General
We purchased Lactobacillus plantarum from Yaxin Biotechnology in Taiwan. The saccharomyces cerevisiae (active dry yeast) was purchased from Angel Yeast Co.Ltd. Acetic acid bacteria (Hu Niang No. 1.01 Acetic acid bacteria) was purchased from Shanghai Difa Brewine-Biotechnology Co., Ltd. Gas chromatograph (Trace1300; Thermo Fisher Scientific, Waltham, MA, USA). LC-MS analysis was conducted using the 5600+ qTOF ESI MS system, which was manufactured by AB Sciex Pte. Ltd, located in MA, USA. Adenosine, gastrodin, 4-hydroxybenzyl alcohol, parishin A, B, C, and E were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Pentylenetetrazole (PTZ) was procured from Macklin Biochemical Technology Co. Ltd. (Shanghai, China). Oxazepam was bought from Yimin Pharmaceutical Factory (Beijing, China). AB wild adult zebrafish were procured from Shanghai FishBio Co. Ltd. (Shanghai, China). Energy Chemical Co. Ltd (Shanghai, China) supplied the organic solvents for the UPLC-QE-MS/MS.
Sample preparation of GE at different processing stages
After fresh GE was cleaned, it was put into the fermentation tank, decocted with an appropriate amount of water for 90 min, and filtered to obtain GE extract (WGE). Next, the lactic acid bacteria strain solution was added to the sugar-sweetened WGE at the inoculation rate of 3% (V/V), after anaerobic fermentation at 37 °C for 24 h, And then the yeast strain solution was added. The prime fermentation liquid (PFL) was obtained after anaerobic co-fermentation at 28 °C for 15 days and sterilization at 60 °C for 30 min. Finally, an acetic acid bacteria strain solution was added to the PFL. After about 50 days of aerobic fermentation at 30 °C, the fermentation was completed and the secondary fermentation liquid was prepared. The supernatant of fermentation liquid at different processing stages was collected, and all water was removed by freeze-drying to obtain GE samples at different processing stages.
Zebrafish locomotor behavior investigation
Zebrafish maintenance and embryo collection were carried out according to a reported protocol36,37. The Institutional Animal Care & Use Committee of Northwest A&F University approved the animal experimental protocol (Protocol Permit Number: XN2023-0902, 15 March 2022). The investigation of zebrafish mobility was conducted in accordance with the previously established procedure outlined by our research group 37. The zebrafish larvae at 5 dpf were subjected to treatment with varying quantities (20 µg/ml and 80 µg/ml) of WGE, PFL, and SFL samples for 4 hours. The zebrafish groups were individually allocated into separate compartments of a 6-well plate, followed by the addition of 2.5 mM PTZ. Following 10 minutes of adaptation, the larvae were observed and their behavior was recorded for durations of 30 and 50 minutes, respectively. The video was subjected to analysis using ethovision software, which was developed by Noldus Information Technology bv. in the Netherlands.
Assessment of neuronal apoptosis
The method described in the literature was utilized to carry out our experiment 38. In brief, zebrafish larvae at 5 dpf were subjected to treatment with varying quantities (20 µg/ml and 80 µg/ml) of WGE, PFL, and SFL samples for 4 hours. The zebrafish were segregated into distinct groups and thereafter introduced into individual compartments of a 6-well plate. Following this, a concentration of 2.5 mM PTZ was administered to each group for 10 minutes. Following that, the larvae were subjected to two rinses using phosphate-buffered saline (PBS) and subsequently exposed to a concentration of 5 mg/L acridine orange (AO) in a light-restricted setting for 30 minutes. After undergoing three rounds of washing with phosphate-buffered saline (PBS), the subjects were anesthetized with the administration of 0.016% tricaine in culture water. The Nikon SMZ25 fluorescent stereoscope, manufactured by Nikon Corporation in Tokyo, Japan, was used to observe apoptotic nerve cells.
Quantitative analysis of the main medicinal components of fermented GE
Briefly, the fermentation broth (5 ml) was centrifuged in a sterile centrifuge tube at 5,000 r/min for 10 min. The supernatant was collected and filtered through a 0.22 μm nylon filter, and 1 mL of the supernatant was analyzed by high-performance liquid chromatography (HPLC). Detection was performed using an Ultimate3000 HPLC-UV (Thermo Fisher Scientific, USA) with a Century SIL C18 column (250 × 4.6 mm, 5 μm). The mobile phase consisted of 0.1 % phosphoric acid in water for phase A and acetonitrile for phase B. The injection volume was set at 20 μL, the flow rate was 1 mL/min, the column temperature was set at 30 °C, and the detection wavelength was 220 nm for quantification of adenosine, 4-hydroxybenzyl alcohol, parishin B, parishin E, gastrodin, parishin A, and parishin C in WGE, PFL, and SFL. The elution gradient was as follows: 0-10.0 min, 3%-10 % B; 10.0-15.0 min, 10 %-12 % B; 15.0-25.0 min, 12 %-18 % B; 25.0-40.0 min, 18 % B; and 40-40.1 min, 18%-3 % B; 40.1-50.0 min, 3 % B.
