Live Bacillus subtilis preparation (TY7210 strain), veterinary grade API, is a spore-enriched, non-sterile powder intended for formulation into tablets, injections after downstream sterilisation or aseptic processing, capsules, powders, granules, premixes, and oral solutions. The material is produced by submerged fermentation, concentrated by continuous centrifugation, and spray-dried with a low-moisture protective carrier matrix. Standardisation targets viable spore count of not less than 1.0×10¹⁰ CFU/g after heat activation at 80°C for 10 min and enumeration by ISO 4833-1:2013. Spray-dryer conditions are typically inlet 150–180°C and outlet 70–85°C; residual moisture is controlled to ≤ 8.0% by USP <731>. Particle size distribution shows 90% below 180 µm by laser diffraction USP <429>. The dry spore form remains stable at 25°C and 60% RH for 24 months in sealed aluminium-laminated pouches; open-container storage under humid conditions can trigger germination and must be avoided. Microbial contamination control is verified by absence of coagulase-positive staphylococci and Pseudomonas aeruginosa in 1 g by USP <62>, run in parallel with spore count release.

How Does TY7210 Differ from Generic Bacillus subtilis Feed-Grade Strains?

TY7210 is a single, defined strain with confirmed 16S rRNA identity and a sporulation efficiency above 95% under production fermentation, whereas many commodity Bacillus subtilis feed additives are untyped strain mixtures. The specific strain influences spore dimensions 0.8–1.2 µm length, germination temperature optima, and survival in dry concentrates. TY7210 releases vegetative cells after germination at 37°C on tryptic soy agar within 24 h, forming off-white irregular colonies; this morphology is part of identity testing. Compared with vegetative probiotic strains such as lactobacilli, TY7210 spores tolerate spray drying and pelleting. In dry powder held at 25°C for 24 months, TY7210 loses typically ≤ 0.5 log₁₀ viable count, while non-sporulating probiotic powders commonly lose more than 2 log₁₀ within 3 months when stored without refrigeration. Another operational distinction is stability in high-mineral premixes: TY7210 can be blended with choline chloride and trace minerals up to 10% by weight without immediate viability loss, although prolonged storage above 35°C with trace minerals can accelerate spore coat oxidation. Generic untyped strains may show variable recovery under the same conditions.

Viable Spore, Moisture, and Elemental Impurity Limits Are Established by Pharmacopoeial Methods

The release specification is based on pharmacopoeial and ISO methods. Because the API is a live spore preparation, water activity is more informative than loss-on-drying alone; water activity is specified at ≤ 0.35 at 25°C to prevent premature germination. Table 1 lists the core release parameters.

Microbial absence tests use 25 g or 1 g test portions as listed; positive release requires no growth on selective agar after prescribed incubation. For elemental impurities, the API is intended for veterinary use, and limits are aligned with Ph. Eur. methods; residual solvents are controlled under VICH GL18 when relevant to the spray-dried carrier. Sampling follows ISO 7218:2007 for random sample plans; a minimum of 3 samples per batch is tested for release, and out-of-specification results trigger retest on 6 additional samples.

Dry Granulation, Direct Compression, and Premix Dilution Characteristics

The spray-dried powder has bulk density 0.45–0.60 g/mL and tapped density 0.60–0.80 g/mL; Carr’s index ranges 15–25%, indicating acceptable flow for direct compression after blending with glidant. Direct compression tablet formulations containing microcrystalline cellulose and crospovidone at 10–15 kN compression force yield hardness 60–90 N and disintegration time <15 min in water at 37°C by USP <701>. Wet granulation should limit water addition to ≤ 20% w/w because the spore coat is hydrophobic and excess water produces non-uniform granule adhesion. For premixes, a stepwise dilution of 1:10 then 1:100 in a double-ribbon mixer at 12 rpm for 10 min yields spore-count coefficient of variation below 5%; single-stage addition of undiluted API to feed has shown segregation due to particle density differences.

