Optimization of Fermentation Conditions of Endophytic Bacterium ZW21 Isolated from Stylosanthes and Stability Analysis of Antimicrobial Substances

doi:10.13560/j.cnki.biotech.bull.1985.2024-1043

Abstract

Abstract:

Objective The antagonistic strain Bacillus cereus ZW21 has significant potential for biological control against the pathogen causing stylo anthracnose. To improve the yield of active secondary metabolites and the bio-control effect of strain ZW21, the optimal fermentation conditions were investigated. Method The medium composition were optimized by single factor test and orthogonal test, and the fermentation conditions (pH, rotating speed, temperature, inoculation amount, liquid volume, and fermentation time) were optimized by single factor test. The stability of fermentation filtrate under the treatment of temperature, ultraviolet irradiation, acid-base and protease was determined. The optimization effect was tested by pot biological control test. Result The optimized results showed that the optimal medium composition was 0.5% soluble starch, 1% phenylalanine, and 0.5% magnesium sulfate. The optimal fermentation conditions were as follows: initial pH 4, rotating speed 180 r/min, temperature 28 ℃, liquid volume 200 mL, inoculation amount 7%, and fermentation time 96 h. The results of stability showed that the fermentation filtrate was resistant to the high temperature of 100 ℃, ultraviolet irradiation for 8 h, pepsin and trypsin treatment and strong acid, but susceptible to strong alkali. Under the optimized culture conditions, the antagonistic activity and bio-control effect of fermentation filtrate increased by 10 times and 4 times, respectively. Conclusion The fermentation broth of strain ZW21 has fine stability. The optimized fermentation broth has an OD₆₀₀ value of 7.05 and an inhibition rate of 73.18%, with a control efficacy of 62.5% against stylo anthracnose.

Key words: Colletotrichum gloeosporioides, biological control, Bacillus, anthracnose, anti-fungal activity, stability, biological control effect

ZHANG Ji-chang, XU Yun-feng, JIANG Ling-yan. Optimization of Fermentation Conditions of Endophytic Bacterium ZW21 Isolated from Stylosanthes and Stability Analysis of Antimicrobial Substances[J]. Biotechnology Bulletin, 2025, 41(5): 280-289.

Figures/Tables 8

Table 1 Factors of orthogonal test

编号 No.	因素 Factor
编号 No.	可溶性淀粉 Soluble starch（A）（%）	苯丙氨酸 L-phenylalanine（B）（%）	硫酸镁 MgSO₄（C）（%）
1	0.25	0.5	0.5
2	0.5	1	1
3	1	1.5	1.5

Fig. 1 Growth curve of ZW21 strain

Fig. 2 Effects of carbon sources, nitrogen sources and types of inorganic salts on the growth of strain ZW21 and the antibacterial activity of its sterile fermentation brothDifferent lowercase letters indicate significant difference between treatments at 0.05 level. The same below

Table 2 Results of orthogonal test on nutritional conditions of strain ZW21

编号 No.	因素 Factor			抑菌率 Inhibition rate （%）	OD₆₀₀
编号 No.	可溶性淀粉 Soluble starch （A）（%）	苯丙氨酸 L-Phenylalanine （B）（%）	硫酸镁 MgSO₄（C）（%）	抑菌率 Inhibition rate （%）	OD₆₀₀
1	0.25	0.5	0.5	41.53±4.65^d	5.5±0.18^b
2	0.25	1	1.5	46.82±2.14^cd	4.96±0.11^b
3	0.25	1.5	1	51.181.62^bcd	4.44±0.14^b
4	0.5	0.5	1	52.82±0.71^bcd	5.49±0.21^b
5	0.5	1	0.5	65.76±1.45^a	7.44±0.23^a
6	0.5	1.5	1.5	61.18±2.78^ab	6.25±0.14^a
7	1	0.5	1.5	44.94±4.4^d	5.8±0.07^b
8	1	1	1	58.24±1.7^abc	7.19±0.16^a
9	1	1.5	0.5	57.41±0.68^abc	7.06±0.44^a
K1	418.59	417.87	494.1
K2	539.28	512.46	486.72
K3	481.77	509.31	458.82
k1	46.51	46.43	54.9
k2	59.92	56.94	54.08
k3	53.53	56.59	50.98
R	13.41	10.51	3.92
K1′	44.7	50.37	60
K2′	57.54	58.77	51.37
K3′	60.15	53.25	51.03
k1′	4.97	5.6	6..67
k2′	6.39	6.53	5.71
k3′	6.68	5.92	5.67
R	1.72	0.93	1

Fig. 3 Effects of different fermentation conditions on the growth of strain ZW21 and the inhibitory activity of sterile fermentation filtrate

Fig. 4 Stability analysis of sterile fermentation filtrate of strain ZW21

Fig. 5 Comparison of inhibition activity of strain ZW21 fermentation filtrateA: Blank control; B: fermentation filtrate before optimization; C: optimized fermentation filtrate

Fig. 6 Comparison of control effects of potted plant sprayed with fermentation liquids before and after ZW21 optimizedA: Control (inoculation of C. gloeosproioides only); B: treatment of unoptimized fermentation filtrate before inoculation of C. gloeosporioides; C: treatment of optimized fermentation filtrate before inoculation of C. gloeosporioides

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