梨储藏期病害生防菌及防腐保鲜的研究
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摘要
梨是我国主栽水果之一,营养价值很高。但是在储藏过程中很容易造成一些病害,除潜伏侵染的轮纹病、褐腐病等病害在储藏期继续发病腐烂应及时清理外,其他的主要病害有黑心病、果柄基腐病、青霉病、黑皮病、褐斑病、黑斑病等,严重影响梨果的储藏时间以及品质,从而使经济价值大大降低。因此在储运中,及时全面把握梨果常见病害并对其进行综合防治十分重要。本文以储藏期梨果为对象,在分析果实微生物区系基础上,分离了梨果储藏期病原菌,并筛选出优势生防菌,以优势生防菌发酵液进行生物保鲜研究,主要结果如下:
1、通过组织分离法及柯赫氏法则获得三种梨果腐烂菌,经形态学以及分子生物学方法鉴定为:疖葡萄座腔菌(Botryosphaeria dothidea)、间座壳属(Diaportheoncostoma)、淡色生赤壳菌(Bionectria ochroleuca)。稀释平板法分离出疑似生防菌(真菌、细菌、放线菌),用对峙培养法筛选优势生防菌,经形态学及分子生物学鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。
2、用正交设计试验方法对解淀粉芽孢杆菌的发酵条件进行优化,确定其最佳C源为麦芽糖,最佳N源为(NH_4)_2SO_4。伸最适发酵条件:温度为25℃,初始pH值为6,瓶装量为125mL,C源的量为2%,N源的量为0.3%。
3、梨果储藏期病害保鲜研究显示:无膜处理方法相对于有膜处理方法效果更好,菌剂浓度稀释至20倍时,其相对防效仍能达到43.76%。稀释至10倍时,相对防效能达到62.51%。稀释至5倍时,相对防效为75%。原液的相对防效为87.51%。在有膜处理方法中,菌剂浓度稀释至20倍时,相对防效仅为17.81%。而当稀释浓度为10倍时,相对防效提高到46.4%。当稀释至5倍时,相对防效能突破50%达到57.14%。原液的相对防效高达89.29%。
4、生化测定显示:与储藏前水平比较,无膜处理后的梨果内可溶性糖基本不减少,仍能保持很好的糖含量。有膜处理后的梨果内可溶性糖的含量均呈现减少的趋势,不同浓度的处理液处理过的梨果之间可溶性糖含量差异不大。而无膜处理和有膜处理后的梨果内可溶性蛋白的含量均出现减少趋势。但减少的规律并不明显,既没有有无膜的明显差异。也没有不同发酵液浓度之间的明显差异。
Pear is one of the main fruits grown in China, which has high nutritional value. However, a number of diseases easily occurred in the course of storage, such as physalospora and brown rot disease which was latent infection and could be cleared in time to stop rotting. But the other major diseases such as black heart disease, rotting disease in fruit base, Green mildew, black disease, brown spot, black spot and so on, could seriously affect the storage time as well as the pome quality, thus greatly reduce the economic value. Therefore, it's very important to know the Pear Diseases and grasp integrated control techniques timely and fully in pear storage. In this paper, pears during storage period were used for study. Pome pathogens during storage were separated based on the analysis of the fruits microbial flora, and the best biocontrol microbes were screened. This paper did some research in biological preservation using zymotic fluid of the best biocontrol microbes. The main results were as follows:
1. Three types of Pear rot fungi were attained throμgh the organizational separation and the Koch assumption, which were identified as: Botryosphaeria dothidea, Diaporthe oncostoma and Bionectria ochroleuca with morphological and molecular biology methods. The suspected biocontrol microbes (fungi, bacteria, actinomycetes) were isolated by dilution plate method. The best biocontrol microbes were screened by confrontation culture and were identified as Bacillus amyloliquefaciens with morphological and molecular biology methods.
2. The fermentation condition of Bacillus amyloliquefaciens was optimized with orthogonal design test method. It determined the best C source as maltose, and the best N source as the (NH4) 2SO4. The optimum fermentation conditions were: temperature of 25℃, initial pH value of 6, cylinders of 125mL, C source was 2%, N source was 0.3%.
3. The results of study on fresh-keeping in pome storage period showed: treatment methods with non-membrane were relatively better than that with membrane. When the concentration diluted to 20 times, the relative control effect would be able to reach 43.76%; when diluted to 10 times, it was 62.51%; when diluted to 5 times, 75%; and the stock solution, 87.51%. In the treatments with membrane, when the concentration diluted to 20 times, the relative control effect was only 17.81%; when diluted to 10 times, reached to 46.4%. When diluted to 5 times. 57.14%. The relative control effect of the stock solution was up to 89.29%.
