广谱高效微生物防腐剂产生菌的筛选及应用.rar

摘  要

微生物导致食品的腐败变质不仅会造成巨大经济损失，还会引起严重的食品安全问题。而现有的食品防腐保鲜方法在实际生产应用中具有一定的局限性，迫切需要寻求更安全有效的防腐保鲜新技术，将微生物及其代谢产物应用于食品防腐保鲜的生物防治方法是现阶段研究的热点。

从轮马热泉、北海温泉、大塘热泉等腾冲温泉附近采集土壤样品23种，采用稀释分离法分离纯化得到细菌菌株142株。以大肠杆菌、黑曲霉、禾谷镰刀菌等为指示菌，采用滤纸片法或平板对峙法从142株细菌菌株中初步筛选得到具有广谱抑菌效果的菌株7株，其中菌株 LM2303广谱抑菌效果最好。通过形态特征分析、生理生化特性分析和16S rDNA序列比对，菌株LM2303应为枯草芽孢杆菌(Bacillus subtilis)。

采用紫外线诱变、硫酸二乙酯诱变和微波诱变对菌株LM2303进行选育。其中，经硫酸二乙酯诱变处理得到突变菌株D59，其抑菌效果较原始菌株提高了11.4%。以抑菌物质产量为指标，从8种基础培养基中筛选出菌株D59最佳发酵培养基M2。采用单因素实验和响应面分析对菌株D59进行发酵条件优化。单因素试验结果表明培养时间、温度和转速对菌株D59抑菌物质产量有显著影响(p<0.05)。利用响应面确定菌株D59的最佳培养条件为：接种量5%、装液量100mL/250mL、培养时间43h、温度33℃、转速177rpm。在上述发酵条件下培养，100mL发酵液可得到抑菌物质粗提物2.8560 g(湿重)，是原发酵条件下的1.4倍，有效提高了抑菌物质产量。

采用盐酸沉淀、甲醇萃取，从拮抗菌株发酵液中得到抑菌物质粗提物。利用薄层色谱对抑菌物质进行分析和初步纯化，紫外及茚三酮显色结果表明抑菌物质含有苯环和闭合肽键。红外光谱扫描结果表明菌株LM2303抑菌物质为环状脂肽类物质。该脂肽类物质理化性质稳定，抑菌谱广，对大肠杆菌、金黄色葡萄球菌等细菌和黑曲霉、黄曲霉、扩展青霉、禾谷镰刀菌等真菌均有较强的抑制作用。

从不同腐烂病症的冬枣上分离得到根菌索菌、细极链格孢、橄榄色盾壳霉、扩展青霉四种致腐真菌。平板对峙试验结果表明菌株LM2303可有效抑制上述四种真菌的生长，菌株LM2303具有冬枣贮藏期致腐真菌的生物防控应用潜力。

…………………

关键词：枯草芽孢杆菌；拮抗；抑菌物质；诱变选育；发酵条件优化

ABSTRACT

Food spoilage caused by pathogenic microorganisms not only results in great financial losses, but also serious food safety problems. And existing food antisepsis method in practical application has certain limitations, to explore an obvious safe and effective anticorrosion technology is particularly important. Microorganisms and their metabolites work for food conservation is a research focus of biological control at the present stage.

142 strains of bacteria were isolated from 23 soil samples which collected form Tengchong hot spring, such as Lun-ma hot spring, Bei-hai hot spring, Da-tang hot spring and so on. Filtering paper method and dual culture technique were employed to test antagonistic effect of bacteria strains, and seven of them showed efficient inhibitory, strain LM2303 has the most obvious inhibitory effect. Strain LM2303 was identified as Bacillus subtilis through the analyses of morphological and biochemical properties and 16S rDNA sequencing.

Bacillus subtilis LM2303 was used as the start strain and the mutation breeding was carried out through UV light, DES and microwave radiation. Inhibition ratio of strain D59 isolated following DES mutagenesis enhance 11.4%. The production of biocontrol bubstance was taken as indexes, M2 medium was used for fermentation screened from 8 different medium. The factors and its levels influencing the production of biocontrol bubstance produced by strain D59, was determined by using single-factor test, and the optimization of fermentation conditions was approached by RSM. The optimum fermentation conditions were as follows: temperature was 32.6℃, inoculation was 5%, medium volume was 100mL/250mL, rotary speed was 176.9 rpm and fermentation time was 43.02 h. Under the new fermentation condition, 2.8560 g biocontrol bubstance can be obtained from 100 mL fermentation broth and is the 1.4 times of original fermentation conditions.

Biocontrol bubstance were obtained with hydrochloric acid precipitate and methanol extraction method from fermentation broth, and purified by thin layer chromatography. Purified biocontrol bubstance was detected by FT-IR. The results showed that the biocontrol bubstance contained lipopeptides. Biocontrol bubstance produced by strain LM2303 has stable physical and chemical properties and wide antibacterial spectrum, exhibit strong inhibitory effect on a number of bacteria and multiple pathogenic fungi.

