Agrobacterium tumefaciens KYC55 (pJZ372)(pJZ384)(pJZ410) (Zhu et al., 2003) and Chromobacterium violaceum CV026 (McClean et al., 1997) were kindly offered by Zhigang Zhou (Chinese Academy of Agricultural Sciences, Beijing, China) and used for the detection of AHLs as previous described (Ravn et al., 2001) A. tumefaciens KYC55 was grown in LB medium supplemented with 1 μg/mL tetracycline,100 μg/mL tetracycline, and 100 μg/mL spectinomycin. C. violaceum CV026 was gown in LB medium supplemented with 20 μg/mL kanamycin. N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL), N-(3-oxohexanoyl)-D-homoserine lactone (3-oxo-C8-HSL), and N-(3-hydroxydodecanoyl)-DL-homoserine lactone (3-OH-C8-HSL) were purchased from Sigma-Aldrich (St. Louis, MO, United States). 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) was bought from Promega (Madison, WI, United States). All other chemicals were analytical grade.

Psychrotrophs, Enterobacteriaceae, and Aeromonas were identified as the dominant spoilage bacteria of vacuum packaged minced sturgeon (Acipenser schrenckii) stored at 4°C (Gui et al., 2014). Psychrotrophs were isolated using plate count agar (PCA, code HB0101, Qingdao Hope Biol-Technology Co., Ltd., China) after culture at 7°C for 7 days. Enterobacteriaceae were isolated using violet red bile glucose agar (VRBGA, code HB0176) after culture at 37°C for 24 h. Aeromonas were isolated using ampicillin macconey agar base (AMA, code HB8576) after culture at 28°C for 2 days; 32, 25, and 30 colonies of Psychrotrophs, Enterobacteriaceae, and Aeromonas, respectively, were selected and purified in nutrient agar plates by streaking, and then identified by 16S rDNA sequencing. For the sequencing, bacterial genomic DNA was extracted using Bacteria Genomic DNA extraction kit (Tiangen, Beijing, China). 16S rDNA was amplified by PCR using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1495r (5′-CTACGGCTACCTTGTTACGA-3′). The PCR program was as follows: 30 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 30 s, and extension at 72°C for 1.5 min, with a final elongation step at 72°C for 5 min. The PCR products were sequenced by the Beijing Biomed Biological Technology Co., Ltd. (Beijing, China). The sequences of 16S rDNA were searched against database GenBank by BLAST.

Twelve strains of dominant spoilage bacteria, which were list as Q1–Q4, C1–C4, and N1–N4 (Table 1), were inoculated into nutrient broth (at 1% inoculum size) and cultured at 30°C for 12–18 h to reach 10⁹ CFU/mL. Bacteria were collected by centrifugation at 12,000 g, 4°C for 10 min, resuspended in sterile saline solution, and diluted to 1.0 × 10⁸ CFU/mL; 400 mL of sterile fish muscle juice was inoculated with 20 μL of prepared bacteria and stored at 4°C to evaluate the spoilage ability of inoculated bacteria. Spoilage ability was evaluated by the microbial growth, total volatile base nitrogen (TVB-N) and sensory score. The sturgeon muscle juice was prepared according to Gu et al. (2013). TVB-N was measured as previously described (Gui et al., 2014) and expressed as g TVB-N per kg minced sturgeon muscle. Quantitative descriptive analysis (QDA) was applied to evaluate the flavor of sturgeon juice. Seven trained people were selected for the sensory panel. Surgeon juice was scored on a 0–7 point scale with 6–7 meaning fresh, 4–6 no distinct odor, 2–4 incipient rancidity, and 0–2 rancid.

Agrobacterium tumefaciens KYC55 (pJZ372)(pJZ384)(pJZ410) and Chromobacterium violaceum CV026 were used for the detection of AHLs in “T” streak assay as previously described by Piper et al. (1993).

Aeromonas veronii LP-11 was cultured in LB at 30°C, 200 rpm for 24 h. And AHLs were extracted from the supernatant of culture using method previously described (Ravn et al., 2001) with some modifications. A sample (600 mL) was centrifuged (4°C, 12,000 g for 10 min) and passed through 0.22 μm filter to remove cells. The supernatants were extracted three times with equal volumes of acidified ethylacetate (0.1% formic acid). The combined extract was first dried at 30°C using a rotatory evaporator .The small amount of residual liquid was dried by nitrogen gas flow. Residue was dissolved in 2 mL of HPLC-grade methanol and stored at −20°C until analysis.

