Development of simple separation method of lactopholin from cheese whey and their functional evaluation

乳蛋白（Lactophorin，LP）是牛乳乳清蛋白中的一种主要糖蛋白，由27和17 kDa的糖肽和3 ~ 5个小肽组成。根据LP中27 k肽的一级结构，从LP中27 k肽的高含量和在疏水氨基酸残基中的定位，认为LP可能具有更高的乳化功能。本研究旨在建立一种从干酪生产乳清中分离LP的简便方法，并对分离得到的LP样品进行乳化活性和对乳脂蛋白脂酶的抑制活性评价。本研究获得了以下结果。1.我们建立了一个简单的和经济的分离方法，从奶酪乳清中的中试规模的LP。用超滤膜将363 kg乳清浓缩至体积的1/16左右（对应于500 k分子量）并反渗透至约1/60-70体积的乳清，然后将含有LP的浓缩物调节至pH 4.0，在95-100°C下加热30分钟，并在3000 rpm下离心15分钟，将上清液冷冻干燥。获得的富含LP的级分为54 g。将分离的LP富集部分通过Sephacryl S-300凝胶过滤制备高度纯化的LP.在pH7.0.4的条件下，1%LP和25%乳脂制备的乳状液中，富含LP的组分具有最高的乳化活性、乳状液稳定性、比表面积和最小的小球中值直径。以部分损伤的乳脂肪球为底物，以合成的乳状液为底物，对乳脂蛋白脂酶的抑制率分别为52%和70%。增加纯化LP的抑制作用增加到90%。

Lactophorin(LP)is a major glycoprotein in the proteose-peptone fraction of whey fraction from bovine milk and constituted of two major glycopeptides(27 and 17 kDa)with other 3 to 5 minor peptides. From high content and localization in hydrophobic amino acid residues on the basis of the primary structure of 27 k peptide in LP, it is considered that LP may have a higher emulsifying functionality. This study was to develop a simple isolation method of LP from whey of cheese manufacturing and to evaluate the emulsifying activity and inhibitory activity to milk lipoprotein lipase of the obtained LP specimen. The following results were obtained in this study.1. We have established a simplified and economic isolation method of LP from cheese whey in the pilot plant scale. Cheese whey(363 kg)was concentrated to about 1/16 in its volume by passing through ultra-filter-membrane(corresponding to 500 k molecular weight)and reversed osmosis to about 1/60-70 of whey in volume, and then the concentrates containing LP were adjusted to pH4.0, heated for 30min at 95-100°C and centrifuged for 15min at 3000 rpm and the supernatant was freeze-dried. The obtained LP-enriched fraction was 54g.2. The highly purified LP was prepared by gel filtration on Sephacryl S-300 of the isolated LP-enriched fraction.3. LP-enriched fraction had the highest emulsifying activity, emulsion stability, specific surface areas and smallest median diameter of globules in emulsions prepared from 1% LP and 25% milk fat at pH7.0.4. LP-enriched fraction and purified LP had the inhibitory activity of 52% and 70%, respectively, to milk lipoprotein lipase in partly injured milk fat globule and synthetic emulsion as the substrate. Increasing purified LP increased inhibitory effect to 90%.