.⁶⁰ It has been shown to have anti-inflammatory properties and may alleviate glycolipid metabolism disorders.⁶¹ Zhang's study⁶² found that improving diarrhea symptoms with an aqueous extract of Epiphyllum officinale increased Prevotellaceae UCG-001's relative abundance in feces while restoring bile acid homeostasis. These studies collectively suggest a strong link between changes in intestinal flora and host metabolism, with the physiological characteristics of strains themselves playing a crucial role.

In summary, B. longum demonstrates significant potential in alleviating constipation through its robust probiotic functions. By constructing a severe constipation model using loperamide hydrochloride, we elucidated the differences in constipation relief among various Bifidobacterium species, focusing on both the strain-specific characteristics and host interactions. At the strain level, B. longum exhibited a broad-spectrum capacity for oligosaccharide utilization, rapid growth rates, and a universal ability to hydrolyze bile salts. These attributes enable B. longum to proliferate efficiently in the gut, enhance SCFAs and bile acid metabolism, and accelerate colonic transit, thereby mitigating constipation. At the host level, among the six Bifidobacterium species studied, only B. longum effectively ameliorated constipation-related symptoms in mice. This was achieved by altering the composition and structure of the host intestinal flora, increasing the abundance of bacteria associated with SCFA production and bile acid metabolism, and restoring SCFA and bile acid levels. Consequently, these changes enhanced gastrointestinal peristalsis and alleviated constipation.

Ethical approval and consent to participate

The animal study protocol was approved by the Ethics Committee of Implementation Methods of Laboratory Animal Management in Jiangsu Province (qualified no: jn. no. 20221030b1350515[463]).

Author contributions

Conceptualization: Wang L. and Wang G.; methodology: Zhao J.; software: Wang L.; validation: Zhang C.; formal analysis: Wang L.; investigation: Zhang C.; resources: Wang L., Liu X., Wang G., Zhao J., and Chen W.; data curation: Zhao J.; writing – original draft preparation: Zhang C.; writing – review and editing: Wang L.; visualization: Zhang C.; supervision: Wang L. and Wang G.; project administration: Wang G.; funding acquisition: Wang L., Wang G., and Chen W.

Data availability

All data generated or analyzed during this study are included in this published article.

Conflicts of interest

All authors declared that there are no conflicts of interest.

Acknowledgements

This research was financially supported by the National Key R&D Program of China (2022YFF1100400).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4fo04660a

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