Spectrophotometric analysis of bioactive metabolites and fermentation optimisation of Streptomyces sp. HU2014 with antifungal potential against Rhizoctonia solani

Figures & data

Table 1. Experimental variables at two levels used for the antifungal effect of the cell-free filtrates from HU2014 culture using PB design.

Parameters	Code	Levels
		−1	+1
Dextrin (g/L)	A	30	40
Yeast extract (g/L)	B	5	10
KNO3 (g/L)	C	1.5	2.5
pH	G	6.8	7.2
Inoculum size (%)	H	10	20
Temperature (°C)	D	20	30
Agitation speed (rpm)	E	120	180
Incubation time (days)	F	8	12

Table 2. PB design for the optimisation of parameters influencing antifungal activity of the cell-free filtrates from HU2014 culture.

Run	A	B	C	D	E	F	G	H	IR (%)
1	−1	−1	−1	−1	−1	−1	−1	−1	70.3
2	1	−1	−1	1	1	1	−1	−1	75.6
3	−1	1	1	1	−1	1	−1	1	88.6
4	−1	−1	−1	1	−1	−1	1	1	75.4
5	−1	1	1	1	1	−1	−1	−1	88.4
6	1	1	−1	−1	1	−1	−1	1	90.2
7	1	1	1	−1	−1	−1	1	−1	93.8
8	−1	−1	1	−1	1	1	1	−1	74.9
9	−1	1	−1	−1	1	1	1	1	83.7
10	1	−1	1	−1	−1	1	−1	1	84.5
11	1	1	−1	1	−1	1	1	−1	92.3
12	1	−1	1	1	1	−1	1	1	83.6

A represents dextrin; B represents yeast extract; C represents KNO₃; D represents pH; E represents inoculum size; F represents temperature; G represents agitation speed; H represents incubation time. IR represents inhibition rate.

Table 3. Input factors with their ranges and levels.

Input parameters	Minimum value (g/L)	Maximum value (g/L)	Level
Dex	30	40	3
YE	5	10	3
KNO3	1.5	2.5	3

DEX represents dextrin; YE represents yeast extract.

Table 4. RSM-based FCCD experimental design matrix for three factors.

Run	C.S: Dextrin (g/L)	N.S: yeast extract (g/L)	I.S: KNO3 (g/L)	IR (%)
R1	40.0	10.0	1.5	89.6
R2	30.0	10.0	1.5	81.3
R3	30.0	10.0	2.5	86.5
R4	35.0	10.0	2.0	90.2
R5	40.0	10.0	2.5	93.0
R6	30.0	5.0	2.5	74.9
R7	35.0	7.5	2.5	96.4
R8	35.0	7.5	2.0	88.8
R9	35.0	7.5	2.0	87.1
R10	40.0	5.0	2.5	82.5
R11	30.0	5.0	1.5	72.3
R12	35.0	7.5	2.0	89.2
R13	35.0	7.5	2.0	93.1
R14	35.0	7.5	1.5	94.5
R15	40.0	7.5	2.0	96.2
R16	40.0	5.0	1.5	76.7
R17	35.0	7.5	2.0	90.4
R18	35.0	7.5	2.0	94.7
R19	35.0	5.0	2.0	80.2
R20	30.0	7.5	2.0	87.0

C.S represents carbon sources; N.S represents nitrogen sources; I.S represents Inorganic salt; IR represents inhibition rate.

Figure 1. Scanning broad spectrum of the cell-free filtrates of HU2014 culture broth by spectrophotometer. IA1-10 represent the Abs lines of different sampling times.

Figure 2. Scanning broad spectrum of four fractions extracted from the cell-free filtrates of HU2014 culture broth by spectrophotometry (A) and the antifungal activity of four fractions against R. solani YL-3 on day 3 (B). Values are means ± SD of three independent experiments. Means with the same letter for inhibition rate are not significantly different (p＜0.05). F2, F4, F6 and F8 represent one of fractions, respectively.

Figure 3. Antifungal activities of cell-free filtrates of HU2014 culture broth against R. solani YL-3 and biomass growth of HU2014 in different conditions: Different basal media (A) and different culture conditions (B-F). Each treatment was repeated three times with three biological replicates, and the data show Means ± SD. G1 represents Gause’s No.1 medium. Cza represents Czapek’s medium.

Figure 4. The antifungal activities of cell-free filtrates of HU2014 culture broth with different medium components against R. solani YL-3 and the biomass of the strain (ACE), and variation of the R. solani YL-3 (BDF) inhibition rate with different concentration of medium components.

Figure 5. Pareto chart of the standardised effects for eight medium factors on the antifungal activities of the cell-free filtrates of HU2014 culture broth.

Table 5. ANOVA for the experimental result of PB design.

Source	DF	SS	MS	F value	P value
Model	8	646.284	80.785	23.74	0.012
Linear	8	646.284	80.785	23.74	0.012
Dextrin	1	126.425	126.425	37.15	0.009
Yeast extract	1	439.593	439.593	129.19	0.001
KNO3	1	57.685	57.685	16.95	0.026
pH	1	3.597	3.597	1.06	0.380
Inoculum size	1	6.035	6.035	1.77	0.275
Temperature	1	0.304	0.304	0.09	0.785
Agitation speed	1	3.050	3.050	0.90	0.414
Incubation time	1	9.594	9.594	2.82	0.192
Error	3	10.208	3.403
Total	11	656.492

Table 6. ANOVA for Quadratic RSM model details.

Source	Sum of Squares	df	Mean Square	F-value	p-value
Model	879.03	9	97.67	11.60	0.0003	Significant
A：DEX	129.04	1	129.04	15.32	0.0029
B：YE	290.34	1	290.34	34.48	0.0002
C：KNO3	35.38	1	35.38	4.20	0.0675
AB	0.8910	1	0.8910	0.1058	0.7517
AC	0.2651	1	0.2651	0.0315	0.8627
BC	0.0074	1	0.0074	0.0009	0.9770
A²	13.59	1	13.59	1.61	0.2327
B²	206.01	1	206.01	24.46	0.0006
C²	7.04	1	7.04	0.8358	0.3821
Residual	84.21	10	8.42
Lack of Fit	44.46	5	8.89	1.12	0.4526	Not significant
Cor Total	963.23	19

DEX represents dextrin; YE represents yeast extract.

Figure 6. 3D surface plot for the inhibition rate. (A) Nitrogen source and Carbon source; (B) Nitrogen source and inorganic salt; (C) Carbon source and inorganic salt. (D) Actual vs. predicted values plot for the inhibition rate. Abbreviation: DEX represents dextrin, YE represents yeast extract.

Figure 7. Optimisation result for the inhibition rate using RSM (A) and the inhibition rate of the cell-free filtrates of HU2014 cultured in GPY and DYK to R. solani YL-3 (B). DEX represents dextrin, YE represents yeast extract; DYK represents the optimised medium (dextrin, yeast extract and KNO₃).

Data availability statement

The data supporting the findings of this study are available from the authors upon reasonable request.