The Extraction, Isolation, And Tenderization Of Beef By Protease In Actinidia Arguta

In this experiment, Actinidia arguta from Changbai Mountain was used as material and the ammonium sulfate precipitation method was adopted to extract crude protease. The crude enzyme was studied in the decomposition of actomyosin and tenderization of beef. In order to get pure Actinidia arguta protease, we used the method of SephdexG-25gel filtration chromatography and DEAE FF ion exchange chromatography to separate and purify crude enzyme. Finally we determined the molecular weight of the enzyme, qualitative identification by mass spectrometry and N-terminal sequencing. The main experimental results were as follows:1. Select ripeness. With the increase of the actinidia arguta maturity, extractable juice content, acidity, sugar degree and protease activity increased gradually. At the maturity stage IV, all indexes reached maximum and the decomposition effect of actinidia arguta crude protease on actomyosin was best. So adopting the maturity stage IV of actinidia arguta was the best raw material extraction.2. Select salting-out condition. With the increase of ammonium sulfate saturation, protease activity in supernatant fluid was reduced gradually. When the saturation reached70%, there was almost no enzyme activity in the supernatant fluid. It indicated that protease almost completely precipitated. Thus70%saturation of ammonium sulfate was the salting-out condition.3. The time of actomyosin extraction.180minutes after the chicken slaughter was the best time for extracting actomyosin, but meat quality was poor and not suitable for processing into products.4. Decomposition of actomyosin. Actinidia arguta protease had strong decomposition effects on actomyosin. The optimal pH value was7.0, the best reaction temperature was60℃and the optimum reaction time was15minutes.5. Effect of beef tenderization. Actinidia arguta protease had significant effect on shearing force of beef, water loss rate in cooking, water holding capacity and had better tenderization effect. Through the orthogonal experiment, the best technological conditions of beef tenderization by actinidia arguta protease were dosage of enzyme 0.05%, treatment temperature50℃, treatment time1h. The orders of significance factors were as follows:temperature, dosage of enzyme and time. Sensory evaluation also proved that under the condition of the beef tenderization by actinidia arguta protease treatment, color of beef was good, soft and juicy, mouthfeel was obviously improved. Electronic microscopy results showed that compared with the untreated meat samples, the Z line of treated meat samples broke. The interval of muscle became bigger. The material of muscle increased. The length of sarcomere became larger. So the tender of meat increased.6. Separation and purification. SephdexG-25gel filtration chromatography and DEAE FF ion exchange chromatography were used to separate and purify actinidia arguta protease. And after3KD hollow fiber column desalting, we could get the purification protease. To reach electrophoretic purity, we tried to get the single protease stripe by SDS-polyacrylamide gel electrophoresis. After purification, the specific activity of the protease reached53428U/mg, the purified multiple was25.5times, activity recovery was9%.7.Mass spectrometry identification and N-terminal sequencing:After mass spectrometry identification and N-terminal sequencing, actinidia arguta protease was analysed by online software BLAST in NCBInr database. The results showed that Actinidia arguta protease was the kiwi protease with N-terminal sequence of vlpdy vdwrs agavv. And molecular weight was23.8KD.