Materials and general methods
Grass carp of 2.0 ± 0.5 kg body weight and 60 ± 5 cm body length was purchased from a local market (Ningbo, China). Immediately after sacrificed, fresh grass carp were removed their scales, head, and gut. Then, they were rinsed in sterile deionized water, and cut into fillets approximately 40 mm thick using sterilized scissors under sterile conditions. After 15 min of UV irradiation, grass carp muscles were used for the subsequent fermentation experiment.
Pyridine (> 99.8% purity), methoxylamine hydrochloride (> 99.8% purity), BSTFA + 1% TMCS (> 99.0% purity), n-heptane (> 99.0% purity) and n-docosane (> 99.0% purity) were purchased from Sigma-Aldrich (St. Louis, MO, USA), in which pyridine and n-heptane were chromatographic grade, while other reagents were analytical grade. Then, the methoxyamine hydrochloride was dissolved in pyridine at a concentration of 15 mg/mL.
LAB was isolated from the traditional pickles in Ningbo City, and initially screened by using the skimmed milk treadmill test, followed by further rescreening and performing a series of protease activity assays experiment. Subsequently, the strain with high productivity of protease was determined by 16S rRNA sequencing and the alignment using the BLAST search program (NCBI), and 16S rRNA gene sequence of primer was shown in the supplementary material (Additional file 5). The primers used for sequencing were universal primers (27F: 5′-CAG CGG TAC CAG AGT TTG ATC CTG GCT CAG-3′; 1492R: 5′-CTC TCT GCA GTA CGG CTA CCT TGT TAC GAC TT-3′).
In the experiment of two 2-DE pH 4–7 IPG gel strips, 2-DE electrophoresis equipments and a variety of reagents were Bio-Rad company products and American Sigma company products.
Preparation of biosamples
L. plantarum in 20% (v/v) glycerol was stored at − 80 °C, and was revitalized in de Man Rogosa and Sharpe (MRS) broth at 35 °C on a shaker at 120 rpm for 24 h before use. Then, the pH and bacterial density at OD600nm were measured according to Liu et al . Subsequently, L. plantarum for the control group was incubated in MRS broth at 35 °C for 24 h with shaking. Grass carp fillets, pickled with 50 g salt kg− 1 and 30 g sucrose kg− 1 for 4 h at room temperature under the sterile conditions, were split into three batches. Each batch was wrapped with 100 mesh gauze of four layers, and tied with sterile cotton thread. Then, they were inoculated with L. plantarum to a concentration of approximately 107–8 cfu/mL and fermented at 35 °C for 24 h. At the end of the fermentation, the fermentation broth was immediately centrifuged at 4 °C and 1200 rpm for 10 min to remove of the pellet, and then the supernatant was centrifuged at 12,000 rpm for 10 min at 4 °C to harvest the cells. Then, the cells in each of the two groups were washed three times with sterile water and used for metabolomic and proteomic analysis.
Extraction and derivatization of metabolites
Metabolome samples of L. plantarum were prepared with some modifications according to a previously described method . Cells were inactivated with liquid nitrogen and fully ground, and metabolites were extracted by addition of 15 mL of 60% methanol (w/v, − 20 °C) to the cell pellet . After centrifugation at 12,000 rpm for 10 min at 4 °C, supernatants were dispensed into centrifuge tubes and dried for approximately 2 h by using nitrogen to remove the excess methanol. Thereafter, the metabolites were completely frozen overnight (approximately − 40 °C), and then freeze-dried for approximately 48 h and preserved in the dryer for analyzing the metabolite levels by GC-MS.
For derivatization, a 200 μL of 15 mg/mL methoxyamine pyridine hydrochloride was added, and then mixed for 30 s prior to incubation at 37 °C for 90 min. Then, the samples were further derivatized with the addition of 200 μL BSTFA with 1% TMCS and incubated at 70 °C for 30 min, following by incubating at room temperature for 30 min . After adding 100 μL of n-heptane (containing 0.10 mg/mL of n-docosane, internal standard) and vortexing, followed by centrifugation (12,000 rpm, 4 °C, 10 min), the derivatized samples were transferred to sample bottle for GC-MS analysis.
