Materials
Bacillus coagulans (BC) was purchased from BeNa Culture Collection. Rosmarinic acid (RA) was obtained from Shanghai Macklin Biochemical Technology Co., Ltd. Silk fibroin (SF) was extracted from tussah silkworms. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. Enterobacteriaceae selective MacConkey agar plates were obtained from Qingdao Rishui Bio-Technologies Co., Ltd. 2,2’-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO) were purchased from Shanghai Titan Scientific Co., Ltd. Pepsin and trypsin were obtained from Shanghai Macklin Biochemical Technology Co., Ltd. Dextran sulfate sodium (DSS, M.W. 36,000–50,000) was purchased from Regent Science Industry Limited. Institute of Cancer Research (ICR) female mice were obtained from Hunan SJA Laboratory Animal Co., Ltd. Fluorescein isothiocyanate (FITC) was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. Enzyme-linked immunosorbent assay (ELISA) kits were obtained from MultiSciences Biotech Co., Ltd. FreeZol Reagent R711 kit was purchased from Nanjing Vazyme Biotech Co., Ltd. Evo M-MLV RT Mix Kit with gDNA Clean for qPCR Ver.2 and SYBR Green Premix Pro Taq HS qPCR Kit were obtained from Accurate Biotechnology (Hunan) Co., Ltd.
Spore preparation
Spores were prepared using MRS medium supplemented with 0.05% MnSO4·H2O as sporulation medium. 100 mL of sporulation medium was inoculated with 200 µL of activated BC and incubated with shaking at 150 rpm for 48 h at 37 °C. After 48 h, the medium was heated in an 80 °C water bath for 30 min to ensure the killing of vegetative cells. The spores were collected by centrifugation at 10,000 rpm for 30 min at 4 °C and washed three times with 1 M KCl/0.5 M NaCl solution, then resuspended in ultrapure water and stored at -20 °C.
Preparation of Spore-RA and Spore-RA-SF
RA was dissolved in ultrapure water, mixed with EDC and NHS at a ratio of 3:3:1, and incubated at 150 rpm for 1 h at 37 °C to activate carboxyl groups. Then, the purified spores were added to the mixed solution and incubated with shaking at 150 rpm for 24 h at 37 °C. After 24 h of reaction, the mixed solution was centrifuged at 4000 rpm for 5 min at 4 °C to collect Spore-RA, then Spore-RA was washed three times with phosphate buffer (pH 6.0) containing 0.01 M Na+. To prepare Spore-RA-SF, 0.1% (w/v) SF was added to the resuspended Spore-RA under shaking at 35 rpm for 10 min. The mixed solution was centrifuged at 4000 rpm for 3 min at 4 °C, and cells were washed once with ultrapure water. Then, the cells were resuspended in phosphate buffer (pH 5.5) containing 0.1 M K+ and shaken vigorously at 1000 rpm for 10 min. After centrifuging the mixed solution and washing cells again, Spore-RA-SF was collected by centrifugation at 10,000 rpm for 5 min at 4 °C. After repeating the above steps four times, the Spore-RA-SF with multi-layer coating was obtained. Spore, Spore-RA, and Spore-RA-SF were characterized using scanning electron microscopy (SEM), atomic force microscopy (AFM), nanoparticle size, zeta potential, Fourier transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD), and fluorescence microscopy. The activities of Spore, Spore-RA, and Spore-RA-SF were determined by the plate-counting method.
Measurement of growth curves
100 mL of MRS medium was inoculated with equal amounts of Spore, Spore-RA, or Spore-RA-SF. Beginning at 0 h, 0.1 mL of the medium was taken and diluted 10 times every 2 h, and its absorbance at 600 nm was measured.
Bacteriostatic test
100 mL of LB medium was inoculated with equal amounts of Spore, Spore-RA, or Spore-RA-SF and incubated statically for 10 h at 37 °C, and then 200 µL or 2 mL of Escherichia coli (E. coli) DH5α was inoculated into the medium. After incubation for 12 h at 37 °C, the medium was diluted 100,000 times and spread on Enterobacteriaceae selective MacConkey agar plates, which were then incubated for 12 h at 37 °C. The colonies on the plates were observed and counted to evaluate the inhibitory effect of spores on E. coli DH5α.
