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甲壳素的微生物法制备及发酵液的利用研究
Alternative TitleMicrobiological preparation of chitin and utilization of fermentation supernatants
周忆
Subtype硕士
Thesis Advisor邢荣娥
2024-05-19
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研 究所
Degree Name理学硕士
Keyword甲壳素,微生物法,虾壳,蟹壳,发酵液活性成分
Abstract

甲壳素经过脱乙酰、降解等处理可得到壳聚糖及壳寡糖,具有抑菌、抗氧化、可生物降解及生物相容等多种生物活性,目前已广泛应用于食品、农业、生物医药等领域。作为壳聚糖等衍生物的源头材料,甲壳素的获取具有巨大的研究意义与价值。海洋甲壳类生物的外壳中富含甲壳素,以虾蟹壳为主的加工副产物多以废弃物的形式被倾倒或填埋,存在严重的环境污染与资源浪费。目前甲壳素的产业化提取方法为强酸强碱法,生产过程中不仅会产生大量废水,所得甲壳素也存在结构降解及均一性差等问题,因此,如何以绿色环保的方法高效获取甲壳素具有重要的研究意义。微生物法作为一种绿色提取方式,已经被证明可去除虾蟹壳等来源中的碳酸钙及蛋白质,获得结构破坏较小的甲壳素,同时得到富含多种活性物质的发酵液,可二次加工利用。目前大多数研究局限于从虾壳中提取甲壳素,因碳酸钙或蛋白质的脱除效率较低一直未实现规模化应用,且从蟹壳中提取甲壳素的相关研究较少,有关于微生物法的提取效果及产物性质的研究较为匮乏。

本研究采用微生物法,利用筛选得到的脱钙菌株植物乳杆菌(Lactiplantibacillus plantarum和脱蛋白菌株铜绿假单胞菌(Pseudomonas aeruginosa),分别从虾壳及蟹壳中制备得到了纯度较高的甲壳素,并进一步探究了发酵液的活性成分及性质,以期为甲壳素的绿色制备提供参考。主要结果如下:

1.分别以虾(Litopenaeus vannamei)壳和蟹(Chionoecetes opilio)壳作为发酵底物,优化筛选得到的植物乳杆菌(L. plantarum, LA01)及铜绿假单胞菌(P. aeruginosa, PS01)的发酵条件,采用先接种PS01再接种LA01的发酵顺序可获得纯度较高的甲壳素,过程中仅在LA01发酵时添加葡萄糖,而未添加酵母粉等其他额外营养物质。发酵结果显示,虾壳甲壳素的脱钙率及脱蛋白率分别为96.92±0.07%78.46±1.29%,蟹壳甲壳素的脱钙率和脱蛋白率分别为97.55±0.15%73.49±0.25%。傅里叶变换红外光谱、X-射线衍射、扫描电镜及热重分析结果表明,采用微生物法所得的甲壳素表现出与商用甲壳素相似的结构及性质,说明所获得的微生物在发酵过程中的底物适用性较好。

2.对发酵液的成分及活性进行分析,结果表明:发酵液含有以乳酸为主的有机酸、氨基酸、总酚等多种活性物质,LA01发酵液中含有胞外多糖,其抗氧化活性显著高于PS01发酵液,并表现出普遍高于目前研究的抗氧化活性,说明该菌株发酵液具有较高的应用潜力。PS01发酵液的抑菌效果优于LA01发酵液,具有明显的抑制金黄色葡萄球菌(革兰氏阳性菌)活性,在发酵过程中可抑制杂菌的生长,维护发酵环境。

Other Abstract

Chitin can be deacetylated and degraded to obtain chitosan and chito-oligosaccharides, which have a variety of activities such as antimicrobial, antioxidant, biodegradable and biocompatible, and have been widely used in food, agriculture, biomedicine and other fields in the form of additives, tissue scaffolds, films and others. As the source of chitosan, the extraction of chitin is of great research importance and value. Marine crustaceans are rich in chitin, and most of the processing by-products, mainly shrimp and crab shells, are dumped or landfilled as waste, resulting in serious environmental pollution and waste of resources. At present, the main industrial method of extracting chitin from natural sources is the chemical method, which not only produces a large amount of waste water, but also has the problems of structural degradation and poor homogeneity of chitin, so how to efficiently obtain chitin in an environmental-friendly way is quite valuable. The microbiological method, as a green extraction method, has been shown to be efficient in removing calcium carbonate and protein from sources such as shrimp and crab shells, obtaining chitin with less structural damage, and at the same time obtaining fermentation supernatants rich in various active ingredients, which can be processed for secondary purposes. Shrimp shells are currently the main material being researched, but have not been realized on a large scale due to low demineralization or deproteinization efficiencies. Furthermore, there are fewer studies on the extraction of chitin from crab shells, also the effect of microbiological extraction methods and the properties of the fermentation supernatants.

