Vol.1 No.1 2008

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Research paper : Mass preparation and technological development of an antifreeze protein (Y. Nishimiya, et al.)−10−Synthesiology - English edition Vol.1 No.1 (2008) based on experience and knowledge of the manufacturing company, while the latter requires stringent tests before approval. This implies that highly purified AFP cannot be used for all technologies utilizing AFP. On the other hand, highly purified AFP is indispensable for preparation of cell preservation fluid or AFP-assembled ice nucleation plate (see below). Any initial examination of AFP, even in food technology, requires highly purified AFP to obtain detailed information of its effects. Therefore, in this study, we focused on the technological developments of both crude and highly purified AFP. Commercial use of the crude form would require production capacities in tons. Technological developments are in progress according to the information shown in Figure 4. The columns show the species of fish (source materials) that contain AFP as mixture of isoforms, and the rows (such as activity and resource availability) are the factors needed for technological developments. The initial step of development is to deduce the activity of AFP sample from “A” (indicated by thick-framed box). In this step, sequential determinations and activity assay are done by preparing minute amount of sample, which provides evaluations for rows in column A (excellent [++], good [+], poor [−], and very poor [−−]) with regard to technological developments. Further examinations for molecular size, 3D structure, acid/alkaline tolerance, heat stability, as three examples, are undertaken. Smaller molecular size, rigid structure, high acid/alkaline tolerance, and high heat stability are preferred in AFP technologies. This approach is similar to the one used in biochemical evaluations of sample. A noticeable point is that Figure 4 contains information that are irrelevant to academic research such as evaluations regarding the availability of natural resources and infrastructure (e.g. fishermen’s union, fish sausage factory, storage warehouse, and marketing channel). However, all such information are important determinants for technological developments. In case of Figure 4, AFP from A will not become a target of development since its resource availability is very poor even though it has excellent activity. In contrast, AFP from B becomes the target although its activity is inferior to AFP-A, since sufficient resource availability compensates for the inferiority. For AFP-B, mass preparation method as well as AFP technology can be developed by utilizing its high thermal stability. AFP-C and AFP-D can also be developed since their resource availabilities are adequate. For AFP-C, poor infrastructure availability may raise the product cost. Although the significance for development of various factors differs, it may be concluded that availability of source material as well as activity are the principle determinants of technological development.We tried to update Figure 4 based on the increasing knowledge of AFP, which is reflected in the increasing number of columns with time. The factors in the row will also be appended with time concerning safety (toxicity), shelf life, and recyclability of AFP as three examples. 5 ResultsFigure 5 summarizes the elements relevant to mass preparation of AFP, such as AFP species (source materials), Fig. 5 Procedure for obtaining mass amount of AFP (thick arrows), which can also be used for collaborative studies and industrialization. Open circles (〇) shown in Process 2 indicate detailed methods to purity AFP such as heat denaturation, centrifugation, filtering, and chromatography. Initial methods in Process 2 are common for both partially and highly purified samples of AFP.AFP species (resources)Figure 5Highly purified AFPPartially purified AFPPlantFishFungiInsectCell culturingPurification fromnatural resourcesChemical synthesisMuscleBloodType of techniqueProcess 1Process 2Final Product (mass AFP)

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