Dissolved oxygen assay
Beginning at 0 h, the medium of Spore or Spore-RA-SF was transferred to dissolved oxygen bottles every 2 h. 1 mL of 2.15 M MnSO4 solution and 2 mL of 0.90 M alkaline KI solution were added below the liquid level successively using pipettes, and then the bottles were inverted and mixed 15 times. When the brown flocculent sediment in the bottles dropped to half, the bottles were inverted and mixed several times again. Before analysis, the bottle caps were opened gently, and 2 mL of sulfuric acid (pH 1.84) was added below the liquid level using pipettes. After all the sediment was dissolved, the bottles were placed in the dark for 5 min. 100 mL of the above solution was transferred into 250 mL conical flasks and then titrated with 0.0129 M Na2S2O3 standard solution until it turned slightly yellow. Finally, 1 mL of 1% starch solution was added to the solution, which was then titrated continually with Na2S2O3 solution until the blue just faded away. Dissolved O2 (mg L− 1) = 1.032 × V (V represents the volume (mL) of Na2S2O3 standard solution consumed by titration).
ROS/RNS scavenging abilities
ABTS assay: Firstly, 7 mM ABTS solution was mixed with 2.45 mM K2S2O8 in the dark for 12–16 h to obtain ABTS•+ solution, which was then diluted until the absorbance at 734 nm reached about 0.7 ± 0.2. Then, 100 µL of different materials with the same concentration were mixed with 3.9 mL of ABTS•+ working solution. After 20 min of incubation at room temperature, the absorbance at 734 nm of the reaction solution was determined. ABTS•+ scavenging capacity was assessed through Equation:
ABTS•+ scavenging ratio (%) = (Ab – As) / Ab × 100% (Ab and As represent the absorbance at 734 nm of the original ABTS•+ solution and reacted ABTS•+ solution, respectively.)
DPPH assay: Firstly, 2 mg of DPPH was dissolved in 24 mL of absolute ethanol and sonicated for 5 min for sufficient dissolution. Then, 100 µL of different materials with the same concentration were mixed with 3.9 mL of DPPH• solution. After 30 min of incubation in a 37 °C water bath, the absorbance at 519 nm of the reaction solution was determined. DPPH• scavenging capacity was assessed through Equation:
DPPH• scavenging ratio (%) = (Ab – As) / Ab × 100% (Ab and As represent the absorbance at 519 nm of the original DPPH• solution and reacted DPPH• solution, respectively.)
PTIO assay: Firstly, 3 mg of PTIO was dissolved in 20 mL of ultrapure water and sonicated for 5 min for sufficient dissolution. Then, 100 µL of different materials with the same concentration were mixed with 3.9 mL of PTIO• solution. After 2 h of incubation in a 37 °C water bath, the absorbance at 557 nm of the reaction solution was determined. PTIO• scavenging capacity was assessed through Equation:
PTIO• scavenging ratio (%) = (Ab – As) / Ab × 100% (Ab and As represent the absorbance at 557 nm of the original PTIO• solution and reacted PTIO• solution, respectively.)
O2•– assay: Firstly, 20 µM riboflavin, 12.5 mM methionine, 75 µM nitroblue tetrazolium (NBT), and different materials with the same concentration were mixed in PBS (pH 7.4). Then, the mixed solution was constantly illuminated for 15 min at room temperature. After the reaction, the mixed solution was centrifuged at 4000 rpm for 3 min, and the absorbance at 560 nm of the supernatant was determined. O2•– scavenging capacity was assessed through Equation:
O2•– scavenging ratio (%) = (A0 – An) / (Ap – An) × 100% (A0, An, and Ap represent the absorbance at 560 nm of the sample, negative control, and positive control, respectively.)