The GC-MS system consisted of an Agilent 7890/M780EI gas chromatograph (GC, Agilent Technologies, Palo Alto, CA, USA) coupled with a PERSEE mass spectrometer (MS, Shimadzu, Kyoto, Japan) and an Agilent AS-2912 autosampler. A sample of 1.0 mL was injected into a deactivated and fused-silica Agilent DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm film, Agilent J&W Scientific, Folsom, CA) with a split ratio of 8:1. After sample preparation, the electron impact (EI) ion source temperature was set to 250 °C with a 70-eV electron beam. Then, the injector temperature was 280 °C with the detector voltage of 0.96 kV and a solvent delay of 6 min. The helium carrier gas (99.999%) was set at a flow rate 1.0 mL/min. The GC oven was held at 90 °C for 3 min, and ramped at 3 °C/min to 160 °C, then further ramped to 220 °C at 2 °C/min, where it was held for 1 min, eventually ramped at 10 °C/min to 290 °C . Masses were acquired in a full scan mode over the range from 45 to 550 m/z with a 0.2 s scan velocity.
Protein extraction and quantification
The lysis buffer (8 M Urea, 2 M Thiourea, 4% (w/v) CHAPS, 1% DTT, 0.25% (w/v) Tris) was added (volume ratio of lysis buffer to the mass of cells = 5:1) into an equal amount of cells in the two groups. The cells were resuspended and lysed in lysis buffer for 30 min at 4 °C, and subsequently disrupted by sonication at 200 W for 20 min (Ningbo Scientz Biotechnology, China). The protein preparation was performed according to previously described . Protein pellets were dissolved with rehydrating buffer solution (8 M Urea, 4% (w/v) CHAPS, 2 M thiourea, 10 mg/mL DTT), and collected by centrifuging at 12,000 rpm for 10 min at 4 °C. Eventually, proteins were purified using the 2-D Clean-Up kit (GE healthcare, USA), and the protein concentration was measured by using the Bradford assay .
After determining the protein concentration from the control and experimental groups, the proteins were diluted with IEF buffer (8 M Urea, 4% (w/v) CHAPS, 2 M thiourea, 65 mM DTT, 0.2% (v/v) Bio-lyte (3/10, Bio-Rad, USA)). After centrifugation at 12,000 rpm and 4 °C for 10 min, isoelectric focusing (IEF) was performed on PROTEAN IEF cell (Bio-Rad, USA) by rehydrating Ready Strip IPG Strips (pH 4–7, 7 cm) with 150 μL protein solution (300 μg) for 14 h with a maximum current setting of 50 mA/strip at 20 °C. After rehydrating, the voltage program of IEF was followed by a linear ramp to 250 V over 1 h, linear ramp to 500 V over 2 h, another linear ramp to 1000 V over 1 h, then linear ramp to 4000 V over 4 h and a constant 4000 V hold for 5.6 h (thus yielding a total of 20,000 V/h). Afterwards, the immobilized pH gradient (IPG) strips were equilibrated by 2.5 mL of equilibration buffer solution I (6 M Urea, pH 8.8, 75 mM Tris-HCl, 20% Glycerol, 2% (w/v) SDS, 2% (w/v) DTT, 0.002% (w/v) bromophenol blue) for 15 min, followed by a second 15-min equilibration step in 2.5 mL of equilibration buffer solution II (6 M Urea, pH 8.8, 75 mM Tris-HCl, 20% Glycerol, 2% (w/v) SDS, 0.002% (w/v) bromophenol blue, 2.5% (w/v) iodoacetamide). After equilibrating, the IPG strips were then loaded onto 12% SDS-PAGE gels and sealed with 0.5% agarose. Then, 2D SDS-PAGE was performed by using a PowerPac basic electrophoresis system (Bio-Rad, California, USA) at 100 constant volts and 15 °C. Hereafter, gels were rinsed twice by using the double distilled water (ddH2O), and the protein spots were visualized by using Coomassie Brilliant blue R-250 staining. Finally, these gels were rinsed several times with ddH2O on a shaker.
