摘  要

微生物導(dǎo)致食品的腐敗變質(zhì)不僅會造成巨大經(jīng)濟(jì)損失，還會引起嚴(yán)重的食品安全問題。而現(xiàn)有的食品防腐保鮮方法在實(shí)際生產(chǎn)應(yīng)用中具有一定的局限性，迫切需要尋求更安全有效的防腐保鮮新技術(shù)，將微生物及其代謝產(chǎn)物應(yīng)用于食品防腐保鮮的生物防治方法是現(xiàn)階段研究的熱點(diǎn)。

從輪馬熱泉、北海溫泉、大塘熱泉等騰沖溫泉附近采集土壤樣品23種，采用稀釋分離法分離純化得到細(xì)菌菌株142株。以大腸桿菌、黑曲霉、禾谷鐮刀菌等為指示菌，采用濾紙片法或平板對峙法從142株細(xì)菌菌株中初步篩選得到具有廣譜抑菌效果的菌株7株，其中菌株 LM2303廣譜抑菌效果最好。通過形態(tài)特征分析、生理生化特性分析和16S rDNA序列比對，菌株LM2303應(yīng)為枯草芽孢桿菌(Bacillus subtilis)。

采用紫外線誘變、硫酸二乙酯誘變和微波誘變對菌株LM2303進(jìn)行選育。其中，經(jīng)硫酸二乙酯誘變處理得到突變菌株D59，其抑菌效果較原始菌株提高了11.4%。以抑菌物質(zhì)產(chǎn)量為指標(biāo)，從8種基礎(chǔ)培養(yǎng)基中篩選出菌株D59最佳發(fā)酵培養(yǎng)基M2。采用單因素實(shí)驗(yàn)和響應(yīng)面分析對菌株D59進(jìn)行發(fā)酵條件優(yōu)化。單因素試驗(yàn)結(jié)果表明培養(yǎng)時(shí)間、溫度和轉(zhuǎn)速對菌株D59抑菌物質(zhì)產(chǎn)量有顯著影響(p<0.05)。利用響應(yīng)面確定菌株D59的最佳培養(yǎng)條件為：接種量5%、裝液量100mL/250mL、培養(yǎng)時(shí)間43h、溫度33℃、轉(zhuǎn)速177rpm。在上述發(fā)酵條件下培養(yǎng)，100mL發(fā)酵液可得到抑菌物質(zhì)粗提物2.8560 g(濕重)，是原發(fā)酵條件下的1.4倍，有效提高了抑菌物質(zhì)產(chǎn)量。

采用鹽酸沉淀、甲醇萃取，從拮抗菌株發(fā)酵液中得到抑菌物質(zhì)粗提物。利用薄層色譜對抑菌物質(zhì)進(jìn)行分析和初步純化，紫外及茚三酮顯色結(jié)果表明抑菌物質(zhì)含有苯環(huán)和閉合肽鍵。紅外光譜掃描結(jié)果表明菌株LM2303抑菌物質(zhì)為環(huán)狀脂肽類物質(zhì)。該脂肽類物質(zhì)理化性質(zhì)穩(wěn)定，抑菌譜廣，對大腸桿菌、金黃色葡萄球菌等細(xì)菌和黑曲霉、黃曲霉、擴(kuò)展青霉、禾谷鐮刀菌等真菌均有較強(qiáng)的抑制作用。

從不同腐爛病癥的冬棗上分離得到根菌索菌、細(xì)極鏈格孢、橄欖色盾殼霉、擴(kuò)展青霉四種致腐真菌。平板對峙試驗(yàn)結(jié)果表明菌株LM2303可有效抑制上述四種真菌的生長，菌株LM2303具有冬棗貯藏期致腐真菌的生物防控應(yīng)用潛力。

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關(guān)鍵詞：枯草芽孢桿菌；拮抗；抑菌物質(zhì)；誘變選育；發(fā)酵條件優(yōu)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.

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

目  錄

第一章 前言 1

1.1 研究背景 1

1.2 生防微生物概況 2

1.2.1 用于生物防治的微生物種類 2

1.2.2 生防微生物的作用機(jī)制 2

1.3 生防芽孢桿菌 4

1.4 芽孢桿菌抑菌物質(zhì)研究概況 4

1.5 本研究的目的和意義 6

第二章 廣譜拮抗菌株的篩選及其鑒定 8

2.1 材料 8

2.1.1 樣品 8

2.1.2 試劑 9

2.1.3 儀器 10

2.1.4 培養(yǎng)基 10

2.1.5 供試有害微生物 11

2.2 方法 11

2.2.1 微生物培養(yǎng) 11

2.2.2 細(xì)菌菌株分離純化 11

2.2.3 菌懸液制備 11

2.2.4 拮抗菌株初篩 12

2.2.5 拮抗菌株無細(xì)胞培養(yǎng)濾液制備 12

2.2.6 拮抗菌株的復(fù)篩 12

2.2.7 拮抗菌株形態(tài)特征觀察 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 結(jié)果與分析 14