Four putrefying fungi were isolated from the decayed tissues of Winter Jujube and identified as Rhizomorpha Roth.ex Fr, Alternaria tenuissima, Coniothyrium olivaceum and Penicillium expansum. Strain LM2303’s prominent inhibition on four putrefying fungi lay the foundation for putrefying fungi biocontrol of Winter Jujube.

…………………

Key words: Bacillus subtilis; antagonistic action; biocontrol bubstance;    mutation breeding; optimization of fermentation conditions

目  录

第一章 前言 1

1.1 研究背景 1

1.2 生防微生物概况 2

1.2.1 用于生物防治的微生物种类 2

1.2.2 生防微生物的作用机制 2

1.3 生防芽孢杆菌 4

1.4 芽孢杆菌抑菌物质研究概况 4

1.5 本研究的目的和意义 6

第二章 广谱拮抗菌株的筛选及其鉴定 8

2.1 材料 8

2.1.1 样品 8

2.1.2 试剂 9

2.1.3 仪器 10

2.1.4 培养基 10

2.1.5 供试有害微生物 11

2.2 方法 11

2.2.1 微生物培养 11

2.2.2 细菌菌株分离纯化 11

2.2.3 菌悬液制备 11

2.2.4 拮抗菌株初筛 12

2.2.5 拮抗菌株无细胞培养滤液制备 12

2.2.6 拮抗菌株的复筛 12

2.2.7 拮抗菌株形态特征观察 13

2.2.8 拮抗菌株生理生化特性分析 13

2.2.9 拮抗菌株16S rDNA序列分析 13

2.3 结果与分析 14

2.3.1 细菌菌株的分离纯化 14

2.3.2 拮抗菌株初筛 15

2.3.3 拮抗菌株复筛 15

2.3.4 拮抗菌株LM2303鉴定结果 17

2.4 结论与讨论 19

第三章 菌株LM2303诱变选育 21

3.1 材料 21

3.1.1 微生物菌株 21

3.1.2 试剂 21

3.1.3 培养基 21

3.1.4 仪器设备 22

3.2 方法 22

3.2.1 菌悬液制备 22

3.2.2 紫外线诱变 22

3.2.3 硫酸二乙酯(DES)诱变 22

3.2.4 微波诱变 23

3.2.5 正突变株筛选 23

3.2.6 突变株遗传稳定性 24

3.3 结果与分析 24

3.3.1 紫外线(UV)诱变 24

3.3.2 硫酸二乙酯(DES)诱变 25

3.3.3 微波诱变 26

3.3.4 突变菌株的遗传稳定性 27

3.4 结论与讨论 27

第四章 拮抗菌株发酵条件优化 29

4.1 材料 29

4.1.1 微生物菌株 29

4.1.2 试剂 29

4.1.3 仪器 30

4.1.4 培养基 30

4.2 方法 31

4.2.1 种子液制备 31

4.2.2 生物量测定 31

4.2.3 抑菌物质粗提物制备 31

4.2.4 基础发酵培养基筛选 31

4.2.5 菌株D59生长曲线测定 32

4.2.6 单因素试验 32

4.2.7 响应面优化 33

4.3 结果与分析 33

4.3.1 菌株D59生长曲线 33

4.3.2 基础发酵培养基筛选结果 33

4.3.3 单因素试验结果 34

4.3.4 响应面优化 36

4.4 结论与讨论 38

第五章 菌株LM2303抑菌作用研究 40

5.1 材料 40

5.1.1 微生物菌株 40

5.1.2 试剂 40

5.1.3 仪器 41

5.1.4 培养基 41

5.2 方法 42

5.2.1 菌悬液制备 42

5.2.2 菌株LM2303对有害微生物生长的影响 42

5.2.3 抑菌物质粗提物制备 42

5.2.4 抑菌物质抑菌谱测定 43

5.2.5 真菌孢子悬液制备 43

5.2.6 抑菌物质对真菌孢子萌发的影响 43

5.2.7 抑菌物质对真菌菌丝形态的影响 43

5.2.8 抑菌物质稳定性研究 44

5.2.9 抑菌物质分离纯化及定性研究 44

5.3 结果与分析 46

5.3.1 菌株LM2303对有害微生物的抑制作用 46

5.3.2 菌株LM2303抑菌物质抑菌谱 47

5.3.3 对真菌菌丝形态的影响 48

5.3.4 对真菌孢子萌发的抑制作用 49

5.3.5 抑菌物质的稳定性研究结果 50

5.3.6 抑菌物质分离纯化及鉴定 51

5.4 结论与讨论 54

第六章 生防菌株在冬枣贮藏中的应用 55

6.1 材料 55

6.1.1 菌株 55

6.1.2 培养基 55

6.1.3 试剂 56

6.1.4 仪器 56

6.2 方法 56

6.2.1 冬枣贮藏期致腐真菌的分离 56

6.2.2 回接验证试验 57

6.2.3 冬枣致腐真菌的种属鉴定 57

6.2.4 冬枣致腐真菌拮抗菌株的筛选 57

6.3 结果与分析 57

6.3.1 冬枣致腐真菌的分离鉴定 57

6.3.2 冬枣致腐真菌拮抗菌株的筛选结果 59

6.4 结论与讨论 60

第七章 结  论 61

参考文献 62

致  谢 69