Microbial Enzyme Laboratory

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Here is a proposed in-house microbial inoculant lab workflow tailored for Crown MycoResin Biotech Inc., focusing on the research, development, and production of microbial inoculants for Agarwood (Aquilaria spp.) resin induction.

Crown MycoResin Biotech Inc. – In-House Microbial Inoculant Laboratory Workflow

Purpose: To isolate, culture, formulate, and quality-test microbial and enzyme-based biotic inducers for the BarIno™ Series

I. LABORATORY SECTIONS

  1. Sample Reception & Isolation Zone
  2. Microbial Culture Room (Biotic Agent Propagation)
  3. Enzyme Extraction & Fermentation Room
  4. Inoculant Formulation & Mixing Area
  5. Quality Control & Bioassay Lab
  6. Packaging & Labeling Room
  7. Cold Storage Facility

II. WORKFLOW PROCESS

Step 1: Source Acquisition & Isolation

  • Source materials: Agarwood rhizosphere soil, decayed wood, naturally infected trees
  • Sterile Processing: Under laminar flow hood, isolate microbes on PDA, MEA, or Czapek-Dox agar
  • Selection Criteria:
    • Resin-inducing potential
    • Compatibility with Aquilaria malaccensis
    • Enzyme production ability
  • Tools: Autoclave, Petri dishes, inoculating loops, biosafety cabinet

Step 2: Culture Maintenance & Expansion

  • Pure Culture Development: Sub-culturing of target fungi (e.g., Fusarium oxysporum) on agar
  • Liquid Propagation: Inoculate broth media (e.g., PDB or custom microbial enzyme media)
  • Incubation: Shaking incubator (25–30°C, 120–150 rpm) for 3–7 days
  • Spore Suspension Harvesting: Filter, centrifuge (if needed), and quantify spore count
  • Enzyme Co-culture Option: Introduce co-fermentation microbes (Bacillus, Trichoderma, etc.)

Step 3: Enzyme Extraction & Assay

  • Fermentation Setup: Bioreactors or shake flasks for enzyme-rich cultures
  • Enzyme Types Targeted:
    • Laccase
    • Peroxidase
    • Cellulase
    • Lipoxygenase
  • Crude Extraction: Filter broth through cheesecloth or vacuum filtration
  • Assay & Quantification: Enzyme activity using spectrophotometer-based assays (e.g., ABTS for laccase)

Step 4: Formulation & Additives Integration

  • Carrier Selection: Buffered water, molasses, or plant extract base
  • Additive Integration:
    • MnO₂ or NaCl for combined bio-chemical blends (e.g., BarIno™ FusaTrinity™)
    • Stabilizers or surfactants for shelf-life
  • Mixing Tank: Stainless or HDPE with mechanical stirring
  • Form Types:
    • Liquid Spore Suspension (primary)
    • Gel Stick or Capsule Form (advanced option)

Step 5: Quality Control Testing

  • Microbial Count: Plate count method (CFU/mL or CFU/g)
  • Spore Viability Test: Using hemocytometer or staining
  • Sterility Check: Ensure absence of contaminants
  • pH & Shelf-Life: Stability testing at 25°C & 4°C
  • Bioassay: Test on Aquilaria seedlings or branches (resin induction observed over 2–4 weeks)

Step 6: Packaging & Labeling

  • Primary Packaging: 1-liter HDPE bottles with tamper-proof cap
  • Label Includes:
    • Product name & batch ID
    • Active ingredients
    • Storage & handling
    • Expiry date
  • Secondary Packaging: Cartons for bulk transport
  • Storage: Refrigerated or cool dry room (16–25°C)

III. DATA RECORDING & TRACEABILITY

  • Lab notebook or LIMS (Lab Info Mgmt System)
  • Batch logbooks for all formulations
  • QR-coded label system for traceability
  • Sample archival of all batches

Optional R&D Add-ons

  • DNA barcoding for strain identity verification
  • Pilot fermentation tank for enzyme optimization
  • Formulation trials using nanoemulsion or microencapsulation
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