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Upstream processing represents a critical phase in biomanufacturing, wherein biological systems such as microorganisms, mammalian cells, or insect cells are cultivated to produce therapeutic proteins, vaccines, enzymes, or other biologically derived products. This phase encompasses all steps from the selection and genetic manipulation of the production organism to the cultivation of cells in bioreactors under tightly controlled environmental conditions.Host Selection and Genetic OptimizationThe...
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Downstream Processing, Formulation Development and Antithrombotic Evaluation of Microbial Nattokinase.

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    Researchers developed acid-stable nattokinase loaded chitosan nanoparticles (sNLCN) for oral delivery. These nanoparticles effectively managed thrombotic disorders in rats, showing significant anti-thrombotic potential comparable to warfarin.

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    Area of Science:

    • Biotechnology
    • Pharmacology
    • Biochemistry

    Background:

    • Nattokinase (NK), an enzyme produced by Bacillus subtilis, possesses fibrinolytic properties beneficial for managing thrombotic disorders.
    • Efficient oral delivery of enzymes like NK is challenging due to gastrointestinal degradation.
    • Developing stable oral formulations is crucial for enhancing NK's therapeutic efficacy.

    Purpose of the Study:

    • To describe the downstream processing of Bacillus subtilis-derived nattokinase (NK).
    • To develop and evaluate an efficient oral formulation of purified NK for managing thrombotic disorders.
    • To investigate the anti-thrombotic potential of the novel oral formulation in vivo.

    Main Methods:

    • Solid-state fermentation was used for NK production, followed by purification and characterization (28 kDa, 504.4 FU/mg specific activity).
    • Acid-stable nattokinase loaded chitosan nanoparticles (sNLCN) were fabricated using Box-Behnken design for optimization.
    • Enzyme integrity and activity were confirmed using SDS-PAGE, CD analysis, and in vitro clot lysis assays.
    • In vivo efficacy was assessed using a rat tail thrombosis model and coagulation tests (PT, aPTT).

    Main Results:

    • Purified NK maintained its integrity, conformational stability, and fibrinolytic activity in the sNLCN formulation.
    • Peroral administration of sNLCN significantly reduced thrombosis frequency in Wistar rats compared to controls.
    • sNLCN demonstrated significantly higher anti-thrombotic potential than free NK, comparable to warfarin.
    • Coagulation analysis confirmed the superior anti-thrombotic effect of sNLCN.

    Conclusions:

    • Acid-stable nattokinase loaded chitosan nanoparticles (sNLCN) provide an effective strategy for oral enzyme delivery.
    • sNLCN exhibits significant anti-thrombotic activity, offering a promising alternative for managing thrombotic disorders.
    • This formulation holds potential for improving NK-based therapies for thrombosis.