Chemicals and reagents
Skim milk powder were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Primary screening medium was solid casein medium (pH 6.8) containing casein peptone 2.5 g/L, skim milk powder 25 g/L, casein 10 g/L, glucose 10 g/L, yeast extract powder 1 g/L and agar 20 g/L. Seed liquid medium was modified TYC medium containing casein peptone 15 g/L, Na2HPO4·12H2O 2 g/L, NaCl 1 g/L, NaHCO3 2 g/L, L-cystine 0.2 g/L, glucose 50 g/L and yeast paste 5 g/L. The fermentation medium was wheat bran shorts (30 g/L) containing carbon 39.28% (w/w), nitrogen 3.12% (w/w) and the moisture 6.51% (w/w). Wheat bran was purchased from Qingdao Yutafeng Trading Co. Ltd. (Shandong, China). The bacterial genomic DNA extraction kit, rTap and related primers were purchased from Takara Biomedical Technology Co., Ltd. (Beijing, China). The other chemicals used in the study were analytical grade from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
Screening and isolation of MCE-producing strain
The bacterial strains were isolated from soil samples taken from different local regions including sports fields, barns, milking places, drinking areas and producing areas in the open pastures in Heilongjiang. Top soil samples (10–15 cm) were collected, filtered through a 3–4 mm mesh screen and kept at 4 °C. Suspending 1 g of soil sample in 9 mL of sterile saline to create a soil suspension. Executing tenfold serial dilutions to get a final dilution of 10–4. A 120 μL aliquot of each diluted sample was cultured in the solid casein medium at 37 °C for 36 h. Each strain was assigned a unique identification number and the width of hydrolysis and precipitation rings were measured every 12 h. The colony that formed a hydrolysis ring and a precipitation ring was regarded as a potential strain and used for further study.
Identification and characterization of strain
On a modified TYC medium, prospective MCE-producing strains were cultivated and examined utilizing physiological and biochemical characteristics testing. Strain cultures were also evaluated using Bergey’s manual of systematic bacteriology for starch hydrolysis (Xu et al. 2021), Voges-Proskauer reaction (VP), methyl red reaction (MR), hydrogen sulfide gas production, citrate utilization, glucose oxidative fermentation, catalase reaction according to standard protocols (Alippi et al. 2019).
The isolates (n = 15) with high ratios of (i) precipitation ring diameter to hydrolysis ring diameter and (ii) precipitation ring diameter to colony zone diameter were chosen and further certified by the 16S rRNA sequencing. For species-level identification, 16S rRNA gene sequences were compared to those in the GenBank database using the BLAST tool (NCBI). Our data were aligned with a 16S rDNA sequence dataset for phylogenetic analysis using the BioEdit tool. The MEGA 7.0 program was used to construct the phylogenetic analysis. The strains were kept at – 80 °C in 20% (v/v) of glycerin solution. The subsequent fermentation test with the selected strains executed MCA and PA measurements.
Measurement of MCA and PA
The fermentation broth was centrifuged at 5000 × g for 10 min. The crude MCE collected from the supernatant was utilized to measure MCA and PA according to the method of Arima et al. (1967) with some modifications. The 10% (w/v) skimmed milk substrate containing 10 mM CaCl2 was incubated at 35 °C for 10 min. Observing curd production by rotating the tube frequently until visible discrete particles appeared on its inner surface. The MCA is expressed in Soxhlet units (SU), which represents the amount of enzyme that can clot 1 mL of the milk substrate at 35 °C. The MCA was determined using the following formula: SU = (2400 × VS × N)/(T × VE), where VS is the milk volume (mL), N is the dilution of MCE, T is the milk-clotting time (s) and VE is the MCE volume (mL). For the measurement of PA, the amount of MCE that releases 1 g of tyrosine per 1 mL in 1 min is defined as one unit (1 U).
