*2.8. Fungicidal Activity of Nystatin A1, A3, and Polyfungin B towards Saccharomyces cerevisiae ATCC 2061*

Single ingredients of nystatin A1, A3, and polyfungin B were extracted and isolated using semi-preparative HPLC, and their fungicidal activity was evaluated according to the experimental process in 3.3.

The results are summarized in Table 6. It can be seen that polyfungin B displayed a better fungicidal activity than nystatin A1 and A3, which is a novel result in nysfungin activity research.


**Table 6.** Results of nystatin A1, A3, and polyfungin B inhibiting *Saccharomyces cerevisiae* ATCC 2061.

\*\* *p* < 0.05, highly significant.

#### **3. Materials and Methods**

*3.1. Chemicals, Strains, Culture Media, and Growth Conditions*

Due to the lack of a nysfungin standard, domestic commercial nysfungin tablets (Nysfungin, Zhenyuan Pharmaceuticals Co. Ltd., Shaoxing City, Zhejiang, China) were purchased and processed to be used as a reference. All other chemicals were analytically pure, unless expressed otherwise.

The starting *streptomyces noursei* D-3-14 was isolated and stored in our lab and had a chemical potency of 3464 U/mL and a biotic potency of 2703 U/mL. Strains were cultured at 28 ◦C for 7 days on Gause's synthetic solid medium slant (GA media). A loop of the freshly grown lawn was inoculated into 50.0 mL seed culture medium, and cultured at 150 rpm, 28 ◦C for 24 h. Then, 10% of this seed culture was inoculated into 50 mL fermentation culture medium and cultured at 200 rpm, 28 ◦C, for 86 h. The resultant culture could be used to extract nystatin.

Gause's synthetic solid medium consisted of (g/L) soluble starch, 20.0; KNO3, 1.0; NaCl, 0.5; K2HPO4, 0.5; MgSO4·7H2O, 0.5; FeSO4·7H2O, 0.01 and agar, 15, pH 7.2. Seed culture medium consisted of (g/L) peanut meal, 20.0; soluble starch, 10; glucose, 10; CaCO3, 6.0; soybean oil, 3.0; (NH4)2SO4, 2.0; peptone 2.0; MgSO4·7H2O, 0.5; K2HPO4, 0.2 and pH 7.2. Fermentation culture medium consisted of (g/L) glucose, 55.0; peanut meal, 25.0; CaCO3, 10.0; (NH4)2SO4, 3.0; peptone 3.0; dried silkworm chrysalis meal, 2.0; soybean oil, 1.0; KH2PO4, 0.02 and pH 7.2.

*Saccharomyces cerevisiae* ATCC 2061 was purchased from China General Microbiological Culture Collection Center (CGMCC), Beijing, China. It was cultured on a YM medium slant at 28 ◦C for 3 d. When was mature, 6.0 mL sterilized NaCl solution (0.9%) was added, and the lawn was scraped to obtain a suspension. The suspension was then transferred into Erlenmeyer flasks with glass beads and incubated 28 ◦C, 180 rpm for 30 min. The final suspension concentration was adjusted and calculated using a hemocytometer.

The YM culture medium consisted of (g/L) glucose, 10.0; peptone 5.0; yeast extract, 5.0; malt extract, 5.0; agar 15.0 and pH 6.2. The biological potency detection culture medium consisted of (g/L) maltose, 40.0; peptone 20.0; NaCl, 3.0; agar 15.0 and pH 7.0–7.2. The Gauss's culture medium was sterilized at 121 ◦C for 20 min, all other media were sterilized at 115 ◦C for 30 min.

## *3.2. UV Mutagenesis, Nysfungin Crude Extraction, and HPLC Detection*

First, 3 mL of *Streptomyces noursei* single spore suspension was added to the 75 mm culture dishes and radiated with UV light (15 W, 30 cm) for 60 s. The dish covers were then opened and radiated for 20 s, 40 s, 60 s, 80 s, 100 s, and 120 s. Next, 12 mL seed culture medium was supplemented, and the dishes were cultured in black bags at 28 ◦C for 2 h. Both the UV radiated (Mutagenesis group) and untreated spore (Control group) suspensions were serially diluted and calculated using a hemocytometer. Then, 100 uL of spore suspension with 10−<sup>3</sup> , 10−<sup>4</sup> , and 10−<sup>5</sup> concentrations were spread on the Gauss plates, each concentration was spread in three replicates, the plates were cultured at 28 ◦C for 5~7 days, the colony numbers were counted, and the lethal rate was calculated.

