深海出芽短梗霉（Aureobasidium sp. 3A00493）菌株特征与胞外多糖特性分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0119

摘要/Abstract

摘要：

普鲁兰糖（pullulan）是短梗霉属（Aureobasidium spp.）的一些菌产生的极具高附加值、开发价值的天然微生物多糖。然而不同来源的菌株在产糖量、抑菌活性、副产物含量等方面有很大的差异,因此,寻找产糖量高、抑菌活性强、副产物少的优良菌株一直是研究人员关注的焦点。本研究的出发菌株3A00493是一株从太平洋深海沉积物中分离的产胞外多糖海洋微生物。通过形态学和显微镜观察、分子鉴定确定其分类归属;改变培养条件确定了其生长特性、最佳产糖条件;选择5种常见微生物作为指示菌,滤纸片法测定其抑菌活性;薄层层析和红外光谱分析了多糖结构。出发菌株3A00493是一株菌落乳白,细胞和菌丝体可转换生长,ITS和18S rDNA部分序列与报道短梗霉属（Aureobasidium sp.）同源性≥ 98%的海洋源出芽短梗霉菌;其最适生长温度为26℃,最适生长pH值为4,最适条件下产糖量达24.58 g/L;3A00493的代谢产物对4种指示菌具有较强抑菌活性;薄层层析和红外光谱测定结果表明3A00493产生的胞外多糖与普鲁兰糖标准品一致,确定为普鲁兰糖。出发菌株3A00493为一株产普鲁兰糖、代谢物抑菌活性较强、色素副产物少的海洋源出芽短梗霉新菌株。本研究为进一步研究海洋源产普鲁兰糖新菌3A00493奠定了理论基础,为寻找海洋源微生物多糖提供了新菌株,也为筛选和开发海洋资源微生物提供研究借鉴。

关键词: 出芽短梗霉, 普鲁兰糖, 结构鉴定, 抑菌活性

Abstract:

Pullulan is a natural microbial polysaccharide with high developed value produced by fungus Aureobasidium sp. But different strains present great differences in polysaccharide yield,biological activities and by-products. Therefore,it has always been the focus of researchers to find the excellent strains with high sugar production,strong bacteriostatic activity and less by-products. The original strain is an extracellular polysaccharide-producing microorganism isolated from deep seabed sediments in the Pacific Ocean. On the basis of its morphological and microscopic observation as well as molecular identification,the strain was identified as its genus. The growth characteristics and optimal polysaccharide production conditions were determined via changing the culturing conditions. Five common microorganisms were selected as indicator bacteria,and the antibacterial activity was determined by filter paper method,and the structure of polysaccharides was analyzed by thin layer chromatography and infrared spectroscopy. The original strain 3A00493 was marine-sourced Aureobasidium pullulans,its colony was milky white,its cell and mycelium grew in converted way,and its ITS and 18S rDNA partial sequences shared homology of ≥98% with reported Aureobasidium sp. Its optimal growth temperature was 26℃ and optimal growth pH was 4,under which the polysaccharide yield reached 24.58 g/L. The metabolites of the strain 3A00493 had well antibacterial activity against 4 pathogenic indicator microorganisms. TLC and IR spectra showed that the extracellular polysaccharide produced by the strain 3A00493 was consistent with the pullulan standard sample,thus it was determined as pullulan. In conclusion,original strain 3A00493 is a new marine-sourced A. pullulans producing pullulan,being strong antibacterial activity of its metabolite,and having little pigment byproduct. This work lays a theoretical foundation for further studying marine-sourced pullulan-producing 3A00493,providing a new strain in seeking marine-sources microbial polysaccharide,also providing references for screening and developing marine resources microorganisms.

Key words: Aureobasidium pullulans, pullulan, structure identification, antibacterial activity

潘镜宇, 陈佳乐, 钱玙呈, 刘鑫, 杨昊宁, 刘立, 魏步云, 赵洪新. 深海出芽短梗霉（Aureobasidium sp. 3A00493）菌株特征与胞外多糖特性分析[J]. 生物技术通报, 2021, 37(12): 71-81.

PAN Jing-yu, CHEN Jia-le, QIAN Yu-cheng, LIU Xin, YANG Hao-ning, LIU Li, WEI Bu-yun, ZHAO Hong-xin. Characteristics of Aureobasidium sp. 3A00493 from Deep Sea Sediment and Characteristic Analysis of Its Extracellular Polysaccharide[J]. Biotechnology Bulletin, 2021, 37(12): 71-81.

图/表 7

图1 菌株3A00493的特征 A:单菌落;B:菌落及菌丝体;C:酵母样单细胞;D:菌丝体细胞（C/D:1 000×显微照片）

Fig. 1 Characteristics of strain 3A00493 A:Single colony. B:Colony and mycelium. C:Yeast like single cells. D:Mycelium cells（C/D:1 000× micrograph）

图2 菌株3A00493系统发育进化树 A:内转录间隔区D1/2区;B:18S rDNA基因部分序列

Fig. 2 Phylogenetic tree of strain 3A00493 A:Internal transcribed spacer D1/2. B:Partial sequence of 18S rDNA gene

图3 菌株3A00493生长曲线及产糖曲线（n=3）

Fig. 3 Growth curve and sugar production curve of strain 3A00493（n=3）

图4 不同培养条件对菌株3A00493的产糖及菌株生长的影响（n=3） A:pH;B:温度;C:装液量;D:转速,下同

Fig. 4 Effects of different culture conditions on the sugar production and growth of strain 3A00493（n=3） A:pH. B:Temperature. C:Cultivation volume. D:Rotation speed. The same below

图5 不同条件培养菌株3A00493发酵液的pH值变化曲线（n=3）

Fig. 5 pH variation of fermentation broth of strain 3A00493 under different cultivation conditions（n=3）

表 1 菌株 3A00493发酵产物的抑菌活性

Table 1 Antibacterial activity of fermentation products of strain 3A00493

指示菌 Indicator	革兰氏染色特性 Gram staining features	抑菌圈直径 Diameter of inhibition zome/mm
指示菌 Indicator	革兰氏染色特性 Gram staining features	A. pullulans 3A00493	PDA（Ck）	卡那霉素 Kanamycin/（50 μg·mL^-1）	氨苄青霉素 Ampicillin（50 μg·mL^-1）
大肠杆菌（E. coil ATCC8091）	G^-	12.47±0.82	-	12.42±2.39	10.02±2.19
铜绿假单胞菌（P. aeruginosa）	G^-	-	-	16.87±0.90	8.65±1.38
枯草芽孢杆菌（B. subtilis ATCC6051）	G⁺	12.70±1.65	-	13.28±0.73	6.95±0.65
金黄色葡萄球菌（S. aureus ATCC6538）	G⁺	11.91±0.36	-	15.88±0.93	18.53±0.79
粪肠球菌（E. faecalis ATCC29212）	G⁺	14.91±0.55	-	11.48±5.86	-
白色念珠菌（M. albican ATCC10231）	无None	-	-	14.78±3.73	9.72±0.83

图6 多糖薄层层析 A. 1/2/3/4:标准品/样品/标准品酶解液/样品酶解液,箭头示麦芽三糖;B:样品红外光谱鉴定

Fig. 6 Polysaccharide TLC A:1/2/3/4:Standard sample/Tested sample/Hydrolysate of standard sample/Hydrolysate of tested sample,arrow shows maltotriose. B:Identification of tested sample with IR spectrogram

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