發(fā)布時間：2018-12-08 09:12

【摘要】：ε-聚賴氨酸(ε-poly-L-lysine,簡稱ε-PL)是一種綠色、安全的生物防腐劑。2014年,我國正式批準ε-PL及其鹽酸鹽為新的食品添加劑品種。經(jīng)過多年的菌種選育和發(fā)酵策略優(yōu)化,發(fā)酵產(chǎn)量已達到工業(yè)生產(chǎn)要求,然而,國內(nèi)關于ε-PL分離提取方面的研究尚不完善,嚴重制約了ε-PL產(chǎn)業(yè)化。因此,研發(fā)能夠用于工業(yè)生產(chǎn)的ε-PL分離提取技術對促進ε-PL產(chǎn)業(yè)化具有十分重要的現(xiàn)實意義。在前期研究工作的基礎上,本研究對離子交換的動態(tài)條件進行系統(tǒng)優(yōu)化,建立了以離子交換技術為核心的提取工藝,并對工藝的穩(wěn)定性進行了考察,最后成功的將該提取工藝應用到試生產(chǎn)。取得的研究成果如下:(1)基于對鈉型和銨型Amberlite IRC-50樹脂動態(tài)條件的優(yōu)化和比較研究,確定鈉型IRC-50樹脂更適合工業(yè)化應用。鈉型樹脂最適動態(tài)條件為:上樣pH為7.0,上樣速度為5 BV·h-1,洗脫劑為0.25 mol·L-1 NaOH,洗脫速度為2 BV·h-1。在最適操作條件下,鈉型IRC-50樹脂解吸率達到95%以上,工作交換容量為303.25 mg·g-1,ε-PL純度從初始的24.60%提升至75.86%,蛋白和色素去除率均在60%以上。(2)針對離子交換洗脫液中存在的雜質(zhì),探索了利用離子交換手段去除該雜質(zhì)的可能。通過分析雜質(zhì)的來源和性質(zhì),初步判斷該雜質(zhì)來源于發(fā)酵,帶電性質(zhì)與ε-PL近似,分子量在900-2000 Da之間。采用鈉型IRC-50樹脂梯度洗脫、負吸附結合正吸附和分部收集、強酸樹脂等方法,只能部分去除雜質(zhì)。由此可知,雜質(zhì)等電點和ε-PL十分接近,依靠離子交換方法難以實現(xiàn)其有效去除。(3)研究了鈉型IRC-50樹脂在不同放大規(guī)模下,對ε-PL分離提取的影響。在十個批次的提取工藝中樹脂工作交換容量沒有明顯下降、解吸率均在95%以上、洗脫液純度在70%以上,工藝相對穩(wěn)定。對離子交換單元進行8倍和27倍實驗室規(guī)模的放大,放大后離子交換分離性能良好。在離子交換柱總容為5.5 m3的試生產(chǎn)提取工藝中,離子交換雜質(zhì)去除情況與實驗室的結果一致,ε-PL損失率僅為2.62%。試生產(chǎn)所提取樣品外觀為接近白色的粉末,液相檢測純度為99.21%,對提取樣品的理化指標進行測定和分析,結果均符合國家標準要求。質(zhì)譜分析結果表明提取樣品為L-賴氨酸聚合物,聚合度在27-34。
[Abstract]:蔚-poly-L-lysine, (蔚-PL) is a green and safe biological preservative. In 2014, 蔚-PL and its hydrochloric acid were approved as a new food additive in China. After years of strain selection and fermentation strategy optimization, the fermentation yield has reached the requirements of industrial production. However, the domestic research on the isolation and extraction of 蔚-PL is not perfect, which seriously restricts the industrialization of 蔚-PL. Therefore, the development of 蔚-PL separation and extraction technology which can be used in industrial production is of great practical significance to promote the industrialization of 蔚-PL. Based on the previous research work, the dynamic conditions of ion exchange were systematically optimized, and the extraction process with ion exchange technology as the core was established, and the stability of the process was investigated. Finally, the extraction process was successfully applied to trial production. The results obtained are as follows: (1) based on the optimization and comparison of dynamic conditions of sodium and ammonium Amberlite IRC-50 resins, it is determined that sodium IRC-50 resin is more suitable for industrial application. The optimum dynamic conditions of sodium resin were as follows: the sample pH was 7.0, the sample rate was 5 BV h-1, and the eluent was 0.25 mol L-1 NaOH,. The elution rate was 2 BV h-1. Under the optimum operating conditions, the desorption rate of sodium IRC-50 resin was over 95%, the working exchange capacity was 303.25 mg g-1, and the purity of 蔚-PL was raised from 24.60% to 75.86%. The removal rate of both protein and pigment is over 60%. (2) the possibility of removing the impurity in the ion exchange eluate by ion exchange is explored. By analyzing the source and properties of impurity, it was preliminarily determined that the impurity originated from fermentation. The charged property was similar to 蔚-PL, and its molecular weight was between 900-2000 Da. Sodium IRC-50 resin gradient elution, negative adsorption combined with positive adsorption and partial collection, strong acid resin and other methods, can only partially remove impurities. It can be seen that impurity isoelectric point is very close to 蔚-PL, and it is difficult to achieve its effective removal by ion exchange method. (3) the influence of sodium IRC-50 resin on the separation and extraction of 蔚-PL was studied in different scale. In the extraction process of 10 batches, the working exchange capacity of the resin was not obviously decreased, the desorption rate was above 95%, the purity of the eluent was over 70%, and the process was relatively stable. The ion exchange unit was amplified by 8 and 27 times of laboratory scale, and the ion exchange separation performance was good after amplification. The results of ion exchange impurity removal are in agreement with the results of the laboratory, and the loss rate of 蔚-PL is only 2.62% in the trial process of ion exchange column with a total capacity of 5.5 m ~ 3. The appearance of the extracted samples was close to white powder, the purity of liquid phase detection was 99.21. The physicochemical indexes of the extracted samples were determined and analyzed, and the results met the requirements of the national standard. The result of mass spectrometry showed that the extracted sample was L- lysine polymer with polymerization degree of 27-34.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：TS202.3;TQ028.33

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