Screening, Identification, and Optimization of Culture Conditions of a Melanin High-yielding Strain of Hypoxylon sp.

doi:10.13560/j.cnki.biotech.bull.1985.2022-0119

Abstract

Abstract:

In order to screen a strain with strong melanin-producing ability and optimize its culture conditions, 11 tested strains were identified by ITS sequencing, and a ash-producing fungus with strong black-producing ability was screened based on mycelial growth rate, plate L value and other indicators, and the carbon source, nitrogen source, pH and other culture conditions required for its growth were optimized. The study showed that all 11 test strains were Hypoxylon sp. Among them, Hp.sp0006 had faster mycelial growth, larger and more uniform balls, the lowest L value, and the highest melanin content in the fermentation broth. The optimal culture conditions for this strain were glucose as the carbon source, beef extract as the nitrogen source, a 20∶1 carbon to nitrogen ratio and the addition of 10 mg vitamin B₁, with a high yield of melanin up to（1.21±0.17）g/L melanin. Hp.sp0006 is a strain producing higher melanin. The optimized medium is more conducive to the synthesis of melanin, which lays the foundation for the development and utilization of melanin in Hypoxylon sp.

Key words: Hypoxylon sp., melanin, culture optimization, single factor optimization, orthogonal test

HUANG Hai-chen, LI Xiao-min, XUE Fan-zheng, WU Xiao-ping, ZHANG Jun-li, FU Jun-sheng. Screening, Identification, and Optimization of Culture Conditions of a Melanin High-yielding Strain of Hypoxylon sp.[J]. Biotechnology Bulletin, 2023, 39(1): 284-294.

Figures/Tables 13

Table 1 Factors and levels of orthogonal experiment

水平 Level	因素Factors
水平 Level	碳源 Carbon source	氮源 Nitrogen source	碳氮比 Carbon nitrogen ratio	pH
1	葡萄糖 Glucose	牛肉浸膏 Beef Extract	5∶1	5
2	果糖 Fructose	硝酸铵 Ammonium Nitrate	10∶1	5.5
3	蔗糖 Sucrose	胰蛋白胨 Tryptone	20∶1	6

Fig. 1 Phylogenetic tree of 11 tested strains The tree-building method of the neighbor-joining method was used, and the Bootstrap test was repeated 1 000 times. Annulohypoxylon stygium KP995421.1, Annulohypoxylon stygium FJ848865.1 and Annulohypoxylon sp. MW862791.1 in the figure were the reference bacteria for comparison

Fig. 2 Analysis of mycelial morphology and mycelial growth rate and chromaticity of 11 strains of Hypoxylon sp. A: Mycelial morphological characteristics of different strains（a: Hp.sp0001; b: Hp.sp0002; c: Hp.sp0003; d: Hp.sp0006; e:ChuanHui; f: Gutian AS（New）; g: Gutian AS; h:Henan Xianghui; i: Xianghui 3; j:Baihui; k: Huang′er Xianghui）. B: Mycelial growth rate of different strains. C: Morphology of micro ball. D: Plate L value of different strains. Different letters of a, b, c represent significant differences. The same below

Fig. 3 Melanin content and spheroid morphology in fermentation broth of 11 Hypoxylon sp. A: The content of melanin in the fermentation broth of different Hypoxylon sp. B: The shape of the spheroids of different strains（a: Hp.sp0001; b: Hp.sp0002; c: Hp.sp0003; d: Hp.sp0006; e: Chuanhui; f: Gutian AS（New）; g: Gutian AS; h: Henan Xianghui; i: Xianghui 3; j: Baihui; k:Huang'er Xianghui））

Fig. 4 Effects of different carbon sources on the produced melanin by mycelium of Hp.sp0006 A: Growth rate of mycelium. B: L value. C: Colony front and back（a: CK. b: Fructose. c: Mannitol. d: Starch. e: Lactose. f: Sucrose. g: Glucose. h: Maltose. i: Xylose. j: Corn flour）。Compared with the CK, *P<0.05, **P<0.01. The same below

Fig. 5 Effects of different nitrogen sources on the produced melanin by mycelium of Hp.sp0006 A: Growth rate of mycelium. B: L value. C: Colony front and back（a: CK. b: Yeast extract powder. c: Tryptone. d: Beef extract. e: Potassium nitrate. f: Ammonium nitrate. g: Ammonium tartrate. h: Urea）

Fig. 6 Effects of different carbon nitrogen ratio on the produced melanin by mycelium of Hp.sp0006 A: Growth rate of mycelium. B: L value. C: Colony front and back（a：5∶1；b：10∶1；c：20∶1；d：30∶1；e：40∶1；f：50∶1；g：60∶1；h：70∶1）

Fig. 7 Effects of different vitamin on the produced melanin by mycelium of Hp.sp0006 A:Growth rate of mycelium. B: L value. C: Colony front and back（a：CK；b：VB1；c：VB2；d：VB6；e：VB9；f：VC；g：VH；h：VPP）

Fig. 8 Effects of different pH on the produced melanin by mycelium of Hp.sp0006 A: Growth rate of mycelium. B: L value. C: Colony front and back（a：CK；b：5.0；c：5.5；d：6.0；e：6.5；f：7.0；g：7.5；h：8.0）

Table 2 Analysis of the orthogonal test result of the L value of Hp.sp0006

序号No.	碳源Carbon source	氮源Nitrogen source	碳氮比Carbon nitrogen ratio	pH	L值L value
1	葡萄糖 Glucose	牛肉浸膏 Beef extract	5∶1	5	24.00 ±1.63
2	葡萄糖 Glucose	硝酸铵 Ammonium nitrate	10∶1	5.5	30.33± 2.62
3	葡萄糖 Glucose	胰蛋白胨 Tryptone	20∶1	6	26.33 ±0.47
4	果糖 Fructose	牛肉浸膏 Beef extract	10∶1	6	25.33±1.70
5	果糖 Fructose	硝酸铵 Ammonium nitrate	20∶1	5	26.00±0.82
6	果糖 Fructose	胰蛋白胨 Tryptone	5∶1	5.5	36.33±3.68
7	蔗糖 Sucrose	牛肉浸膏 Beef extract	20∶1	5.5	26.00±2.16
8	蔗糖 Sucrose	硝酸铵 Ammonium nitrate	5∶1	6	29.33±2.62
9	蔗糖 Sucrose	胰蛋白胨 Tryptone	10∶1	5	34.33±3.68
k₁	26.887	25.110	29.887	28.110
k₂	29.220	28.553	29.997	30.887
k₃	29.887	32.330	26.110	26.997
极差	3.000	7.220	3.887	3.890

Fig. 9 Growth status of the mycelium of Hp.sp0006 in orthogonal test Serial number in the Figure as in Table 2

Table 3 Analysis of variance

方差来源Source of variance	平方和Sum of squares of deviations	自由度df	均方Mean square	F值F value	显著性Significance level
修正模型	440.000^a	8	55.000	6.346	0.001
截距	22 188.000	1	22 188.000	2 560.154	0.000
碳源	44.667	2	22.333	2.577	0.104
氮源	234.889	2	117.444	13.551	0.000
碳氮比	88.222	2	44.111	5.090	0.018
pH	72.222	2	36.111	4.167	0.033
误差	156.000	18	8.667
总计	22 784.000	27
修正后总计	596.000	26

Fig. 10 Orthogonal experiment for optimal combination of liquid fermentation for melanin content Compared with the pre-optimization, * P <0.05

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