Strains and culture conditions
Lactobacillus strains isolated from traditional Sichuan pickle were identified by the 16s rRNA methods and further culture in MRS broth. The other two traditional strains S. thermophilus and L. bulgaricus were also stored and cultured in our laboratory.
Symbiotic growth characteristics of L. plantarum
Exploring the effects of L. plantarum under symbiotic culture, L. plantarum and L. bulgaricus and S. thermophilus were cultured in 37°C MRS medium under the same conditions with different strain inoculation ratio. Growth and acid production of the strains in the symbiotic growth media were measured at 600 nm every 2 hours and the curves were plotted in 24 hours’ culture time.
Fermented milk preparation
- bulgaricus and S. thermophilus combined with L. plantarum in a ratio of 1:1:0, 1:1:1, 1:1:2, 1:1:3 were screened as the mixed starters and inoculated with fresh pasteurized milk at a concentration of 3% (v/v) for 7 h at 42 ° C, and then ripening at 4 ° C for 24 h before further use.
Texture, acidity, water holding capacity analysis
The firmness, consistency, cohesiveness and index of viscosity of the yoghurt were determined by using a texture analyzer (TA, UK), and set the cylindrical probe: 70mm height, 65mm drop, 3mm/s descent speed, 500 PPS speed, 5g contact force; return distance 70mm, return speed 10mm/s. The measuring cup and probe should be cleaned after each test. Acidity of the yoghurt was performed with the acid-base titration method: Two drops of phenolphthalein were added to a 10 g sample and then titrated with 0.1 mol/L NaOH. The data were recorded and the acidity was calculated according to the following formula.
X2 = C2 × (V2 - V0) × 100 / m2 × 0.1
X2: acidity of the sample in degrees (°T);
C2: concentration of the sodium hydroxide standard solution;
V2: the volume of the sodium hydroxide standard solution;
V0: the volume of the sodium hydroxide standard solution used in the blank experiment;
100:100g sample; m2: the weight of the sample.
GC-MS analysis of volatile compounds
The fermented milk was sealed in a 5ml to 20mL headspace vial (CNW Technologies, Germany) with a Teflon/silicone septum in an aluminum cap. 10 μL of 10 ng/μL 2-methyl-3-heptanone was added as an internal standard. Extraction of Volatile Compounds by Solid Phase Micro extraction (SPME) with 75μm Carboxy/Polydimethylsiloxane (CAR/PDMS) Fibers from Supelco Inc (Bellefonte, PA, USA). The constant flow rate of the carrier gas (helium) was 3 mL/min, with an inlet temperature of 210°C and split less injection mode. The initial temperature of the column oven was 40°C, which was maintained for 3 min, increased to 140°C at 4°C/min, held for 1 min, and then increased to 200°C at 10°C/min for 20 min. The signal acquisition of the mass spectra was in full scan mode, ionization method EI, electron bombardment energy of 70 eV, interface temperature of 220°C, ion source temperature of 230°C, quadrupole rod temperature of 150°C, scan mass range of m/z 40 to 600, and scan frequency of 3.6 scans/s.
Fatty acid composition analysis
The fermented milk sample were mixed with methanol/CH2Cl2 (1:3) for 10 min at room temperature and oil components were ultrasonic extracted after centrifugation procedure (1800 rpm, 10 min). The supernatant was blow-dried with nitrogen and dissolved in methanolic KOH (6%) followed by 4N HCl. After BF3-MeOH treatment, fatty acid compositions were extracted with n-hexane for 3 times and the extraction solution was transferred to 2ml sample bottle, and blow-dried with nitrogen for further analysis.
The fatty acid composition was analyzed using a TG-5MS 30m×0.25mm×0.25μm column, the heating procedure as follows: starting temperature at 80℃, maintained for one minute, then the temperature rises to 200℃ at 10℃/min, 225℃ at 5℃/min, and 250℃ at 2℃/min for 5min. Flow rate is 1.2 mL/min, with helium as the carrier gas. Scanning range 30-400, injection volume 1 μL. The content was calculated by external standard method and internal standard method, and all the samples were done in triplicate.
Amino acid analysis
The homogenized fermented milk samples were hydrolyzed with 6 H HCl solution in a drying oven at 110 ° C for 22 hours. The next day, the hydrolyzed sample was filtered, dried and dissolved with 1 ml citric acid buffer solution (pH 2.2). Finally, the amino acid profiles of the different samples were determined by L-8900 amino acid automatic analyzer (Physics and Chemistry Test Center, Jiangsu Province, China).
Sensory profiling evaluation
The Alpha Astree II potentiometric electronic tongue system (Isenso, New York, NY) with an Ag/AgCl electrode was adopted for the sensory profiling analysis. The prepared fermented milk samples were homogenized and poured into an electronic tongue special cup and data were collected at room temperature. For all the samples, five replicates were carried out and three stable data were collected for principal components analysis (PCA) and discriminant factor analysis.
β-galactosidase and lactate dehydrogenase analysis
Cells were suspended in PBS buffer and lysozyme was added and bathed at 37 ° C for 1 h. After that, the crude enzyme solution was dissolved in 0.4 M NaCl solution before the enzyme activity analysis. The activity of β-galactosidase and lactate dehydrogenase under symbiotic culture conditions were assayed according to the assay kit and the method of Vasiljevic [21].
Data Analysis
All statistical analyses were performed using SPSS (SPSS Inc./IBM Corp., Chicago, IL) and the plots were drawn with Origin 8.5 (Origin Lab, Northampton, MA) software. The differences between the mean values of the groups were analyzed using a one-way ANOVA with Duncan’s multiple range test and P-values < 0.05 were considered statistically significant.