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枯草芽孢杆菌CCTCC M207209抗扩展青霉特性研究——活性物质的提取、稳定性与应用
孙卉1, 师俊玲
西北农林科技大学 食品科学与工程学院,陕西 杨凌 712100
摘要:
【目的】 考察枯草芽孢杆菌CCTCC M207209抗扩展青霉活性蛋白在不同条件下的稳定性,评价其对苹果扩展青霉污染的抑制作用,为该菌的实际应用提供理论依据。【方法】 在确定出最佳硫酸铵饱和度的基础上,用50%的硫酸铵饱和度沉淀发酵液中的活性蛋白,将所得粗蛋白用pH 7.0的磷酸盐缓冲液溶解,用截留分子质量为8.0~10.0 ku的透析袋透析后,分别在121 ℃下处理30 min、经不同pH及0.05 mol/L不同金属离子处理8 h和紫外线照射不同时间后,用杯碟法测定其对扩展青霉的抑菌活性;在接种有15 μL扩展青霉孢子悬液的苹果上分别接种30 μL蛋白提取物、混合液体发酵物、无菌体发酵液和121 ℃灭菌30 min的带菌体发酵液,28 ℃培养7 d后,观察各孔处的发病率,并测量接种处病斑直径。【结果】 提取发酵液中抗扩展青霉活性蛋白的最适硫酸铵饱和度为50%;粗蛋白提取物的抑菌活性在121 ℃处理30 min后无减小;pH为7时蛋白稳定性最高,处理8 h后抑菌活性无降低;紫外线照射前3 h,蛋白活性迅速降低,此后变化较小;不同pH和紫外线照射8 h后,蛋白的活性保持率均大于50%;0.05 mol/L的Na+和Mg2+对抑菌活性有促进作用,Ca2+、Zn2+和Fe3+则使其完全失活。含菌体(1×108 /mL)发酵物能够完全抑制低含量扩展青霉(1.5×105 /mL)对苹果的侵染,活性蛋白可将其在苹果上的侵染率降低至55.6%,并可有效抑制其在苹果中的生长。【结论】 枯草芽孢杆菌CCTCC M207209所产抗扩展青霉活性蛋白在广泛的pH、紫外线、温度条件下,具有良好的稳定性,能够有效抑制扩展青霉对苹果的侵染,在防治大田及采后苹果贮藏过程中扩展青霉的污染方面,有很好的应用前景。
关键词:  枯草芽孢杆菌  扩展青霉  抗菌蛋白  蛋白稳定性  苹果贮藏
DOI:
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基金项目:国家科技支撑计划项目(2006BAK02A24);国家“863”高新技术研究与发展计划项目(2007AA10Z428)
Antifungal characteristics of Bacillus subtilis CCTCC M207209 against Penicillium expansum ——Extraction,stability,and application of the antifungal substances in the liquid culture
SUN Hui,SHI Jun-ling
Abstract:
【Objective】 The paper aimed at investigating the stability of the antifungal protein produced by Bacillus subtilis CCTCC M207209 under different conditions and evaluating its antifungal activity on apples against Penicillium expansum,and thus providing information on the application of strain CCTCC M207209 in practice.【Method】 Based on the greatest antifungal activity,ammonium sulfate saturation degree 50% was used to precipitate the active protein from the cell-free liquid culture of strain CCTCC M207209.After dissolved in a 0.2 mol/L pH 7.0 sodium phosphate buffer,the precipitated protein was dialyzed by a membrane of molecular weight of 8-10 ku against the same sodium phosphate buffer.The protein buffer solution retained by the membrane was used in all the other tests on the antifungal stability under different conditions.A cup-plate method was used to test the antifungal activity.Treatments for testing the active stability included holding at 121 ℃ for 30 min,under different pH and in 0.05 mol/L different metal ion solutions for 8 h,and certain UV strength for different periods.After the treatments,the protein solution was adjusted to its original pH and temperature.The antifungal activity on apples was tested by injecting 15 μL P.expansum spores suspension in the same hole after 30 μL antifungal substances were inoculated in apples.The tested antifungal substances included the antifungal protein,the liquid culture of strain CCTCC M207209 with cells,the autoclaved liquid culture with cells,and the cell-free liquid culture.The above inoculated apples were cultivated at 28 ℃ for 7 d.At the end of the cultivation,the ratio of P.expansum contaminated holes in apples was calculated and the diameter of the rotten spots was measured.【Result】 Protein precipitated at 50% ammonium sulfate saturation showed the greatest antifungal activity.The antifungal activity of the crude protein extracts did not reduce after treatment at 121 ℃ for 30 min and pH 7 for 8 h,but decreased quickly at the first 3 h under UV irradiation followed by very slow drop with more than 50% antifungal activity left after 8 h.Na+ and Mg2+ stimulated,but Ca2+,Zn2+ and Fe3+ destroyed the antifungal activity of the active protein at concentration of 0.05 mol/L The liquid culture with 1×108 cells/mL B.subtilis completely inhibited the growth of P.expansum (1.5×105 spores /mL)in apples.The antifungal protein reduced the occurrence rate of P.expansum contamination by 55.6% and significantly inhibited the growth of P.expansum in apples.【Conclusion】 The antifungal protein produced by B.subtilis CCTCC M207209 showed good stability under different environmental conditions and kept activity after autoclave,and thus had good potential in biological control of P.expansum in practice.
Key words:  Bacillus subtilis  Penicillium expansum  antifungal protein  protein stability  apple storage