4.Results on chemical and biological measuration showed that: compared with pre-stored, the soluble sμgar in pome basically did not reduce by non-membrane treatment, in which good sμgar content was maintained. The soluble sμgar content showed a decreasing trend after membrane treated, but the difference among these treatments was not significant. The soluble protein content was also declined no matter with membrane or without. However, the law of the reduction was not significant, neither between with and without the membrane, nor among the deferent fermentation concentration.
1、通过组织分离法及柯赫氏法则获得三种梨果腐烂菌,经形态学以及分子生物学方法鉴定为:疖葡萄座腔菌(Botryosphaeria dothidea)、间座壳属(Diaportheoncostoma)、淡色生赤壳菌(Bionectria ochroleuca)。稀释平板法分离出疑似生防菌(真菌、细菌、放线菌),用对峙培养法筛选优势生防菌,经形态学及分子生物学鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。
2、用正交设计试验方法对解淀粉芽孢杆菌的发酵条件进行优化,确定其最佳C源为麦芽糖,最佳N源为(NH_4)_2SO_4。伸最适发酵条件:温度为25℃,初始pH值为6,瓶装量为125mL,C源的量为2%,N源的量为0.3%。
3、梨果储藏期病害保鲜研究显示:无膜处理方法相对于有膜处理方法效果更好,菌剂浓度稀释至20倍时,其相对防效仍能达到43.76%。稀释至10倍时,相对防效能达到62.51%。稀释至5倍时,相对防效为75%。原液的相对防效为87.51%。在有膜处理方法中,菌剂浓度稀释至20倍时,相对防效仅为17.81%。而当稀释浓度为10倍时,相对防效提高到46.4%。当稀释至5倍时,相对防效能突破50%达到57.14%。原液的相对防效高达89.29%。
4、生化测定显示:与储藏前水平比较,无膜处理后的梨果内可溶性糖基本不减少,仍能保持很好的糖含量。有膜处理后的梨果内可溶性糖的含量均呈现减少的趋势,不同浓度的处理液处理过的梨果之间可溶性糖含量差异不大。而无膜处理和有膜处理后的梨果内可溶性蛋白的含量均出现减少趋势。但减少的规律并不明显,既没有有无膜的明显差异。也没有不同发酵液浓度之间的明显差异。
Pear is one of the main fruits grown in China, which has high nutritional value. However, a number of diseases easily occurred in the course of storage, such as physalospora and brown rot disease which was latent infection and could be cleared in time to stop rotting. But the other major diseases such as black heart disease, rotting disease in fruit base, Green mildew, black disease, brown spot, black spot and so on, could seriously affect the storage time as well as the pome quality, thus greatly reduce the economic value. Therefore, it's very important to know the Pear Diseases and grasp integrated control techniques timely and fully in pear storage. In this paper, pears during storage period were used for study. Pome pathogens during storage were separated based on the analysis of the fruits microbial flora, and the best biocontrol microbes were screened. This paper did some research in biological preservation using zymotic fluid of the best biocontrol microbes. The main results were as follows:
1. Three types of Pear rot fungi were attained throμgh the organizational separation and the Koch assumption, which were identified as: Botryosphaeria dothidea, Diaporthe oncostoma and Bionectria ochroleuca with morphological and molecular biology methods. The suspected biocontrol microbes (fungi, bacteria, actinomycetes) were isolated by dilution plate method. The best biocontrol microbes were screened by confrontation culture and were identified as Bacillus amyloliquefaciens with morphological and molecular biology methods.
2. The fermentation condition of Bacillus amyloliquefaciens was optimized with orthogonal design test method. It determined the best C source as maltose, and the best N source as the (NH4) 2SO4. The optimum fermentation conditions were: temperature of 25℃, initial pH value of 6, cylinders of 125mL, C source was 2%, N source was 0.3%.
3. The results of study on fresh-keeping in pome storage period showed: treatment methods with non-membrane were relatively better than that with membrane. When the concentration diluted to 20 times, the relative control effect would be able to reach 43.76%; when diluted to 10 times, it was 62.51%; when diluted to 5 times, 75%; and the stock solution, 87.51%. In the treatments with membrane, when the concentration diluted to 20 times, the relative control effect was only 17.81%; when diluted to 10 times, reached to 46.4%. When diluted to 5 times. 57.14%. The relative control effect of the stock solution was up to 89.29%.
4.Results on chemical and biological measuration showed that: compared with pre-stored, the soluble sμgar in pome basically did not reduce by non-membrane treatment, in which good sμgar content was maintained. The soluble sμgar content showed a decreasing trend after membrane treated, but the difference among these treatments was not significant. The soluble protein content was also declined no matter with membrane or without. However, the law of the reduction was not significant, neither between with and without the membrane, nor among the deferent fermentation concentration.
引文
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