H2O2 assay: Firstly, 0.1 g of (NH4)6Mo7O24·4H2O was dissolved in 250 mL of 0.5 M H2SO4 to obtain molybdate chromogenic agent. Then, 100 mM H2O2 and different materials with the same concentration were mixed in PBS. After 30 min of incubation in a 37 °C water bath, the reaction solution was mixed with an equal volume of molybdate chromogenic agent. Then, the absorbance at 330 nm of the mixed solution was determined. H2O2 scavenging capacity was assessed through Equation:
H2O2 scavenging ratio (%) = (A0 – An) / (Ap – An) × 100% (A0, An, and Ap represent the absorbance at 330 nm of the sample, negative control, and positive control, respectively.)
Survival of Spore, Spore-RA, and Spore-RA-SF after simulated gastrointestinal fluid treatment
Preparation of simulated gastric fluid (SGF): 3.2 g pepsin and 2.0 g NaCl were dissolved in 500 mL ultrapure water, and 7 mL concentrated hydrochloric acid was mixed in the solution, which was then adjusted the pH to 2.0 with dilute hydrochloric acid. The mixed solution was supplemented with ultrapure water to a total volume of 1 L, and SGF (pH 2.0) was obtained by filtering the mixed solution with a 0.22 μm filter membrane.
Preparation of simulated intestinal fluid (SIF): 6.8 g KH2PO4 was dissolved in 450 mL ultrapure water, and 77 mL 0.2 M NaOH and 10 g trypsin were mixed in the solution, which was then adjusted the pH to 6.8 with dilute hydrochloric acid. The mixed solution was supplemented with ultrapure water to a total volume of 1 L, and SIF (pH 6.8) was obtained by filtering the mixed solution with a 0.22 μm filter membrane.
Preparation of simulated colonic fluid (SCF): 5.59 g dipotassium hydrogen phosphate and 0.41 g potassium dihydrogen phosphate were dissolved in 1 L ultrapure water, which was then adjusted the pH to 7.8. SCF (pH 7.8) was obtained by filtering the mixed solution with a 0.22 μm filter membrane.
Equal amounts of Spore, Spore-RA, or Spore-RA-SF were incubated in SGF, SIF, or SCF for 1 h at 37 °C. The incubated solution was diluted 1,000,000 times and spread on MRS agar plates, which were then incubated for 24 h at 37 °C. The colonies on the plates were observed and counted to evaluate the survival of Spore, Spore-RA, and Spore-RA-SF after simulated gastrointestinal fluid treatment.
ROS/RNS scavenging capacities in simulated gastrointestinal environment
Equal amounts of Spore-RA or Spore-RA-SF were incubated in SGF, SIF, or SCF for 1 h at 37 °C. After incubation, ROS/RNS scavenging capacities were determined by the above methods.
In vivo targeting test
Forty-five mice were divided into three groups (fifteen mice per group), and the drinking water of two groups was supplemented with 3% DSS for 7 d to induce colitis after adapting to the environment for one week. Next, mice in the normal group were orally administered FITC-labeled Spore-RA-SF (5 × 108 CFU per mouse), and mice in the two groups with colitis were orally administered FITC-labeled Spore or Spore-RA-SF (5 × 108 CFU per mouse), respectively. At 2, 4, 8, 12, or 24 h after oral administration, the mice were sacrificed (three mice per group at each time point), and the intestines were collected for in vivo imaging system (IVIS) imaging.
Biosafety assessment
Six mice were divided into two groups (three mice per group) and orally administered the same volume of PBS or Spore-RA-SF (5 × 108 CFU per mouse) on days 0, 2, 4, and 6. The weight of the mice was observed every day. The mice were sacrificed on day 7, and their blood samples were collected from the orbital venous sinus for the erythrocyte hemolysis test. The major organs (heart, liver, spleen, lung, and kidney) were also collected for histopathological evaluation by hematoxylin and eosin (H&E) staining.
Erythrocyte hemolysis test: Mouse orbital venous sinus blood samples collected with anticoagulant tubes were centrifuged at 3000 rpm for 5 min at 4 °C. After removing the supernatant, the precipitate was washed three times with normal saline. The obtained blood cell solution was mixed with normal saline at a volume ratio of 4:5, and then 0.2 mL of the suspension was mixed with 5 mL of ultrapure water, normal saline, or different concentrations of Spore-RA-SF for 30 min at 37 °C. After additional incubation for 1 h at 37 °C, the solution was centrifuged at 3000 rpm for 5 min, and the absorbance at 545 nm of the supernatant was measured.
