Marchal R, Chaboche D, Marchal-Delahaut L, Gerland C, Gandon J P, Jeandet P
Laboratoire d'Oenologie, URVVC, UPRES EA 2069, Faculté des Sciences, Université de Reims, France.
J Agric Food Chem. 2000 Aug;48(8):3225-31. doi: 10.1021/jf990848a.
We describe here techniques to detect and quantify lysozyme in Pinot noir and Chardonnay Champagne wines. Using a dot-blot technique, lysozyme antibodies were able to recognize their antigens even when the concentration of lysozyme in wine was 75 mg/L. SDS-PAGE was the second technique used. After Coomassie Brilliant Blue (CBB) staining or antibody immunostaining was performed, the wine originating from the lysozyme-treated must gave only one band corresponding to the lysozyme. It is then possible to precisely determine the concentration of lysozyme in a must or a wine by densitometric measurement of this band. The control wine gave no band with the CBB staining, such as with the immunostaining. The quantification of lysozyme with HPLC is another useable technique because the lysozyme elution time is largely superior to that of all of the wine compounds. In wines, losses of lysozyme were higher when the enzyme was added at one time to the must (-34% for the Pinot noir and -37% for the Chardonnay) than when lysozyme is added in 2-fold both in the must and in the wine (around -26% for the two wines). The lowest diminution is observed when lysozyme was added to the wine only (-18%) in comparison to the addition to the must at 300 mg/L (-43%).
我们在此描述了检测和定量黑皮诺及霞多丽香槟酒中溶菌酶的技术。使用斑点印迹技术,即使葡萄酒中溶菌酶的浓度为75毫克/升,溶菌酶抗体也能够识别其抗原。十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)是使用的第二种技术。在进行考马斯亮蓝(CBB)染色或抗体免疫染色后,源自经溶菌酶处理的葡萄汁的葡萄酒仅产生一条对应于溶菌酶的条带。然后通过对该条带进行光密度测量,可以精确测定葡萄汁或葡萄酒中溶菌酶的浓度。对照葡萄酒在CBB染色以及免疫染色时均未出现条带。用高效液相色谱法(HPLC)定量溶菌酶是另一种可行的技术,因为溶菌酶的洗脱时间远长于所有葡萄酒成分的洗脱时间。在葡萄酒中,一次性向葡萄汁中添加溶菌酶时(黑皮诺损失-34%,霞多丽损失-37%),溶菌酶的损失高于在葡萄汁和葡萄酒中均分两次添加溶菌酶时(两种葡萄酒均约为-26%)。与以300毫克/升的量向葡萄汁中添加溶菌酶相比(损失-43%),仅向葡萄酒中添加溶菌酶时溶菌酶的减少量最低(-18%)。