$$\text{(Leftal rate (\%))} = \frac{(\text{cfu of control group} \times \text{dilution ratio}) - (\text{cfu of Matagenesis group} \times \text{dilution ratio})}{(\text{cfu of control group} \times \text{dilution ratio})} \times 100\%$$

After 86 h, the fermentation broth was centrifugated at 3500 rpm for 10 min, to collect the wet mycelia. These wet mycelia were transferred into a brown beaker, 3 times the volume of 98% alcohol (*v/v*) was added and stirred for 40 min, then the solution was centrifugated at 3500 rpm for 10 min, the supernatant was collected and extracted with an equal volume of 98% alcohol (*v/v*) three times. All the supernatants were combined and vapored with reduced pressure to concentrate to 20 mL, and these solutions were kept at 4 ◦C overnight to obtain the nystatin suspension. These suspensions could be further processed at 4000 rpm for 6 min to eliminate the supernatant and to obtain the crude nystatin extract crystal and were stored at −20 ◦C before use.

Subsequently, 50.00 mg of the above crude nystatin crystal was dissolved in 5.00 mL methanol (chromatographic grade) and filtered using a 0.22 µm filter, to produce a 10 mg/mL solution. HPLC was performed using a WondaSil C18 Column (250 × 4.6 mm × 5 µm, Shimadzu, Kyoto, Japan), and the mobile phase consisted of methanol and acetonitrile, acetate buffer = 26:37:37, the detection wavelength was 305 nm, the column temperature was 30 ◦C, the loading speed was 1 ml/min, and the loading volume was 10 µL.

#### *3.3. Determination of the Chemical and Biological Potencies*

First, the nystatin reference was resolved in *N,N*-dimethylformamide to make a 1000 U/mL stock. Then, 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, 1.2 mL, 1.4 mL, 1.6 mL, 1.8 mL, and 2.0 mL stock was dissolved and filled to 25.00 mL, and the absorption values were detected on OD319 to establish a standard curve.

Chemical potency was detected using the following process adapted from the European Pharmacopoeia 8.0 [20]: First, after 86 h, 5 mL well-dispersed culture broth was washed three times with ddH2O and centrifugated to collect the mycelia. Then, the mycelia were well mixed with 10 mL methanol and left to stand for 2 h. Next, the supernatant was collected after 3500 rpm for 10 min, and extracted using 10 mL methanol twice. The combined methanol solution was filled to 50.00 mL. Then, 2.0 mL was used to detect OD319, and the chemical potency was evaluated by comparing with the above standard curve.

The biological potency was detected using the following process adapted from a USP monograph [21]. The first step was to evaluate the proper concentration of *Saccharomyces cerevisiae* ATCC 2061. Then, 1 mL of 10−<sup>1</sup> , 10−<sup>2</sup> , 10−<sup>3</sup> , 10−<sup>4</sup> , and 10−<sup>5</sup> dilution suspension was added to 15 mL biological potency detection culture medium at 48 ◦C and mixed well. When the media become solid, one Oxford cup was placed on the center of the petri dish and 100 uL nystatin reference solution (80 U/mL) was supplemented and the cover was put on. Each concentration was performed in triple replicates and cultured at 28 ◦C for 14–18 h. The diameter of the inhibition zone was measured with a Vernier caliper. Empirically, a diameter of 18–22 mm is acceptable for biological potency detection.

Then, 250 uL of *Saccharomyces cerevisiae* ATCC 2061 was added to the 15 mL biological potency detection culture medium at 48 ◦C and mixed well. When the media became solid, four Oxford cups were symmetrically placed on the petri dish. Two cups on one diagonal

line were supplemented with 100 uL nystatin reference solution (80 U/mL and 40 U/mL), while the other two cups on the other diagonal line were supplemented with 100 uL nystatin sample solution (80 U/mL and 40 U/mL). After being cultured at 28 ◦C for 14–18 h. The diameter of the inhibition zone was measured with a Vernier caliper. And the biological potency of the crude nystatin extracts was calculated using the following formula:

$$\theta = \log^{-1}\left(\frac{T\_2 - S\_2 + T\_1 - S\_1}{S\_2 + T\_2 - S\_1 - T\_1} \times I\right)$$

in which,

S<sup>1</sup> = inhibition zone diameter of the low-dose control solution

S<sup>2</sup> = inhibition zone diameter of the high-dose control solution

T<sup>1</sup> = inhibition zone diameter of the low-dose sample solution

T<sup>2</sup> = inhibition zone diameter of the high-dose sample solution

*I = lg*(high-dose concentration/low-dose concentration)

#### *3.4. Preliminary Screen and Secondary Screen for Mutants*

Preliminary screening was performed using the following process: Single colonies on the UV radiation plates were inoculated onto a fresh Gauss's synthetic media plate and cultured at 28 ◦C for 5–7 days, to obtain viable mutants. A 1 <sup>×</sup> 1 cm<sup>2</sup> lawn was scraped and transferred into 50 mL fermentation broth and cultured at 28 ◦C and 180 rpm for 86 h. Then, the crude nystatin was extracted, to examine the chemical potency. Those strains with higher chemical potency than the starting strains were screened as the preliminary strains.

