一株高产黑色素香灰菌菌株的鉴定、筛选及培养条件的优化

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

摘要/Abstract

摘要：

为筛选一株产黑色素能力强的菌株并优化其培养条件。通过ITS测序鉴定11株供试菌株，以菌丝生长速度、平板L值等指标筛选出一株产黑能力强的香灰菌，并对其生长所需碳源、氮源、pH等培养条件进行优化。研究表明，11株供试菌株均为香灰菌（Hypoxylon sp.），其中Hp.sp0006菌丝生长速度较快、菌球大且均匀、L值最低，并且发酵液黑色素含量最高。该菌株最优培养条件是，以葡萄糖为碳源、牛肉浸膏为氮源、碳氮比20∶1并添加10 mg维生素B₁，黑色素含量可达（1.21±0.17）g/L。香灰菌Hp.sp0006是一株产黑色素较高的菌株，优化后的培养基更有利于黑色素的合成，为香灰菌黑色素的开发利用奠定基础。

关键词: 香灰菌, 黑色素, 培养优化, 单因素优化, 正交试验

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

黄海辰, 李晓敏, 薛帆正, 吴小平, 张君丽, 傅俊生. 一株高产黑色素香灰菌菌株的鉴定、筛选及培养条件的优化[J]. 生物技术通报, 2023, 39(1): 284-294.

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.

图/表 13

表1 正交试验因素及水平

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

图1 11株供试菌株的系统发育进化树采用邻接法的建树方法，自展法（Bootstrap）检验重复1 000次，图中的Annulohypoxylon stygium KP995421.1、Annulohypoxylon stygium FJ848865.1和Annulohypoxylon sp. MW862791.1为比对时的参考菌

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

图2 11株香灰菌菌丝形态及其菌丝生长速度、色度分析 A:不同菌株菌丝形态特征（a:Hp.sp0001; b:Hp.sp0002; c:Hp.sp0003; d:Hp.sp0006; e:川灰; f:古田AS（新）; g:古田AS; h:河南香灰; i:香灰3; j:白灰; k:黄耳香灰）; B:不同菌株菌丝生长速度; C:不同菌株平板L值。a, b, c不同字母代表显著差异（P<0.05）。下同

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

图3 11株香灰菌发酵液黑色素含量及菌球形态 A：不同香灰菌发酵液黑色素含量；B：不同菌株菌球形态（a: Hp.sp0001; b: Hp.sp0002; c: Hp.sp0003; d: Hp.sp0006; e:川灰 f:古田AS（新）; g:古田AS; h:河南香灰;i:香灰3; j:白灰; k:黄耳香灰）

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））

图4 不同碳源对香灰菌 Hp.sp0006菌丝体产黑色素的影响 A:菌丝生长速度; B:平板L值; C: 菌落正反面（a：CK对照；b：果糖；c：甘露醇；d：淀粉；e：乳糖；f：蔗糖；g：葡萄糖；h：麦芽糖；i：木糖；j：玉米粉）。与对照组相比，*P<0.05，**P<0.01。下同

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

图5 不同氮源对香灰菌Hp.sp0006菌丝体产黑色素的影响 A:菌丝生长速度; B:平板L值; C:菌落正反面（a：CK对照；b：酵母提取粉；c：胰蛋白胨；d：牛肉浸膏；e：硝酸钾；f：硝酸铵；g：酒石酸铵；h：脲）

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）

图6 不同碳氮比对香灰菌Hp.sp0006菌丝体产黑色素的影响 A:菌丝生长速度; B:平板L值; C菌落正反面（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. 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）

图7 不同维生素对香灰菌Hp.sp0006菌丝体产黑色素的影响 A:菌丝生长速度; B:平板L值; C:菌落正反面（a：CK对照；b：VB1；c：VB2；d：VB6；e：VB9；f：VC；g：VH；h：VPP）

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）

图8 不同pH对香灰菌Hp.sp0006菌丝体产黑色素的影响 A:菌丝生长速度; B:平板L值; C: 菌落正反面（a：对照；b：5.0；c：5.5；d：6.0；e：6.5；f：7.0；g：7.5；h：8.0）

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）

表2 香灰菌平板L值正交试验结果直观分析

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

图9 正交试验中平板菌丝体生长状况图中序号同表2

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

表3 方差分析

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

图10 正交实验最优组合液体发酵的黑色素含量与优化前相比，*P<0.05

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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