Strain growth and DB219 MCE production
The strain stored at − 80 °C was inoculated to the modified TYC medium and incubated at 37 °C for 12 h. Then, a single colony was inoculated into a 250 mL flask containing 50 mL of modified TYC medium and incubated at 37 °C for 10–12 h under shaking at 180 rpm as seed liquid. Three 250 mL of flasks (30 g/L wheat bran shorts) were autoclaved at 115 °C for 20 min and inoculated with 5% (v/v) inoculum size to produce MCE. The fermentation parameters were as follows: temperature 37 °C, agitation speed 180 rpm, initial pH 6.15 and volume 50 mL. Samples were taken every 12 h from 0 to 60 h and centrifuged for 10 min at 5000×g and 4 °C. The MCA and PA of MCE were determined using the fermentation supernatant.
Optimization of fermentation conditions
The effects of wheat bran concentrations on DB219 MCE production
The wheat bran shorts concentration included 20, 30, 40, 50, 60 and 70 g/L. The initial pH was 6.15 and 5% of inoculum size was added to a 250 mL flask containing 50 mL wheat bran medium without extra carbon and nitrogen sources at 37 °C and 180 rpm. MCA was measured for 12–60 h every 12 h.
The effects of carbon and nitrogen sources on DB219 MCE production
The carbon sources were optimized through adding 10 g/L glucose, sucrose, soluble starch, maltodextrin and lactose to wheat bran medium. The MCA in the wheat bran medium (without extra carbon sources) was the control and taken as 100%. The optimal nitrogen sources were optimized through adding 3 g/L corn steep liquor, casein peptone, urea, yeast extract powder, ammonium sulfate and ammonium citrate to wheat bran medium containing the optimal carbon source. The MCA in the wheat bran medium containing 10 g/L optimal carbon source (without extra nitrogen sources) was the control and considered 100%. The concentration optimization of carbon and nitrogen sources was carried out based on optimal carbon source (2.5, 5, 10, 12.5, 15 and 20 g/L) and optimal nitrogen source (1, 2, 3, 4, 5 and 6 g/L). The 10 g/L optimal carbon source and 3 g/L optimal nitrogen source were the control. The MCA was measured every 12 h from 12 to 60 h.
The effects of bioprocess parameters on DB219 MCE production
The effect of inoculum size (1, 3, 5, 7, 9 and 11%, v/v) and fermentation medium volume (20, 30, 40, 50, 60 and 70 mL) on MCE production was evaluated. The initial pH of the fermentation medium was chose as 4.15, 5.15, 6.15, 7.15, 8.15 and 9.15. The fermentation medium contained the optimal carbon and nitrogen source. The fermentation process was conducted at 37 °C and 180 rpm. The MCA was measured every 12 h from 12 to 60 h.
Plackett–Burman design tested six factors on fermentation including wheat bran concentration, inoculum size, optimal carbon source concentration, optimal nitrogen source concentration, volume and initial pH. A single factor experiment determined the selection of twelve runs of factors (A-F) at two levels (+ 1 and –1). The results were fitted by Mini Tab 2.0.
Path of steepest ascent/descent
The Plackett–Burman design relevant factors over the 95% confidence level (P < 0.05) were chosen and optimized further using the steepest ascent/descent method. Experiments were conducted at predetermined intervals along the steepest ascent/descent. Three factors were selected for the steepest ascent/descent experiment: wheat bran concentration (A), optimal nitrogen source concentration (C) and volume (E).
The DB219 MCE production was optimized through an RSM-based Box Behnken design model (BBD). Based on the above experiment finding, three factors wheat bran concentration (A), optimal nitrogen source concentration (B) and volume (C) were chosen and tuned at three levels to maximize the decolorization process. Analysis of Variance (ANOVA) was used to evaluate the viability of design model.
Each experiment was conducted in triplicate. Means and standard deviations were used to express the findings. The acquired data were analyzed by SPSS 17.0, one-way ANOVA and Minitab 2.0. Box-Behnken design was executed by Design-Export V8.0.6. The statistical significance was established at P < 0.05.