Acetylated homoserine lactones extract was examined by thin layer chromatography (TLC) as described by Ravn et al. (2001) with some modifications. Briefly, 2 μL of AHL extract or synthetic AHL standards was applied to a 10 cm × 10 cm sizes C18 TLC plate (TLC aluminum sheets 20 cm × 20 cm, RP-18 F254_S, Merck 64271 Darmstadt, Germany) and developed in 60:40 (v/v) methanol/water until the front reached the top. The TLC plates were dried for at least 10 min while an agar cover layer of either C. violaceum CV026 or A. tumefaciens KYC55 was prepared. To prepare the agar top layer, 0.5 mL of preculture of C. violaceum CV026 or A. tumefaciens KYC55 was inoculated into 50 mL LB medium and grown at 30°C for 24 h with aeration. The culture was then added with 100 mL of LB-agar (1.2% agar) and immediately poured onto the TLC plates. After the agar was solidified, the TLC plates were transferred to a sealed sterile container and cultured at 30°C for 48 h. AHL standards used included C4-HSL, C6-HSL, C8-HSL, and 3-oxo-C8-HSL.

HPLC/QqQ-MS analysis of AHLs extract was performed as described by Gould et al. (2006) with some modification; 10 μL of AHL extract was injected onto a C18 reverse-phase column (2.0 mm × 150.0 mm, Agilent). Mobile phase A was H₂O containing 0.1% glacial acetic acid. Mobile phase B was methanol containing 0.1% glacial acetic acid. Elution started with 10% mobile phase B for 5 min, which then raised to 90% over 30 min and stayed at 90% for 15 min. The column was re-equilibrated for 10 min, and a blank run was performed between each analysis. The flow rate, column temperature and UV detection wavelength was 200 μL/min, 30°C, and 210 nm, respectively.

Mass spectrometric analysis was performed on Agilent 6460 triple-quadrupole tandem mass spectrometer. Precursor ion-scanning experiments were performed in positive-ion mode to monitor for m/z 102 in the range of m/z 50–400. The ion spray voltage, focusing potential, and collision energy were set at 4200, 200, and 25 V, respectively. To verify the AHLs detected in A. veronii extract, AHL reference standards including C6-HSL, C8-HSL, and 3-oxo-C8-HSL were analyzed in the same way.

10 μL sample was injected onto an Agilent SB-C18 column (2.1 mm × 50.0 mm, Agilent) and eluted at a flow rate of 400 μL/min. Mobile phase A was H₂O containing 0.1% formic acid. Mobile phase B was acetonitrile containing 0.1% formic acid. Elution started with 10% mobile phase B for 3 min, which then raised to 100% over 21 min, stayed at 100% for 2 min, reduced to 10% over 0.1 min, and stayed at 10% for 3.9 min. The column temperature and UV detection wavelength were 30°C and 210 nm, respectively. Mass spectrometric analysis was performed on a Waters XEVO-G2 QTOF of a quadrupole and orthogonal acceleration time-of-flight tandem mass spectrometer. The scan range was m/z 50–600. For positive-ion mode, the capillary and cone voltage were set at 3.5 kV and 40 V, respectively. The desolvation gas was set to 600 L/h at a temperature of 450°C. The ion source temperature was set to 110°C.All data were acquired and processed using Waters MassLynxv4.1 software.

Statistical tests were performed using the SPSS 20.0 computer program (SPSS Statistical Software, Inc., Chicago, IL, United States). Data were reported as mean ± SDs of three measurements. Means were subjected to Duncan’s test and a p-value of <0.05 was considered significant.

A total of 87 strains of dominant spoilage bacteria were selected from selective mediums culture and subjected to identification by 16s rDNA sequencing. Totally, 13 species were identified from the 87 selected strains (Table 1). One strain from each of 12 most common identified species was selected for spoilage ability analysis. The selected 12 stains listed as Q1–Q4, C1–C4, and N1–N4 (Table 1) were inoculated into sterile sturgeon muscle juice. Viable counts, TVB-N values, and flavor of sturgeon muscle juice during storage were determined or evaluated. As shown in Figure 1, the viable count of Q1 rapidly exceeded 8 log CFU/g on day 4, which was significantly higher (P < 0.05) than those of all the other groups. At the end of the storage, the viable counts of Q1, N3, C1, and C4 became significantly higher (P < 0.05) than those of the others. The sensory scores of Q1, C1, and C4 were significantly lower (P < 0.05) than those of the other groups on day 6. In addition, the TVB-N values of Q1 increased rapidly after 2 days of storage and became significantly higher (P < 0.05) than those of all the other groups from day 4 to day8. On the final day 10 of storage, the TVB-N values of Q1, C1, and C4 increased to over 40 mg N/100 mL which were significantly higher (P < 0.05) than those of the other groups. Based on these data, it can be concluded that the spoilage ability of Q1, C1, and C4 was higher than that of the other tested strains. Strains Q1, C1, and C4 were classified as the SSOs of sturgeon in the present study and named as Aeromonas veronii LP-11, C. freundii LPJ-2, and R. ornithinolytica LPC-3, respectively.

