Effect Of Heat-stable Proteases From Raw Milk On The Quality Of UHT Milk

Heat-stable protease can not be completely inactivated during UHT sterilization. The activities ofprotease that survive the heat treatments may cause some quality defects.The objective of this paper wasto carry out comprehensive research in the field of effect of processing on proteases and effect ofheat-stable proteases on the quality of UHT milk. The main results of this paper are as follows:Crude protease from Pseudomanas fluorescens BJ-10was purified by using ammonium sulfatefractionation, ion-exchange, and gel filtration chromatography. The purified enzyme was a monomerwith a molecular weight of47kD by SDS-PAGE. The specific activity of the enzyme increased61.38-fold. The optimum pH and temperature was7.0and30℃, respectively. The purified protease waspartially inhibited by DL-dithiothreitol, with little increase upon Fe2+addition. The protease showedtypical heat-stable behavior. After treatment at130℃for3min, more than47.67%activity wasremained. The purified protease had comparatively high content of glycine, alanine and glutamic acid.Glycine content was the highest, mole fraction up to42%.Growth rate and enzyme production of Pseudomanas fluorescens BJ-10in milk was significantlymore than in nutrient broth medium.When the counts exceeding109cfu/mL, the enzyme activity morethan0.775U/mL at6°C cultivation, milk protein began to be hydrolyzed. When the counts exceeding108cfu/mL, the enzyme activity more than6.49U/mL at28°C cultivation, κ-casein was completelyhydrolyzed. The protease preferentially degrade caseins in the following order κ-casein> β-casein>α-casein and have low activity on whey proteins.Plasmin activity were closely related to the breeds, stage of lactation, feeding and environmentalfactors. Holstein milk showed a high content (7.81U/mL) of plasmin in comparison with milk fromother breeds. The plasmin of buffalo milk such as Murrah, Nili, crossbreed F1, multi-crossbreed FHmilkwere6.49,5.9,6.46,6.07U/mL, respectively. The plasmin activity of yak milk was5.7U/mL. Theplasmin activity of Jersey milk was lowest (4.6U/mL) in the measurement of breeds. Plasmin activity ofHolstein milk was higher in2,3parity than in1,4parity, but the difference was not significantly (p>0.05).Plasmin activity in the Pasture was lower than in the farming community. Upon pasteurization of milk at75°C for15s, plasmin decreased by about25%. Cysteine could inhibited plasmin activity in raw milk.Plasmin (PL), plasminogen (PG), plasminogen activator (PA), which were associated with caseinmicelle. Plasmin hydrolyzes β-, αs2-, and αs1-caseins, but has little or no activity on the whey proteinsβ-LG and α-LA. In fresh milk, PG was the predominant form, where its concentration was4times thatof PL. Pseudomanas fluorescens BJ-10protease had activation effect on PG, but effect of BJ-10proteaseand urokinase (uPA) on PG activation was better than urokinase activate PG alone. Results of this studyconfirmed the interaction of the BJ-10protease with the plasmin system by acting as a PA.The objective of this study was to establish the forecasting model for predicting the shelf life ofUHT milk. The model base on somatic cell count, the number of psychrophilic bacteria, heat-stableprotease activity of raw milk and preservation temperature of UHT milk. Through the quadratic orthogonal rotatable design and analysis, we obtained regression equation:y=225.069167＋0.324417x1－0.026167x2－8.776042x3－5.722143x4－0.000983x1×x1－0.000600x2×x1－0.001083x2×x2－0.004688x3×x1＋0.009375x3×x2＋0.111979x3×x3＋0.004429x4×x1＋0.005143x4×x2＋0.054464x4×x3＋0.034422x4×x4. The results showed that regression equation was significant. It canbe used within the design range forecast. Analysis of variance of showed main factors have extremelysignificant impact on shelf life. Protease addition (x4) and storage temperature (x3) had siginificanteffect on UHT milk shelf life (p-<0.01); while SCC (x1) and psychrophile counts (x2) on the shelf lifewas not significant (p>0.05), there was a certain deviation with desired results, mainly due to thedifficult to control the whole experiment precisely.It can be concluded that heat stable proteases had a greater impact on the quality of UHT milk, itwas difficult to control it in the actual production. So, use of high-quality raw milk was of utmostimportance for achieving a long shelf-life of UHT milk. Storage of raw milk at low temperature (≤4°C)for a minimum period of time (≤48hours) minimizes growth of psychrotrophic bacteria and,consequently, the amount of extracellular bacterial proteinases produced in the milk before heattreatment. Furthermore, the use of milk with a low somatic cell count ensures a minimum level ofplasmin and plasminogen in the milk.