实验海洋生物学重点实验室知识库

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.

第1章 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

第2章 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

第3章 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