摘要
微生物胞外多糖-结冷胶具有凝胶效率高、透明度好、稳定性强等优点,在不同领域有着广泛应用。结冷胶发酵液粘度较高,胶体围绕细胞以粘性聚合物形式构成网状结构,对结冷胶的提取带来了不小的困难。目前研究主要集中在发酵方面,提取工艺报道较少,为此本论文对结冷胶提取工艺进行了初步探索,并得到了低酰基结冷胶的提取工艺路线。
菌体的去除：比较了离心除菌、絮凝过滤除菌以及微滤除菌方式的优劣。结果表明：(1)离心除菌需要将发酵液大量稀释,工业生产成本较高；(2)微孔过滤除菌效率最高,但初始料液菌体含量过大时,微滤膜表面菌体层较厚,膜通量较小。(3)采用CaCl2作为絮凝剂,经板框过滤可以去除94%左右的菌体。最后确定采用先絮凝过滤再微滤的除菌方式。
色素的去除：首先考察了粉末活性炭、双氧水的脱色效果。结果表明：酸性或中性条件下粉末活性炭脱色效果最好,但结冷胶回收率很低；双氧水在高温碱性条件下脱色效果较强,但pH大于8时会使结冷胶分子降解。考虑到色素主要是发酵液中残留葡萄糖在脱酰基过程中发生一系列反应所产生,最终确定先用3%HCl将结冷胶与菌体一同沉淀,然后用乙醇洗涤沉淀以去除其中的色素以及葡萄糖的方式来粗步脱色,然后用双氧水彻底脱色。
蛋白质的去除：考察了等电点除蛋白以及酶法除蛋白。结果表明将料液pH调至4过滤后可以去除大部分蛋白质,但是结冷胶在pH4时也要沉淀,造成其回收率很低；酶法除蛋白效果较好,添加0.3%的碱性蛋白酶,在pH10,55℃下保温4小时蛋白质去除率达90%以上。
钙离子的去除：分别采用一价离子取代法(KCl)、化学沉淀法(Na2C03、多聚磷酸钠、草酸钠)、螯合法(EDTA)去除钙离子。结果表明使用Na2C03去除钙离子效果最好,干燥后的产品在蒸馏水中稍微加热即可溶解。
提取工艺总结：1、向发酵液中加入等体积的3%HCl,过滤后将滤饼压榨脱水,然后加入1.5倍滤饼体积的工业酒精并调pH至7,浸泡40min,过滤除去酒精。2、将脱色后的滤饼按发酵液初始体积的2-3倍重新溶解,调pH为10,80℃下保温25min脱酰基。加入0.05%CaCl2(0.0045mol/L)絮凝菌体后用板框过滤去除菌体,过滤温度为80℃,压力0.25MPa,过滤介质为200目工业滤布,助滤剂为硅藻土,添加量为3%,平均分配到滤布和料液中。3、向滤液中加入0.0045mol/L的Na2C03,再用平板微滤除去CaCO3沉淀以及剩余菌体,过滤压力为0.7MPa,过滤介质为0.45μm的聚醚砜滤膜,过滤温度90℃。4、向滤液中加入0.3%(w/v)的碱性蛋白酶,调pH10,55℃下保温4小时除蛋白。5、向除蛋白后的料液中加入0.5%(w/v)双氧水,pH8、75℃下保温15min彻底脱色。6、加入与料液等体积的3%HCl沉淀结冷胶,过滤后得滤饼,然后加入1.5倍滤饼体积的工业酒精并调pH至7,过滤后回收酒精用于第一步洗涤,滤饼于50℃下干燥得白色产品。产品重均分子量为113万,凝胶强度为890g/cm2,透光率为79.9%,结冷胶总回收率为71%。
Extracellular polysaccharide gellan gum is one of the industrially useful microbial exopolysaccharides due to its stability and high gelling efficiency. Because of its high viscosity, the extraction work is very difficult. Current researches are mainly focusing on its fermentation rather than extraction. In laboratory, fermentation broth is largely diluted to reduce its viscosity, and remove the cells by centrifugation, the method is obviously uneconomical for industrial production. In this paper an extraction process for gellan gum was developed including cell removing, decolorization, deproteinization and Ca2+ removing. The total process was as follow:
The first step decolorization:add 3% HCl (v/v) solution into the broth to precipitate crude gellan gum (gellan gum mixed mainly with cells). The volume of 3%HCl solution is equal to the initial fermentation broth.Then filtrate the precipitate with Plate and Frame Filter(PFF) to get the filter cake(crude gellan gum), and squeeze the cake to remove HCl solution as much as possible. Then the cake was immersed in industrial alcohol of 1.5 cake volumes, and adjusts pH to 7, after 40min, remove alcohol and main pigment by filtration.
The second step cell removing:dissolve the crude gellan gum in water (the ratio of water volume to initial broth was about 2:1).Then adjust pH to 10,and keep the solution at 80℃for 25min to make gellan deacetylated. After that the cells were flocculated by adding 0.0045mol/L CaCl2 into the solution (pH10,80℃)and removed by filtration with PFF using industrial filter cloth of 200 mesh along with diatomite as filter aid at pressure of 0.25MPa.
The third step Ca2+ removing and further removing of cells:add 0.0045mol/LNa2CO3 into permeation liquid, filtrated the liquid by PES membrane of 0.45μm pore at 90℃,0.7MPa using PFF.
The fourth step deproteinization and further decolorization:after cell and Ca2+ removing,0.3% alkaline protease (w/v) was added and the solution was kept at 55℃for 4h to remove protein.after that add 0.5% H2O2 and keep the solution at 75℃,pH8 forl5 min to remove pigment absolutely.
The last step precipitate and drying:add equal volume of 3%HC1 into the solution, then get the precipitate by filtration,and wash it with alcohol and adjust the pH to7, then remove the alcohol by filtration again.At last drying the refined gellan gum at 50℃. The molecular weight, gel strength, transparency, and total recovery of deacetylated gellan gum was 1130000,890g/cm2,79.9%,71% respectively.
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