A secondary screen was performed on those strains with higher chemical potencies. Those strains were further detected by HPLC to determine their biological potencies. Only those strains exhibiting both higher chemical and biological potencies were screened and selected for the next steps.

#### *3.5. Genetic Stability Evaluation*

The selected strains with high chemical and biological potency were cultured for five consecutive generations, and the chemical potency was tested for each generation to evaluate the genetic stability.

#### *3.6. Single Ingredient Evaluation for the Obtained Nysfungin*

The obtained Nysfungin was detected using HPLC, each of the major peaks was further extracted and isolated using semi-preparative HPLC, and the resultant single ingredients were identified by MS and NMR and tested for their fungicidal activities using the cup-plate methods in 3.3.

#### *3.7. Optimization of the Fermentation Conditions*

The culture process was performed as described in 3.1. The glucose concentration was set at 2.5%, 4.0%, 5.5%, 7.0%, and 8.5%. The peanut meal concentration was set at 0.5%, 1.5%, 2.5%, 3.5%, and 4.5%. The starting pH value was set at 6.0, 6.5, 7.0, 7.5, and 8.0. The inoculation volume ratio was set at 6%, 8%, 10%, 12%, and 14%. Each inoculation volume was replicated three times.

The orthogonal experiment with four factors and three levels L<sup>9</sup> (3<sup>4</sup> ) was designed according to the glucose concentration, peanut meal, starting pH, and inoculate volume ratio, determined in a single factor experiment.

#### *3.8. Statistical Analysis*

All experiments were carried out in triplicate and each presented value is the average of three independent experiments. SPSS 20.0 software was used to conduct *t*-tests on the data, to determine the statistical difference, *p* < 0.05 was significant (\*), *p* < 0.01 was extremely significant (\*\*).

## **4. Conclusions and Discussion**

As polyene macrolide antibiotics produced by the *Streptomyces noursei strain,* both nystatin and nysfungin have broad-spectrum antifungal effects, with the strongest inhibition of *Candida albicans*, and have been widely used in clinical practice [3]. In this study, *Streptomyces noursei* D-3-14, a nystatin producing strain, was treated with UV mutagenesis for three rounds and screened for high yield mutants. Finally, *Streptomyces noursei* 72-22-1, a genetically stable strain with enhanced nystatin production, was obtained. Its chemical potency was 8912 U/mL and its biological potency was 5557 U/mL, 2.57 times and 2.06 times of those of the original strain, respectively. After optimizing the fermentation conditions, the chemical potency and biological potency of *Streptomyces noursei* 72-22-1 were 14,082 U/mL and 10579 U/mL, respectively, 4.07 and 3.92 times those of the starting strain.

Admittedly, although antibiotics have been applied for many years, there are only a few clinical options for polyene macrolide ingredients. In the FDA Orange Book dated December 2022, there are only four polyene macrolide fungicidal antibiotics listed. The only discontinued ingredient is Candicidin (Vanobid), which was approved on 1 January 1982 and manufactured by Sanofi Aventis US. The Eyevance Pharmeceuticals-manufactured Natamycin (Natacyn) was approved on the same day as Candicidin and is the only listed and prescribed Natamycin. Ten out of the 15 entries of Amphotericin B have been discontinued and only five manufacturers are still producing it. Nystatin has 123 entries, with 62 discontinued and 61 approved manufacturers [22].

All these data suggest that the diversity of polyene macrolide antifungal antibiotics remains limited. When equimolar single ingredients of nystatin A1, A3, and polyfungin B were tested, polyfugin B exhibited a better fungicidal activity than nystatin A1 and A3. These promising results warrant further extensive metabolic and biological studies of polyfugin B, nystatin A3, and other structurally similar ingredients in the near future.

**Supplementary Materials:** The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/catal13020247/s1, Table S1. Orthogonal experimental factors and level assignments. Table S2. Orthogonal experimental results and analysis.

**Author Contributions:** M.S. (data curation, investigation); W.H. (investigation, methodology); S.C. (data curation); F.W. (resources); W.X. (Weizhuo Xu) and W.X. (Wei Xu) (resources, supervision, writing—review and editing). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** Data are available upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.
