实验海洋生物学重点实验室知识库

甲壳素（Chitin ）广泛存在于甲壳类、昆虫的外壳及真菌的细胞壁中，是自

然界中数量仅次于纤维素、含氮量仅次于蛋白质的的碱性生物多糖。由于其自身

具备的生物可降解性、生物相容性、吸湿性、无毒副作用等优点，被广泛应用到

食品、环保、农业、纺织、化工等领域。而其降解产物壳寡糖（ Chitooligosaccharides

兼备甲壳素优点的同时，具有更好的溶解性和可吸收性，在某些领域表现出更强

的生物活性，因而备受关注。然而，目前甲壳素的生产方式还是传统的酸碱法，

制备过程中需要消耗大量的酸和碱，且蛋白质、钙、磷、虾青素等资源均未利用，

环境污染严重、资源浪费大。因此，甲壳素 壳寡糖的绿色清洁制备技术是甲壳

素行业发展的必然趋势。壳寡糖自从 2009 年被批准为饲料添加剂后，在水产和

畜牧养殖方面进行了较多的研究，但由于 其 分子量、乙酰度等参数的影响，所得

活性结果差异较大，真正应用于养殖的壳寡糖产品很少。 因此本论文针对甲壳素

行业加工转化技 术存在高污染、高耗能等现状，采用微生物发酵法替代传统化学

法生产甲壳素，对制备方法进行了深入研究，同时研究了壳寡糖对凡纳滨对虾生

长、免疫性能、肠道菌群等的影响。结果表明：

1.以虾壳为原料，通过发酵条件的优化以及获得的甲壳素产物的结构表征，制备了高纯度的甲壳素。在最适条件下，产酸菌株体系中 pH 低于 3.4 ，对虾壳的脱钙率达到了 97.5%97.5%；产蛋白酶菌株所产蛋白酶酶活为 701.3 U/mL ，对虾壳的脱蛋白率达到了 96.8%96.8%；终产物甲壳素的灰分含量为 1.2%1.2%，残余蛋白质含量为1.5% 。本方法得到的脱钙率、脱蛋白率等 关键指标明显优于此前的研究，且不需要酸碱进一步处理。

2. 生物发酵制备甲壳素方法中，蛋白酶的分离纯化一直是研究的热点与难点，本实验成功的从脱蛋白发酵液内分离纯化得到目的蛋白酶，并对此蛋白酶进行了质谱分析鉴定，结果显示该蛋白酶分子量在19 kDa左右，为解淀粉芽孢杆菌中的D2M30_3251蛋白酶，酶活性为821.32 U/mL。

3.对发酵液内活性物质及其抗氧化活性分析发现，发酵液内富含 酚类 、糖类等 多种活性物质，且具有一定的抗氧化活性，可进一步开发成功能性食品、饲甲壳素/壳寡糖的生物法制备及其对凡纳滨对虾的促生长作用研究料添加剂等产品。

4. 与对照组相比，特定分子量和脱乙酰度的壳寡糖处理组显著提高了凡纳与对照组相比，特定分子量和脱乙酰度的壳寡糖处理组显著提高了凡纳滨对虾的生长指标、促进了肠道内有益菌的增殖，抑制了有害菌的增殖、上调了滨对虾的生长指标、促进了肠道内有益菌的增殖，抑制了有害菌的增殖、上调了相关抗氧化基因的表达量并且提高了抗氧化酶的活性和胰蛋白酶水平，同时，有相关抗氧化基因的表达量并且提高了抗氧化酶的活性和胰蛋白酶水平，同时，有更好的保护肝胰腺的正常形态的能力，说明壳寡糖可以直接促进凡纳滨对虾的生更好的保护肝胰腺的正常形态的能力，说明壳寡糖可以直接促进凡纳滨对虾的生长和抗病能力，这对壳寡糖在对虾饲料中的开发应用具有指导作用。长和抗病能力，这对壳寡糖在对虾饲料中的开发应用具有指导作用。

Chitin is widely found in crustaceans, insect shells and cell walls of fungi. It is an alkaline biological polysaccharide in nature, second only to cellulose in quantity and second only to protein in nitrogen content. Due to its advantages of biodegradability, biocompatibility, moisture absorption ability and non-toxic side effects, it is widely used in food, environmental protection, agriculture, textile, chemical and other fields. Chitooligosaccharides, the degradation product of chitin, which combines the advantages of chitin, has better solubility and absorbability, and shows stronger biological activity in some fields, which has attracted much attention. However, the current chitin production method is still the traditional acid-base method, which requires a large amount of acids and bases in the preparation process, and the resources such as protein, calcium, phosphorus and astaxanthin are not utilized, resulting in serious environmental pollution and large waste of resources. Therefore, the green and clean preparation technology of chitin/ chitooligosaccharides is an inevitable trend of the development of chitin industry. Since chitooligosaccharides was approved as a feed additive in 2009, more studies have been carried out on aquatic and animal husbandry. However, due to the influence of its molecular weight, acetyl degree and other parameters, the results of the activity obtained are quite different, and few chitosan oligosaccharide products are really applied to aquaculture. Therefore, in this paper, in view of the high pollution and high energy consumption of chitin processing and transformation technology in chitin industry, microbial fermentation method was used to replace the traditional chemical method to produce chitin, and the preparation method was studied in depth. Effect of chitooligosaccharides with different molecular weight and deacetylation degree on growth, immune performance and intestinal flora of Pacific white shrimp (Litopenaeus vannamei) in feed. The results show that:

1. High purity chitin was prepared from shrimp shell by optimizing the fermentation conditions and the structure characterization of chitin products obtained. Under the optimum conditions, the pH of the acid-producing strain system was lower

than 3.4, and the decalcification rate of prawn shell reached 97.5%. The protease activity produced by the protease producing strain was 701.3 U/mL, and the deproteinization rate of prawn shell reached 96.8%. The ash content of the final product chitin was 1.2% and the residual protein content was 1.5%. Compared with the studies on the decalcification and deproteinization of chitin prepared by many fermentation methods, which require further treatment by acid and base, this study overcomes the above shortcomings.In addition, the two strains havelow nutrient requirements, which made it possible to prepare chitin by fermentation on a large scale.

2. Biological fermentation in the preparation of chitin, the separation and purification of protease, has been the research hotspot and difficulty in the fermented liquid from the success of the experiment that the protein purification by protease, and the mass spectrometry identification of the protease, the results showed that the protease at about 19 kDa, molecular weight of starch solution D2M30_3251 of bacillus protease, the enzyme activity of 821.32 U/mL, in numerous studies at a higher level, has the complete removal of protein in shrimp shell of potential applications, provide theoretical support for this protease efficientutilization.

3. Through the analysis of the active substances and their antioxidant activities in the fermentation broth, it is found that the fermentation broth is rich in a variety of active substances and has certain antioxidant activities, which can be further developed into functional food and feed additives.

4.Compared with control group, certain degree and molecular weight of chitooligosaccharides group was significantly improved, and achieved the growth index, promote the beneficial bacteria in the intestines of proliferation, inhibits the proliferation of harmful bacteria, raised the relevant amount of antioxidant gene expression and improve the level of the activity of antioxidant enzymes and trypsin, at the same time, have a better ability to protect liver pancreas normal form, explain chitooligosaccharides can directly promote the growth of Pacific white shrimp (Litopenaeus vannamei) and disease-resistant ability, it is helpful for the development and utilization of chitooligosaccharides in the feed of Pacific white shrimp (Litopenaeus vannamei).