CN201510367541.2 provides a lactoperoxidase extraction method that can be used in industrial production with high recovery rate and purification multiple and can be used in food industry. The object of the present invention is achieved through the following technical solutions: take cow whey as raw material, take ammonium sulfate salting-out method as extraction means, determine saturation, salting-out time, optimal salting-out condition in ammonium sulfate salting-out method The process conditions of rotational speed and system pH were compared, and the effects of ammonium sulfate fractional precipitation and direct single-use 65% ammonium sulfate saturation on the recovery rate of LP enzyme activity and purification times were compared. Ultrafiltration technology is used for further purification, and finally dialysis desalination and freeze-drying are used to obtain the finished product, namely raw whey → ammonium sulfate salting out method for fractional precipitation and extraction of lactoperoxidase → precipitation and dissolution → solution separation by ultrafiltration → collection of retentate solution → dialysis Desalting → freeze drying → crude lactoperoxidase product. Concretely, a kind of method that adopts ultrafiltration to assist ammonium sulfate salting out to extract lactoperoxidase of the present invention, comprises the following steps:
(1) Fractional extraction of lactoperoxidase by ammonium sulfate salting out method:
After removing the fat and casein in the fresh milk, whey is obtained. At room temperature, add 25-85% ammonium sulfate to the whey, centrifuge, remove the precipitate, and keep stirring on a mixer to make ammonium sulfate. Fully dissolve, adjust the pH of the system to 5.5-8.5, leave it for 2-10h to completely precipitate the lactoperoxidase, and centrifuge at 5000-10000r/min at 4°C for 20min until the precipitation is the lactoperoxidase product;
The precipitate obtained in step (1) is reconstituted with an appropriate amount of deionized water and subjected to ultrafiltration separation with an ultrafiltration membrane to remove impurity proteins and collect the intercepted solution;
(3) Desalination treatment:
The retentate solution collected in step (2) is loaded into the dialysis bag for desalination;
The crude lactoperoxidase product is obtained by freeze-drying the desalted solution in step (3).
The crude product of lactoperoxidase extracted by the present invention is light yellow powder, and the enzyme activity recovery rate and purification multiple of lactoperoxidase reach 79.25-85.21% and 4.08-4.58 times respectively. The lactoperoxidase with higher recovery rate can be directly obtained through the above-mentioned extraction process, the natural antibacterial medium lactoperoxidase product can be directly obtained, and the product can also be directly added to food as a natural preservative.
Through the above extraction method, lactoperoxidase with high recovery rate can be directly obtained, and natural antibacterial medium lactoperoxidase products can be directly prepared, and the products can also be directly added to daily necessities such as food and toothpaste as natural preservatives. middle.
2. Horseradish Peroxidase (Horseradish Peroxidase, HRP, E.C.220.127.116.11) is a glycoprotein composed of enzyme protein and iron porphyrin. The enzyme consists of multiple isozymes with a molecular weight of 40kDa. The isoelectric point is pH3.0‑9.0. The maximum absorption spectra of the prosthetic group and enzyme protein of HRP are at 403nm and 275nm, respectively, so people usually use the RZ value (ie the ratio of OD403nm/OD275nm) to represent the purity of the enzyme. The larger the RZ value, the higher the purity.
HRP is one of the most important medical diagnostic enzymes (J. Chromat. B 2004, 803:237‑241). This enzyme is not only used in many biochemical detection items, but also widely used in immunological kits. In addition, HRP can also be used for decolorization of industrial wastewater (Chem. Technol. Biotechnol. 2009, 84: 126–132), removal of peroxides and treatment of phenolic wastewater (Biotechnol. Bioprocess Eng. 2006, 11: 462–465), etc.
In nature, horseradish is the plant with the highest HRP content, so it has become the most attractive raw material for the production of HRP. At present, the main methods for separating and extracting HRP from horseradish are: two-phase extraction technology (Appl. Biochem. Biotechnol. 1995, 53: 147-154), reverse micelle extraction technology (Enzyme Microb. Technol. 1996, 18: 332-339), electrophoresis (J. Biol. Chem. 1966, 241: 2166-2172) and chromatography (J. Membr. Sci. 2003, 211: 101-111), etc. However, these technologies all have certain defects. Among them, the products obtained by aqueous two-phase extraction technology and reverse micelle extraction technology are of low purity, and are prone to inactivation and denaturation of HRP; electrophoresis technology and chromatography technology are expensive, and Difficult to scale up industrially.