Histopathological evaluation: The collected tissues were fixed in 4% paraformaldehyde for 24 h, and then rinsed with running water for 1 h to remove excess fixative. After alcohol dehydration and xylene treatment, the tissues were embedded in paraffin and sectioned. The sections were dewaxed and H&E stained after being fixed to glass slides. Finally, the sections were observed with optical microscopy for histopathological evaluation.
Therapeutic efficacy against mouse colitis
Thirty mice were divided into six groups (five mice per group), and the drinking water of five groups was supplemented with 3% DSS for 7 d to induce colitis after adapting to the environment for one week. Next, mice in the four groups with colitis were orally administered RA (5 mg kg− 1), Spore, Spore-RA, or Spore-RA-SF (5 × 108 CFU per mouse) on days 0, 2, 4, 6, and 8, respectively. Mice in the other group with colitis and in the normal group were orally administered equal amounts of PBS. During the treatment, the weight of the mice was recorded every day. On day 10, the mice were sacrificed, their colons were collected and photographed, and the colon length was recorded.
Disease activity assay
During the treatment, the body weight, stool consistency, and hematochezia of mice with different treatments were recorded every day. Disease activity index (DAI) was calculated according to the scoring schemes of DAI parameters in Supplementary Table 1 (the final score was the average of the scores of various parameters).
Colonic damage assessment
The distal colons of mice were subjected to H&E staining and observed by optical microscopy. The colonic damage score was assessed in a blinded fashion to avoid observer bias, according to the histological grading schemes in Supplementary Table 2 (the final score was the sum of the scores of colonic epithelial damage and inflammatory cell infiltration).
Myeloperoxidase (MPO) activity detection
The MPO activity in the colon tissues of mice was detected by an immunohistochemical test and observed by optical microscopy.
Colon tissue cytokine detection
Colon tissues were soaked in PBS at a w/v ratio of 1:10 and then homogenized. The concentrations of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-4, and IL-10 in colon tissues were determined by ELISA kits.
Gut microbiota analysis
Fresh mouse colon contents were snap frozen in liquid nitrogen and sent to Biomarker Technologies Co., Ltd. for 16 S ribosomal RNA (16 S rRNA) sequencing. The reads of each sample were spliced using FLASH v1.2.11, and the raw Tags sequence data with a minimum overlap length of 10 bp and a maximum allowable mismatch ratio of 0.2 in the overlap area were obtained. After filtering Tags whose length was less than 75% of the tag length after quality control using Trimmomatic v0.33 and obtaining high-quality Clean Tags, chimeras in Clean Tags were removed by UCHIME v8.1 to obtain high-quality tag sequences. Next, sequences were clustered using USEARCH (v10.0) at a level of 97% similarity to filter OTUs at a threshold of 0.005% of all the sequence numbers. Sequencing results were analyzed on the BMKCloud data analysis platform.
Transcriptomic analysis
Total RNA in the colon tissues of mice was extracted by standard kits, and the concentration and integrity of the RNA were detected using Nanodrop 2000 and Agilent 2100. The library construction and purification of the extracted total RNA were conducted using Hieff NGS Ultima Dual-mode mRNA Library Prep Kit for Illumina (Yeasen Biotechnology (Shanghai) Co., Ltd.) and Hieff NGS DNA selection Beads (Yeasen Biotechnology (Shanghai) Co., Ltd.). The mRNA sequencing was performed at Biomarker Technologies Co., Ltd. using the Illumina NovaSeq 6000 platform.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) tests
Total RNA in the colon tissues of mice was extracted by standard kits and quantified. 1 µg RNA from each sample was taken for gDNA removal and reverse transcription. Finally, the cDNA was subjected to qPCR (primers listed in Supplementary Table 3) by the SYBR Green method.
Statistical analysis
All experimental results were presented as the mean ± standard deviation. When two groups were compared, two-tailed Student’s t tests were performed. Statistical significance was expressed as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
