Characteristics of Aureobasidium sp. 3A00493 from Deep Sea Sediment and Characteristic Analysis of Its Extracellular Polysaccharide

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

Abstract

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

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.

Figures/Tables 7

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）

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

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

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

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

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

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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