薄膜改質提升核桃仁脫油餅粕萃取小分子蛋白液分離純化效能研究

Application of separating protein from walnut pressed cake by using PEGMA modified PVDF membrane

彭冠瑋(Kuan-Wei Peng)

指導教授 ： 鍾財王

中原大學/工學院/化學工程研究所/碩士(2020年)

https://doi.org/10.6840/cycu202000166

摘要

核桃是優質植物蛋白來源，核桃蛋白的價值與動物蛋白相近。目前，對核桃的研究主要在其油脂方面，對核桃蛋白的研究較少，因此，為了更好地開發利用核桃資源，對核桃蛋白功能性質的研究極為重要。
有許多核桃油生產廠家，大量核桃渣被廢棄或是低價值的利用。在分析中發現核桃渣中蛋白質達43.8%。為充分利用核桃資源並提高核桃產品附加價值和市場競爭力，對其進行進一步的過濾萃取出小分子蛋白更是重要。
在生化分離技術之中，如何有效的純化混合蛋白質溶液，為目前主要的研究方向。薄膜過濾在過濾系統中為簡單又可以大規模連續操作的分離方式。但利用薄膜過濾法純化蛋白質，常會發生蛋白質與薄膜表面之間的作用而導致過濾速度衰減，進而影響純化效率。因此，本研究藉由改變薄膜表面特性來改善超過濾薄膜的表面選擇性及延緩膜孔堵塞。
實驗初始階段，利用商業用聚偏二氟乙烯(polyvinylidene fluoride，PVDF)薄膜以及利用聚(乙二醇)甲醚甲基丙烯酸酯(Poly(ethylene glycol) methacrylate，PEGMA)在不同濃度下所配製的PVDF-PEGMA薄膜以及作為過濾核桃蛋白溶液的條件進行掃流過濾實驗，並檢測其過濾前後蛋白的表現特性。實驗結果顯示，改質完的薄膜都有改善原始薄膜的缺陷，除了在通量上能維持之外，透過SDS-PAGE檢測可以看到改質薄膜都能篩選至蛋白質顆粒大小為70kDa以下的範圍。其中，選擇使用PVDF-2.5wt%PEGMA配製的改質薄膜作為核桃蛋白超過濾之膜材。
本實驗以多種不同的參數下進行數據一次一因子分析，利用回應曲面法( Response Surface Methodology，RSM)，並找出最佳化之操作參數。本研究主要分為兩個部分進行數據分析，為萃取核桃仁蛋白之最佳參數以及掃流過濾最佳化參數探討。在萃取部分，以兩種因子進行10組數據分析(Central Composite Design)，分別是萃取時間(X1)以及固液比 (X2)，結果顯示在第7組實驗中可獲得最佳萃取數值，在X1為30分鐘與X2為1:10，核桃仁蛋白濃度(Y)為40.825(g/L) ;掃流過濾部分，選擇三種因子，分別為進料濃度(X1)，進料pH值(X2)，以及跨膜壓差(X3)，進行15組數據分析實驗(Box Benhken Design)。結果顯示，在第7組的實驗中，可以達到最高的過濾蛋白含量5.64(g/L)，而在第10組的實驗中，可以達到最高的穩態通量42.83 (kg/m2*hr)。而在進料濃度10 (g/L)，pH值8.5，跨膜壓為20.28psi， 可達到結果數據之最高期望值(desirability)，數值為蛋白濃度4.91 (g/L)，穩態通量為41.59 (kg/m2*hr)。

關鍵字

核桃仁蛋白 ； 回應曲面法 ； PVDF 薄膜 ； 超過濾

並列摘要

Walnut is a high quality albumen originate, the walnut protein value is similar to the animal protein. At present, research on walnut is mainly focused on its lipid aspect, there are less on the walnut protein research. In order to develop the use walnut resources, it is extremely important to study the walnut protein function nature.
Many walnut oil manufacturer has the massive walnut residue been wasted. In the analysis discovered that in the walnut residue contains 43.8% of the protein. To fully enhances the walnut product attachment value and the market competitive power using walnut resources, a further research on filtrating and extracting small molecular protein from walnut is essential.
In biochemistry separation technology, how to effectively purify mix protein solution is the main goal. Membrane filtration is a simple way and can be functioned in large-scale continuous-running system compare to other filtration technique. The use of membrane filtration can often occur issues because the interaction between protein and the membrane surface function which weakens the filtration rate gradually, which also influence the purification efficiency. Therefore, this study wants to improve the membrane superficial selectivity by changing the membrane characteristics.
In the first stage of experiment, the PVDF-PEGMA membrane with different concentration of PEGMA on the commercial PVDF membrane is being conducted. Membranes are tested on the cross-flow filtration system, and the results are examined to see the protein performance. The experiment results show that changing the modified PVDF membrane can improve the flaw from the pristine one, not only can it maintain the flux, the results from SDS-PAGE examination can also observe that the modified PVDF membrane are able to select the size of protein with 70kDa. Among them, the PVDF-2.5wt%PEGMA membrane is selected as the material for the walnut protein ultrafiltration.
This experiment uses lots of parameters to start the statistical analysis by using Response Surface Methodology, in order to achieve optimization. This research mainly divided into two sections for the data analysis, the parameters optimization for extracting walnut protein and cross-flow filtration.
In the extraction part, 10 groups of data with two factors is analyzed by using Central Composite Design, which is the extract time(X1) and liquid-solid ratio(X2), respectively. The results demonstrate that the 7th group obtain the best extraction value, the total walnut protein is 40.825(g/L);The filtration part, 15 group of data with three factors is analyzed by using Box Benhken Design, which is the feeding concentration , the pH value, as well as trans-membrane pressure (TMP), respectively. The results showed that, at the 7th group of experiment can achieve the highest filtrated protein content value with 5.64(g/L), and at the 10th group of experiment it can achieve the highest stable flux value 42.83 (kg/m2*hr).
With the parameter set with feed concentration 10 (g/L), pH values 8.5, trans-membrane pressure (TMP)20.28psi, the results can achieve highest desirability, which the value of protein outlet concentration 4.91 (mg/ml), the stable flux is 41.59 (kg/m2*hr).

並列關鍵字

Walnut protein ； RSM ； PVDF membrane ； Ultra-filtration

參考文獻

[1]姜莉、徐懷德、何玉君(2007, 04)。〈核桃渣中蛋白質的提取工藝及其功能性研究〉。食品科技, 頁255-258.

[2] Szetao KWC, Sathe SK (2000). “Walnut (Juglaus regia) proximate composition, protein solubility, protein amino acid composition and protein in vitro digestibility”. Sci Food Agric, 80 (1), Pages 393-401.

[3] María del Mar Contreras, Antonio Lama-Muñoz (2019). “Protein extraction from agri-food residues for integration in biorefinery: Potential techniques and current status”. Bioresource Technology, Volume 280, Pages 459-477.

[4] Ingmar H.Huisman (2000). “The effect of protein–protein and protein membrane interactions on membrane fouling in ultrafiltration”. Journal of Membrane Science ,Volume 179, Issues 1–2, Pages 79-90.

[5] Sang Hwan Lee , Eli Ruckenstein (1988). “Adsorption of proteins onto polymeric surfaces of different hydrophilicities—a case study with bovine serum albumin”. Journal of Colloid and Interface Science, Volume 125, Issue 2, Pages 365-379.