2.1. Materials
TB, 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl), and flow phase (acetonitrile) were purchased from Sigma-Aldrich Co., Ltd. (Shanghai, China). NM standards were purchased from XINYU Pharmaceutical Co., Ltd. (642 U/mg; Suzhou, China). All other standard reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Deep-well plates were purchased from Shanghai Canvic Bio-Technology Co., Ltd. (Shanghai, China). The microplate reader (Multiskan FC) was purchased from Thermo Fisher Scientific (Shanghai, China). The HPLC system (LC-2010HT) was purchased from Shimadzu (Shanghai, China).
2.2. Strains and Media
S. fradiae GC 6010 (wild-type strain and stored in our laboratory) and 300 mutant strains were generated by ARTP mutagenesis. Strains were grown on solid medium (glucose 10 g, beef extract 1 g, peptone 3 g, corn steep liquor 3 g, NaCl 5 g, and agar 20 g dissolved in 1 L deionized water; pH 7.3–7.8) for 7 days at 30 °C. Single colonies were transferred to seeding medium (soluble starch 10 g, peanut meal 10 g, yeast extract 20 g, (NH4)2SO4 1 g, glucose 30 g, corn steep liquor 10 g, peptone 5 g, Na2HPO4 1 g, CaCO3 10 g, and soybean oil 2 g, pH 7.3–7.8; and made up to 1 L in deionized water) and grown in 24-deep well plates (2 mL volume) at 220 rpm and 35 °C for 30 h. Seed cultures (8% inoculum) were then transferred to fermentation medium (soluble starch 70 g, peanut meal 28 g, yeast extract 6 g, (NH4)2SO4 6 g, glucose 20 g, corn steep liquor 2.5 g, peptone 9 g, soybean meal 5 g, NaCl 4.5 g, and soybean oil 3 g, pH 6.8–7.3; and made up to 1 L in deionized water) in 24-deep well plates (2 mL volume) and grown at 220 rpm and 35 °C for 7 days.
2.3. ARTP Mutagenesis and Screening
The general workflow involved the preparation of
S. fradiae spore suspensions followed by ARTP treatment (Si Qing Yuan Biotechnology Co., Ltd./now Tmax Tree Co., Ltd.; Wuxi, China) and pre- and rescreening (
Figure 1). The ARTP system (model: ARTP-IIS; weight: 95 kg; voltage: 220 V-50/60 Hz 500 VA; size: 73 cm × 65 cm × 69 cm;
Figure 2) needs high-purity helium (>99.99%) as a working gas and a typical electric socket as a power source. Inside the ARTP operation chamber, the helium flowing through the discharge region between the two electrodes is ionized by the radio frequency electric field and then acts on the microbial sample fixed on the metal plate sheet on a regulating platform via the nozzle. Since the breakdown voltage is not high (100–200 V), the plasma maintains discharge consistency, derives little ultraviolet radiation, and combines with the cooling of the cathode to maintain a biocompatible gas temperature. The continuously flowing gas seldom mixes with the surrounding air, thereby minimizing the production of germicidal ozone. It has been reported that the generation of reactive chemical species (He*, He
2*, He
+, He
2+, and N
2+) was considered as the biggest cause of physical plasma mutagenesis [
28,
29], and therefore requires careful adjustment of the plasma-generating parameters. The ARTP manufacturer provides a standard value for each adjustable parameter to meet the conditions of biocompatibility and produce enough active chemical substances [
9]. Firstly, it is recommended to use a gas flow rate of 10 SLPM (standard liters per minute) or above to prevent the gas from combining with the surrounding air to produce germicidal ozone. Further research showed that when the gas flow rate is between 5–30 SLPM, the production of active materials is proportional to the gas flow rate [
8]. Secondly, the suggested value of the distance between the sample and nozzle is 2 mm, which was used in almost all reports. In line with increasing distance (2–10 mm), the active chemical substances generate decreased sharply and not enough to cause damage to the cells [
8]. Thirdly, it was found that the temperature was within a biologically compatible range between 36 °C and 57 °C when the energy was between 40 W and 200 W [
9]. In the early study, the radio frequency power input of 40 W was applied in obtaining high-yield butanol
Clostridium acetobutylicum strain [
30]. Finally, for different species, the recommended values for the treatment time are different, including bacteria (15–120 s),
Actinomycetes (30–180 s), fungi (60–360 s), yeast (30–240 s), and microalgae (5–150 s) [
8]. Early research showed that along with the increasing time (0.5–10 min), more DNA damages in cells was produced under the same conditions as other parameters [
28].