Image analysis and protein identification
Three parallel gels were consistent duplicates for each the control and experimental groups, and scanned with an Image Scanner (Calibrated Densitometer GS800, Bio-Rad, USA) in a transmission mode. Subsequently, PDQuest software (version 8.0.1, Bio-Rad, California, USA) was used for analyzing 2D protein profiles, and these DEPs were extracted for following identification by MALDI-TOF/TOF MS (p < 0.05). The pretreatment of selected protein spots and MS analysis was performed as described previously . Briefly, α-cyano-4-hydroxycinnamic acid as matrix was used to ionize peptides. MS analysis of peptide solutions from trypsin digested proteins was performed on a Bruker UltraReflex™ III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Karlsruhe, Germany) . A maximum accelerating potential of 20 kV was used in this instrument with UV wavelength of 355 nm and repetition rate of 200 Hz. It was operated in reflector mode and scanning quality range was 700–3200 Da, while the best quality resolution was 1500 Da. The pancreatin self-cutting peak was internal standard to correct the mass spectrometer, and the mass spectra of samples were obtained by default mode. Peptide mass fingerprints were processed by using a software flex analysis (Bruker Daltonics, Germany). The MS data were interpreted by BioTools 3.0 (Bruker Daltonics, Germany) combined with the Mascot search engine against Uniprot database for known proteins from firmicutes. A fixed modification of cysteine was implemented by allowing for one missed cleavage site and assuming carbamidomethyl, and with the oxidized methionine as a variable modification. Then, peptide mass tolerance and fragment mass tolerance were set to 50 ppm and ± 0.6 Da, respectively .
All GC-MS data, including peak intensities, retention characteristics and the integrated mass spectra of each cell metabolites sample, were used for the component analysis . Based on the similarity of their retention time, the commensal metabolites of each sample were aligned. Then, in accordance with each GC-MS TICs, the areas of corresponding chromatographic peaks were determined. The identification of peaks-of-interest was performed with their mass spectra by matching with NIST08 MS spectral library of the WILEY workstation (similarity ratio > 80%). The concentrations of metabolites were represented as the relative areas (divided by the area of reference compounds). The preprocessed GC-MS data were subsequently imported into Simca-P software (Demo version 11.0, Umetrics, Umea, Sweden), and subjected to the principal component analysis (PCA) and loadings analysis. Finally, a heatmap of these metabolites was performed using HemI 1.0 software .
UniProt protein sequence database was used to determine the theoretical pI values and molecular weights (MWs) of the identified proteins . Also, the assembled transcripts were searched using BLASTx (NCBI database) with a cut-off E-value of 1E-5 . Then, the identified proteins were distributed over clusters of orthologous groups (COGs), and grouped into the cellular roles according to COGs. Additionally, the proteins were further defined according to Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/).
All the experiments were performed in triplicate. Then, T-test was used to analyze the significance between the control and experimental groups. A p-value of < 0.05 in all replicate experiments represented statistical significance.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted and purified from frozen cell pellets using the RNeasy mini RNA extraction kit (Trizol, Invitrogen, USA) in accordance with the manufacturer’s instructions, and then RNA yields were determined by using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific Inc. South Logan, USA). Total RNA based on the manufacturer’s protocol was reverse-transcribed into the first strand cDNA synthesis by using oligo (dT) primers and the M-MLV first strand cDNA synthesis kit (Invitrogen, USA). A Primer3plus software was used to design qRT-PCR analysis primers based on the genome sequence of L. plantarum (Additional file 6: Table S1).
qRT-PCR was performed by a Rotor-Gene 6000 real-time PCR detection system (Corbett Research, Mortlake, Victoria, Australia) along with fluorescence signal detection (SYBR® Premix Ex TaqTM II) with four biological replicates. A 20.0 μL reaction mixture was used for each PCR reaction, including 10.0 μL of SYBR® Premix Ex TaqTM II (2X), 2.0 μL of properly diluted cDNA (30 ng/μL of cDNA for all genes used for qRT-PCR except for 16S rRNA, where the concentration of cDNA was 0.3 ng/μL), each forward and reverse primer (10 μM) of 1.0 μL, and 6.0 μL of nuclease-free water . The negative control in each run was set to Diethyl pyrocarbonate (DEPC)-treated water. The thermal cycling conditions contained an initial denaturation step for 95 °C for 10 s, followed by 40 cycles of 15 s denaturation at 94 °C, annealing (10 s at 54 °C) and extension (15 s at 72 °C), followed by a single fluorescence measurement .
A specific threshold for each gene was set to analyze the data at the approximate midpoint of exponential phase to the amplification. Each of four biological replicates on the resulting threshold cycle (Ct) values were averaged. Subsequently, the standard curves were established to evaluate the expression levels of target genes, and eventually Ct values were converted to the relative amounts of cDNA .