| Pineapple juice is the second most popular juice in the juice market after orange juice. Fresh pineapple juice is rich in bromelain which possess many biological activities with health benefits, such as anti-platelet aggregation, fibrinolytic, anti-inflammation, detumescence and digestion-promoting properties, but also may induce allergic reactions in persons with allergic constitution. Commercialized pineapple juice needs to satisfy a certain storage period, while the fresh pineapple juice must be sterilized for storage. Traditional thermal sterilization would completely deactivate the bromelain due to its heat-sensitivity, resulting in complete loss of its biological activities, while with non-thermal sterilization, the allergenicity may not be controlled. If functional hypoallergenic pineapple juice could be developed by retaining the health benefit of the fresh pineapple juice and reducing its allergenicity on the premise of effective sterilization, the added value of pineapple could be considerably increased, thus making the best use of the fruit.In this study, non-thermal allergenicity reducing and bioactivity keeping processing was carried out on the fresh pineapple juice using ultra-high hydrostatic pressure (UHP). First, the bromelain extracted from the fresh pineapple juice was analyzed for components and structures by SDS-PAGE, Western-blot and LC-MS/MS, to confirm the main allergenic protein and its components, and the feasibility of concurrent allergenicity reducing and bioactivity keeping of fruit bromelain by UHP and the relationship of UHP with secondary structure of the fruit bromelain were also studied in the experiments. Second, the difference in the effects of UHP on the fresh pineapple juice and fruit bromelain, the effectiveness of UHP for the concurrent allergenicity reducing and bioactivity keeping of the fresh pineapple juice, and the synergistic effect of temperature on the concurrent allergenicity reducing and bioactivity keeping were explored, and the concurrent allergenicity reducing and bioactivity keeping process was optimized. Finally, the effect of the concurrent allergenicity reducing and bioactivity keeping was improved by pH adjustment and addition of NaCl in combination with UHP, and the storage stability of the processed juice was investigated. The results were as follows.1. The allergenicity of the fresh pineapple juice was mainly derived from fruit bromelain complex of 24 kDa. UHP could realize the concurrent allergenicity reducing and bioactivity keeping of the fruit bromelain, and its effect was associated with K value (K=α-helix content/β-sheet content) of the fruit bromelain.The main components of the fruit bromelain had a molecular weight of 24 kDa, and this 24 kDa band was also the main allergenidentified by the serum of people who were allergic to the pineapple using Western-blot method. The results of LC/MSMS analysis showed that the 24 kDa bromelain was a complex enzyme composed of eight cysteine proteases with different components. Although the primary structures of those cysteine proteases were homologous in a certain degree, there were also differences among them.The pressure lower than 300 MPa was favorable for the activity enhancement of the fruit bromelain, but not for the concurrent allergenicity reducing. While in the case of the pressure higher than 300 MPa, proteolytic activity, fibrinolytic activity and allergenicity were all reduced to a varying degree, with the greatest reduction in the allergenicity. The suitable pressure for the concurrent allergenicity lowering and bioactivity keeping was 400 MPa.The results of circular dichroism spectrum analysis indicated that the influence of UHP on the concurrent allergenicity reducing and bioactivity keeping effect of the fruit bromelain would be estimated by the change of K value. With K>1, it was favorable for the activity enhancement, but not for the concurrent allergenicity lowering. While with the K<1, the concurrent allergenicity lowering and freshness keeping could be realized, but the difference between activity and allergenicity was smaller with the decrease in the K value.2. The sterilization, allergenicity reducing and bioactivity keeping process could only be realized when the fresh pineapple juice was treated with a pressure higher than 300 MPa. Both temperature lowering and raising process combined with UHP could improve the concurrent allergenicity reducing and bioactivity keeping effect on the premise of preserving the flavor of the fresh pineapple juice.With the pressure higher than 300 MPa, the decrease rates of the proteolytic activity, fibrinolytic activity and allergenicity in the fresh pineapple juice were lower than that in the pure bromelain system, but the difference between the two systems was not significant (p>0.5). Only higher than 300 MPa could process the sterilization, allergenicity reducing and bioactivity keeping of fresh pineapple juice. The suitable pressure for the concurrent allergenicity lowering and freshness keeping was 400 MPa, but the allergenicity-lowering effect remained to be optimized.When a pressure of 400 MPa was combined with a lowered temperature (by 10～20 ℃) or a raised temperature (by 20～50℃), the allergenicity was significantly reduced,while the proteolytic activity and fibrinolytic activity remained at high levels. Therefore, under the condition of high pressure, both temperature lowering and raising process were favorable to the concurrent allergenicity reducing and bioactivity keeping of the fresh pineapple juice.When the allergenicity was reduced to 52% by the temperature combined with pressure processing, the proteolytic activity and fibrinolytic activity had 81% and 92% remained, respectively. The corresponding process involved a pressure of 490 MPa, a pressure-holding time of 20 min and a temperature of 45℃3. pH and NaC1 combined with UHP processing could remarkably improve the allergenicity reducing and bioactivity keeping effect of the fresh pineapple juice, enabling a long storage period at 4℃.Both pH adjustment and addition of NaCl could improve the concurrent allergenicity reducing and bioactivity keeping effect of the fresh pineapple juice. Considering the flavor of the fresh pineapple juice and the concurrent allergenicity reducing and bioactivity keeping effect, pH3.2～4.2 and addition of NaCl ranged 0.2 to 0.6% were suitable.The process with synergistic effect could reduce the relative allergenicity to about 20%, but the relative proteolytic activity and fibrinolytic activity were only 45% and 56%. However, when the allergenicity was reduced to 32%, the proteolytic activity and fibrinolytic activity had about 70% and 85% remained, respectively. While the allergenicity was low, the difference in reduction rates between bioactivity and allergenicity was significantly increased and the flavor was not influenced by NaCl content The corresponding process condition for synergetic effect involved 478 MPa, 50℃, pH3.2, NaCl 0.50%, and a pressure-holding time of 20 min.With this process, the fresh pineapple juice could be stored for at least 42 days at 4 ℃, the allergenicity was reduced to 28%, the proteolytic and fibrinolytic activity had about 60% and 70% remained, and the quality of the pineapple juice was not significantly changed. |
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