Based on the analysis of aforementioned adjustable parameters, the following ARTP parameters were used: spore suspension = 10 μL (10
6–10
8 cells/mL), helium gas flow rate = 10 SLPM, 2 mm = distance between sample and nozzle, radiofrequency power input = 40 W, and ARTP treatment times = 0–210 s. Untreated spores were used as controls. Several constants were used to evaluate the effects of multiple ARTP mutagenesis rounds, and were calculated based on Equations (1)–(5).
where
U = total colony counts in untreated samples,
T = total number of colonies after ARTP treatment,
M = total colony count of mutant strains which the NM potency different from wild-type strain (the difference was above ±2%),
P = total colony counts of mutant strains with higher NM potencies than the wild-type strain (>2%),
N = NM potency of mutant strains in each mutagenesis round,
W = NM potency of the wild-type strain (
S. fradiae GC 6010), and
A = average NM potency of mutant strains generated in each mutagenesis round.
Then, treated spore suspensions were resuspended in 0.9% NaCl, diluted, and spread onto streptomycin agar plates for prescreening. The concentrations of streptomycin-resistant screening plates were 0, 2, 4, 6, 8, and 10 μg/mL. Plates without streptomycin were used as controls.
Next, single colonies after prescreening were transferred to seeding medium in deep-well plates. Then, seed cultures were transferred to a fermentation medium in deep-well plates. We used 24-deep well plates, 48-deep well plates, and 250 mL shake flask for fermentation, with fermentation correlations used for analysis by fitting data into Origin 9 software.
2.4. Method Development to Assess NM Potency in Fermentation Broth
Fermented media (7 days, 220 rpm, 35 °C) was centrifuged for 10 min at 10,000 rpm and supernatants were collected to determine NM potency. When TB reacts with NM, ions become associated and a blue color is formed [
31]. At a particular wavelength, the NM potency in fermentation broth could be determined using a microplate reader.
2.4.1. Selection of the Detection Wavelength
A 100 μL NM standard solution (25.68 U/mL) was mixed with 100 μL Britton-Robison buffer (pH 6.5), then 300 μL TB solution (1.0 × 10−4 mol/L) added and made up to 1 mL with deionized water. The reaction was incubated at room temperature for 10 min. Finally, the solution underwent a full-wavelength scan in a microplate reader to determine the maximum absorption wavelength peak. As a control, the TB solution was replaced with deionized water.
2.4.2. Optimizing TB Solution (1.0 × 10−4 mol/L) Volumes
Different volumes (50, 100, 150, 200, 250, 300, 350, and 400 μL) of the NM standard solution (25.68 U/mL) were mixed with 100 μL Britton-Robison buffer (pH 6.5), and then different volumes (100, 200, 300, 400, and 500 μL) of TB solution (1.0 × 10−4 mol/L) were added in separate experiments. Deionized water was then added to 1 mL and the reaction was incubated at room temperature for 10 min. Finally, the absorbance of different solutions was determined at the maximum absorption wavelength to determine the optimal TB solution (1.0 × 10−4 mol/L) volume. A standard curve of NM potency versus absorbance was generated.
2.4.3. Spike and Recovery Studies
We divided a fermentation broth of known NM potency into five parts and added NM standard solutions (25.68 U/mL) of different volumes (75, 125, 175, 225, 275, 325, and 375 μL). The NM potency in different solutions was determined by the aforementioned optimized method, and average recovery rates, with relative standard deviation (
RSD), were calculated based on Equations (6)–(10).
where
V1 = NM standard solution volume,
S1 = potency of the NM standard solution (25.68 U/mL),
D = NM standard potency (642 U/mg),
V2 = volume of the reaction system (1 mL),
dS = the measured NM potency minus the NM potency of the known fermentation broth in the reaction system,
E = sum of all recovery rates,
n = number of spike and recovery experiments,
X = average recovery rate, and
SD = standard deviation of all recovery rates.
2.6. Optimization of Fermentation Medium
At first, PB designs were used to screen for key components in the fermentation medium affecting NM potency. In total, 10 components were selected, and it was assumed no interactions occurred between them. Based on PB regression analysis, components with significant p < 0.05) values were selected for further optimization.
Interactions between these significant factors were investigated using Box-Behnken designs. Next, a second-order polynomial equation was obtained using Design-Expert 8.0.2 software based on analysis of variance (ANOVA).
where
Y = the predicted response of NM potency;
= the value of the fitted response at the center point of the design;
,
, and
= linear, quadratic, and cross-product regression terms, respectively;
= independent variables.
Then, optimal values for independent variables and corresponding predicted responses for NM potency were calculated using the second-order polynomial equation. Finally, fermentation was conducted using these optimal independent variables to verify the accuracy of the predicted response.