In this research, chitin of high purity was prepared from shrimp shells and crab shells by microbiological method using Lactiplantibacillus plantarum for demineralization and Pseudomonas aeruginosa for deproteinization, and the active components and properties of the fermentation supernatants were further investigated, with a view to providing theoretical reference for research about extracting chitin by green preparation technology. The main results are as follows:

1. Shrimp (Litopenaeus vannamei) shells and crab (Chionoecetes opilio) shells were used as fermentation substrates, respectively. A strain with high acid-producing activity (L. plantarum, LA01) and the other strain with high protease-producing activity (P. aeruginosa, PS01) were obtained by screening from different sources, and optimized fermentation conditions were used to obtain the best impurity removal effect in two-step fermentation. Inoculation of PS01 followed by LA01 was chosen as the scheme for better purity of products. Optimal impurities removal could be obtained by adding glucose only to the LA01 fermentation process, without adding other additional nutrients such as yeast powder. Results showed that the demineralization (DM) and deproteinization (DP) rates were 96.92±0.07% and 78.46±1.29% for chitin from shrimp shells, while 97.55±0.15% of DM rates and 73.49±0.25% of DP rates were found in the products from crab shells. The results of fourier infrared spectroscopy, X-ray diffraction, scanning electron microscopy and thermogravimetric analyzes showed that the chitin obtained by the microbiological method had similar structure and properties to those of commercial chitin, which indicated that the obtained bacteria were well suited for different substrates..

2. The active components and activities of the fermentation supernatants were analyzed. Results showed that fermentation supernatants by LA01 and PS01 were rich in lactic acid-based organic acids, essential amino acids, total phenols, where the LA01 fermentation supernatant contained exopolysaccharide, performing higher antioxidant activity than PS01 and which had been reported, indicating the potential applications. Better antibacterial activity was found in the fermentation supernatant of PS01 than LA01, which showed obvious suppression on Staphylococcus aureus (Gram-positive bacteria), which could inhibit the growth of other irrelevant bacteria during the fermentation process and maintain the fermentation environment.

Language中文
Table of Contents

1绪论 1

1.1 背景 1

1.2 甲壳素研究现状 1

1.2.1 甲壳素及其衍生物概述及应用 1

1.2.2 甲壳素主要来源 4

1.2.3 甲壳素提取方法 6

1.3 发酵液成分及活性研究进展 11

1.4 选题意义与研究内容 12

2铜绿假单胞菌与植物乳杆菌发酵制备虾、蟹壳甲壳素 14

2.1 实验材料与仪器 14

2.1.1 主要试剂 14

2.1.2 主要实验仪器 15

2.1.3 虾蟹壳原料 15

2.2 实验方法 15

2.2.1 培养基配制 15

2.2.2 产酸菌株及产蛋白酶菌株的筛选 16

2.2.3 生长曲线测定 17

2.2.4 化学指标测定 17

2.2.5 脱钙率及脱蛋白率计算 18

2.2.6 发酵条件单因素优化 18

2.2.7 两步发酵法提取甲壳素 19

2.2.8 甲壳素结构分析 19

2.3 数据分析 20

2.4 实验结果 20

2.4.1 产酸菌株及产蛋白酶菌株的筛选结果 20

2.4.2 生长曲线测定结果 22

2.4.3 化学指标测定结果 22

2.4.4 发酵条件单因素优化结果 24

2.4.5 两步发酵法结果 28

2.4.6 甲壳素结构分析 31

2.4.7 讨论 37

本章小结 38

3微生物法所得发酵液主要成分及抑菌、抗氧化活性研究 40

3.1 实验材料与仪器 40

3.1.1 主要试剂 40

3.1.2 主要实验仪器 41

3.1.3 实验材料 41

3.2 实验方法 41

3.2.1 发酵液活性物质测定 41

3.2.2 发酵液活性检测 42

3.3 实验结果 43

3.3.1 发酵液活性物质测定结果 43

3.3.2 发酵液抑菌和抗氧化活性测定结果 45

3.4 讨论 48

本章小结 50

结论 51

创新点 52

参考文献 53

致谢 67

作者简历及攻读学位期间发表的学术论文与其他相关学术成果 68

 

 

Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/185229
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
周忆. 甲壳素的微生物法制备及发酵液的利用研究[D]. 中国科学院海洋研 究所. 中国科学院大学,2024.
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