2.3.1 細(xì)菌菌株的分離純化 14

2.3.2 拮抗菌株初篩 15

2.3.3 拮抗菌株復(fù)篩 15

2.3.4 拮抗菌株LM2303鑒定結(jié)果 17

2.4 結(jié)論與討論 19

第三章 菌株LM2303誘變選育 21

3.1 材料 21

3.1.1 微生物菌株 21

3.1.2 試劑 21

3.1.3 培養(yǎng)基 21

3.1.4 儀器設(shè)備 22

3.2 方法 22

3.2.1 菌懸液制備 22

3.2.2 紫外線誘變 22

3.2.3 硫酸二乙酯(DES)誘變 22

3.2.4 微波誘變 23

3.2.5 正突變株篩選 23

3.2.6 突變株遺傳穩(wěn)定性 24

3.3 結(jié)果與分析 24

3.3.1 紫外線(UV)誘變 24

3.3.2 硫酸二乙酯(DES)誘變 25

3.3.3 微波誘變 26

3.3.4 突變菌株的遺傳穩(wěn)定性 27

3.4 結(jié)論與討論 27

第四章 拮抗菌株發(fā)酵條件優(yōu)化 29

4.1 材料 29

4.1.1 微生物菌株 29

4.1.2 試劑 29

4.1.3 儀器 30

4.1.4 培養(yǎng)基 30

4.2 方法 31

4.2.1 種子液制備 31

4.2.2 生物量測定 31

4.2.3 抑菌物質(zhì)粗提物制備 31

4.2.4 基礎(chǔ)發(fā)酵培養(yǎng)基篩選 31

4.2.5 菌株D59生長曲線測定 32

4.2.6 單因素試驗(yàn) 32

4.2.7 響應(yīng)面優(yōu)化 33

4.3 結(jié)果與分析 33

4.3.1 菌株D59生長曲線 33

4.3.2 基礎(chǔ)發(fā)酵培養(yǎng)基篩選結(jié)果 33

4.3.3 單因素試驗(yàn)結(jié)果 34

4.3.4 響應(yīng)面優(yōu)化 36

4.4 結(jié)論與討論 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 試劑 40

5.1.3 儀器 41

5.1.4 培養(yǎng)基 41

5.2 方法 42

5.2.1 菌懸液制備 42

5.2.2 菌株LM2303對有害微生物生長的影響 42

5.2.3 抑菌物質(zhì)粗提物制備 42

5.2.4 抑菌物質(zhì)抑菌譜測定 43

5.2.5 真菌孢子懸液制備 43

5.2.6 抑菌物質(zhì)對真菌孢子萌發(fā)的影響 43

5.2.7 抑菌物質(zhì)對真菌菌絲形態(tài)的影響 43

5.2.8 抑菌物質(zhì)穩(wěn)定性研究 44

5.2.9 抑菌物質(zhì)分離純化及定性研究 44

5.3 結(jié)果與分析 46

5.3.1 菌株LM2303對有害微生物的抑制作用 46

5.3.2 菌株LM2303抑菌物質(zhì)抑菌譜 47

5.3.3 對真菌菌絲形態(tài)的影響 48

5.3.4 對真菌孢子萌發(fā)的抑制作用 49

5.3.5 抑菌物質(zhì)的穩(wěn)定性研究結(jié)果 50

5.3.6 抑菌物質(zhì)分離純化及鑒定 51

5.4 結(jié)論與討論 54

第六章 生防菌株在冬棗貯藏中的應(yīng)用 55

6.1 材料 55

6.1.1 菌株 55

6.1.2 培養(yǎng)基 55

6.1.3 試劑 56

6.1.4 儀器 56

6.2 方法 56

6.2.1 冬棗貯藏期致腐真菌的分離 56

6.2.2 回接驗(yàn)證試驗(yàn) 57

6.2.3 冬棗致腐真菌的種屬鑒定 57

6.2.4 冬棗致腐真菌拮抗菌株的篩選 57

6.3 結(jié)果與分析 57

6.3.1 冬棗致腐真菌的分離鑒定 57

6.3.2 冬棗致腐真菌拮抗菌株的篩選結(jié)果 59

6.4 結(jié)論與討論 60

第七章 結(jié)  論 61

參考文獻(xiàn) 62

致  謝 69