Metabolite extraction and LC-MS analysis of GE at different processing stages
Three portions of WGE (WGE1, WGE2, WGE3) and PFL (PFL 1, PFL2, PFL3) 、SFL (SFL 1, SFL2, SFL3) freeze-dried samples were precisely weighed and were respectively dissolved in MeOH solution to prepare the sample solution with the concentration of 100 μg/mL. UPLC-ESI-MS analysis and metabolite extraction procedures were conducted using the protocols outlined in our previously published works37. Furthermore, the PLS-DA analysis was performed using the Metaboanalyst website (https://www.metaboanalyst.ca/, visited on July 2, 2023).
Zebrafish Larvae preparation for transcriptome and metabolism analysis
In brief, the blank group (G1) was given E3 water, the model group (G2) was to add 2.5 mM PTZ based on G1, and the test group (G4) was given SFL (80 µg/ml) for 4h, and then 2.5 mM PTZ was added for 10 minutes. On day five, zebrafish larvales were gathered. Before use, centrifuge tubes were prepared and weighed. Each tube held a single hundred fish, which were then given three rounds of clean water washing before being killed in liquid nitrogen. Lastly, the samples were kept for transcriptomic, metabolomic, and PCR analysis in a refrigerator at ‒80 °C.
RNA sequencing and data analysis of Zebrafish larvae
Three separate biological replicates of the 5 dpf Zebrafish larvae of the G1, G2, and G4 groups were used. For details of the experiment, please refer to the previous paper of our study group 36. The BGI (http://report.bgi.com) processed RNA-seq data and carried out gene set enrichment analysis (GSEA) utilizing the Dr. TOM technique, an internal, specially designed data mining system of the BGI. Enriched gene sets were allocated based on a nominal p-value of less than 0.05 and an FDR q-value of less than 0.25. The use of gene set enrichment analysis (GSEA) was used to uncover trends related to the overrepresentation of biological terms in the transcript.
LC-MS/MS profiling of metabolism
For sample processing methods and condition parameter settings, please refer to the previously published articles by our team36,39. Next, we used the Oebiotech tools, a free online platform for data analysis (https://cloud.oebiotech.cn/task), to do pathway enrichment.
Evaluation of short-chain fatty acids
The separation was performed using a Thermo TG-WAXMS capillary column with dimensions of 30 m in length, 0.25 mm in internal diameter, and a particle size of 5 μm. Gas chromatography equipment was employed for this purpose. The samples were subjected to centrifugation at a velocity of 15,000 revolutions per minute and a temperature of 4 °C for five minutes until a transparent supernatant became visible. Using a 0.45 μm pore size membrane, the supernatant from various fermentation stages was collected in a 1 mL syringe and filtered. The organic phase was gathered and subjected to the following gas chromatography analysis. The temperature was started at 100 °C and increased gradually over the next three minutes to 150 °C at 5 °C/min, 220 °C at 70 °C/min, and finally, to 220 °C, where it remained for three minutes. The carrier gas, helium, was employed at a flow rate of 2.0 mL/min. The injection volume of the sample was 1 μL, and it was divided at a ratio of 1:10 at a temperature of 230°C, with an inlet split flow rate of 20 ml/min. The flame ion detector (FID) used for the GC had a temperature of 250 °C. To create the standard curve, the concentration was utilized as the abscissa and the peak area as the ordinate. The content was then computed using the standard curve.
RT-qPCR Analysis
RT-qPCR assays were utilized to validate the extent of gene expression. The three groups (G1, G2, and G4) were conducted consistently, following the sample preparation procedure outlined in Section 2.7. The RNA extraction process utilized AG RNAex Pro RNA kit, manufactured by Accurate Biotechnology (Hunan) Co., Ltd. The retrotranscription reactions were performed using the EVO M-MLV RT Mix Kit (Accurate Biotechnology Co., Ltd., China). The 5X gDNA Clean Reaction Mix reagent contained in the kit was employed to eliminate DNA, whereas the 5X Evo M-MLV RT Reaction Mix was utilized to perform the reverse transcription of RNA into DNA. The sequences of the primers, which were created using the Shenggong online primer creation tool, are provided in Table S1. Lastly, the quantification of fluorescence was conducted utilizing the Premix Pro Taq HS qPCR kit, manufactured by Accurate Biotechnology Co., Ltd., located in China. The internal reference gene utilized was the β-actin gene from zebrafish. The 2‒ΔΔCT method was utilized to evaluate the fold change of the genes that were being tested.
Statistical analysis
Statistical analysis was performed using the program GraphPad Prism (version 8.0) developed by GraphPad program Inc. (La Jolla, CA, United States) and the R project (version 4.2.1). The data were presented in the form of mean values together with their corresponding standard deviations. A one-way analysis of variance (ANOVA) was employed to perform the analysis of variations among groups.