Acetylated homoserine lactone biosensor C. violaceum CV026 responds well to AHLs with four to eight carbon acyl side chains but responds very poorly to AHLs with acyl chains of C10 or more (Steindler and Venturi, 2007). Another AHL biosensor A. tumefaciens KYC55 is sensitive to 3-oxo-substituted AHLs with acyl chain of lengths from 4 to 12 and 3-unsubstituted AHLs (Zhu et al., 2003). As each biosensor preferentially responds to AHLs with specific structure, using the two biosensors in combination can enlarge the detection range of AHLs. A. veronii LP-11 was able to induce violacein production in C. violaceum CV026 and β-galactosidase production in A. tumefaciens KYC55 (Figures 2A,B). The color of the two AHL biosensors was in gradient, with most strong response observed at the meeting point of the two strains, showing AHL production by A. veronii. In contrast, C. freundii LPJ-2 and R. ornithinolytica LPC-3 did not induce the QS phenomenon in the two biosensors (data was not shown). The AHLs produced by A.veronii LP-11 were further identified in the following study.

Acetylated homoserine lactones can be partially characterized by TLC on C₁₈ reversed-phase plates. Each AHL migrates with a characteristic mobility and results in a spot shape of response detected in a way depending on the reporter of the biosensor strain (Steindler and Venturi, 2007). A. veronii LP-11 induced two spots in the TLC plate coupled with A. tumefaciens KYC55 (Figure 2C). Spots a and b had similar mobility and shape as those of 3-oxo-C8-HSL and C8-HSL, respectively. At the same time, A. veronii LP-11 inducted two spots in the TLC plate coupled with C. violaceum CV026 (Figure 2D). The R_f value of spot c (∼0.49) was similar to that of the short chain AHL standard C6-HSL (R_f∼0.49), while the R_f value of spot d (∼0.37) was smaller than that of 3-oxo-C8-HSL (R_f∼0.41). From these results, it can be concluded that spots a, b, and c were 3-oxo-C8-HSL, C8-HSL and C6-HSL, respectively, but spot d was an unidentified AHL; these results demonstrated that at least four different AHLs were synthesized by A. veronii LP-11.

To accurately identify the AHLs species synthesized by A. veronii LP-11, A. veronii LP-11 extract was analyzed by HPLC/ QqQ-MS. The ions used to identify AHLs are m/z 102, which corresponding to the loss of the acyl chain, and the corresponding m/z molecular ion (Morin et al., 2003). As shown in Figure 3, three AHLs were identified by HPLC/QqQ-MS. The prominent peak at m/z 242.8, 244.7, and 228.7 corresponded to the molecular ion [M + H]⁺ of 3-oxo-C8-HSL, 3-OH-C8-HSL, and C8-HSL, respectively. In addition, the specific retention times of molecular ions of m/z 244.2, 244.7, and 228.7 were in accordance with standards 3-oxo-C8-HSL (26.902 min), 3-OH-C8-HSL (27.678 min), and C8-HSL (32.932 min), respectively. Therefore, the molecular weight, retention time, and characteristic ion m/z 102 consistently confirmed the presence of 3-oxo-C8-HSL, 3-OH-C8-HSL, and C8-HSL in A. veronii LP-11 extract.

Thin layer chromatography analysis indicated that four different AHLs might present in A. veronii LP-11 extract, while QqQ-MS detected only three AHLs. To clarify the discrepancy, qTOF-MS with both high sensitivity and high mass accuracy for both precursor and product ions (Cataldi et al., 2013) was used to search the signaling molecules. The prominent precursor ions [M + H]⁺ at m/z 244.153, 242.1375, and 228.1579 in MS1 spectra corresponded to 3-OH-C8-HSL, 3-oxo-C8-HSL, and C8-HSL, respectively. Their intense characteristic protonated homoserine lactone product ion m/z 102 was observed in the MS2 spectra (Figures 4C–F). However, the precursor ions [M + H]⁺ at m/z 200.1269 which corresponded to C6-HSL were in low abundance in the MS1 spectra and no obvious characteristic product ions at m/z 102 were present in the MS2 spectra (Figures 4A,B). In order to confirm the presence of C6-HSL in the sample, standard C6-HSL was analyzed by the same method and the results showed that their retention time was the same, indicating the four compounds in Figure 4 were 3-OH-C8-HSL, 3-oxo-C8-HSL, and C8-HSL and C6-HSL, respectively. In addition, the typical mass errors of the four compounds were below 2.1 mDa and the elementary compositions of the four compounds calculated by Marker Lynx XS software were consistent with their predicted molecular formulas (Table 2), which substantiated the presence of the four AHLs in A. veronii LP-11extract.