Production of Neoglycoprotein with Neutralizing Activity to Bacterial Toxins from Cheese Whey and Its Characteristic Analysis.

a.研究了用12% TCA直接萃取甜奶酪乳清制备酪蛋白糖肽类（CGP）的新方法。用三种不同的化学或酶的方法对唾液乳糖（n -乙酰神经氨酸(NeuAc) α - 2-x-半乳糖β - 1-4葡萄糖）与霍乱毒素进行了反应，最终得到了新糖蛋白（neoCGP）。c.引入的糖链经碱性硼氢化物裂解后，用甲基化和核磁共振分析分析了其化学结构。d.建立了一种新的检测霍乱毒素中和活性的方法。首先，在聚苯乙烯珠上涂上霍乱毒素b亚基。其次，将微球与样品反应，然后用生物素化的neuac探针孵育。第三，用酶标链亲和素孵育后显色。当样品对霍乱毒素有中和作用时，其显色度比空白样品降低。利用所建立的新评价方法，证实了neoCGP比asialoCGP的中和作用增强。

a.The new preparation method for caseinoglycopeptides (CGP) from sweet cheese whey by direct extraction with 12% TCA was developed.b.The introduction of an acidic trisaccharide ; sialyllactose (N-acetylneuraminil (NeuAc) alpha2-x-galactosylbeta1-4glucose) was tried against CGP by three different chemical or enzymatic technique and finally noeglycoprotein (neoCGP) was produced.c.The chemical structure of the introduced sugar chains was analyzed by methylation and NMR analysis after cleavage by alkaline borohydride treatment.d.A new detection method for neutralizing activity to Cholera toxin was developed by using biotinated-polymer probe. First, polystirene beads were coated with Cholera toxin B-subunit. Second, the beads was reacted with the sample and then incubated with biotinated NeuAc-probe. Third, the beads was color-developed after incubation with HRP-streptoavidin. If the sample has neutralizing activity to Cholera toxin, the coloration level is decreased compared to blank.e.The increase of the neutralizing activity of neoCGP was actually confirmed compared to asialoCGP by using the developed new evaluation method.