These loss ranges reflect typical spore-former behaviour under controlled conditions and should be confirmed lot-wise for the final blended product because excipient type, water activity, and carrier pH can increase or decrease recovery. Published data for TY7210 under every specific formula is limited; validation with the finished dosage form is required. For capsule filling, dosator-type machines set to 10–15 mm dosing depth and tamping force 80–120 N achieve fill weight variation below 3% relative standard deviation when the powder is pre-lubricated with 0.5% magnesium stearate. Higher lubricant levels can impair powder flow and reduce spore recovery from acidic dissolution media.

The API can also be formulated as an oral suspension or soluble powder for drinking water administration. The spores are not truly soluble; a 10 mg/mL dispersion in water buffered to pH 6.5–7.5 shows sedimentation volume below 0.8 after 24 h. Resuspendability is improved by adding 0.1% polysorbate 80, but surfactant levels above 0.5% may alter spore coat hydrophobicity and reduce plate recovery, so surfactant concentration must be validated against ISO 4833-1:2013. Free chlorine at 2 mg/L in drinking water reduces viable spores by approximately 1 log₁₀ within 60 min; dechlorination or stabilised formulation is therefore required in water medication lines. Buffered oral solutions stored at 25°C for 7 days retain about 90% of initial CFU when pH is maintained at 6.5; at pH 4.0, germination is delayed and viability loss is minimal over 48 h, but extended acid contact beyond 7 days can reduce count. Dosing pumps with silicone tubing show no significant spore adsorption after 24 h continuous recirculation at 25°C if tubing is rinsed with buffer pH 6.5; polyvinyl chloride tubing may retain spores on the surface and should be validated by rinse recovery.

When Injectable Administration Is Required, Which Controls Apply?

The API is not sterile, and injectable presentations are not direct compendial uses. Because spores measure 0.8–1.2 µm by 0.6–0.9 µm, sterile filtration through 0.2 µm membranes is not feasible without major viable-loss. Terminal autoclaving at 121°C for 15 min destroys spore viability and is unsuitable for a live preparation. Gamma irradiation at 5 kGy can reduce viable count by 1–2 log₁₀; if the starting count is 1.0×10¹⁰ CFU/g, the irradiated material may remain within specification for oral uses but may not meet an injectable target count without overage. Aseptic processing with gamma-irradiated spore powder at 2–4 kGy and sterilised excipients has been described experimentally, but published data for TY7210 in injectable matrices is limited. Injectable formulations must meet USP <71> sterility and USP <85> bacterial endotoxin acceptance, typically ≤ 0.5 EU/mL or ≤ 5 EU/kg body weight per dose. Depyrogenation of component glassware is performed by dry heat at 250°C for 30 min; the spore powder itself cannot be depyrogenated by dry heat, so alternative endotoxin reduction must be validated. Intravenous administration of live spores is not recommended unless target-species safety, pyrogenicity, and strain-specific biodistribution data are available.

For tablet and capsule dosage forms in companion animals, the spore content can be adjusted by blending with lactose monohydrate and magnesium stearate; the blend is compressed to target weight and enteric coating may be required if gastric pH 2.0 is a concern, although spore forms are inherently acid-tolerant. In poultry pelleted feed, live TY7210 spores survive standard conditioning at 70–85°C for 30–90 s with 0.5–1.0 log₁₀ loss; retention above 90 s at 85°C can exceed 1.5 log₁₀ loss. This distinguishes the product from lactobacilli and yeast cultures, which are largely inactivated during steam pelleting. For piglet and calf oral solutions, in vitro simulated gastric fluid at pH 2.0 with pepsin for 60 min shows ≤ 0.5 log₁₀ loss, but this assay is not a pharmacopoeial standard and should be repeated with the specific formulation. Formulation overage is typically set at 0.5–1.0 log₁₀ to compensate for processing loss. Tablet doses of 1×10⁹ CFU to 1×10¹⁰ CFU per unit are common in companion animal products, but target species and indication define the final dose. In-feed inclusion of 1×10⁶ CFU/g to 1×10⁷ CFU/g final feed is achieved by premix dilution, with intermediate premix counts of 1×10⁹ CFU/g to 1×10¹⁰ CFU/g. Published data for TY7210 in target-species field trials is limited.

Maintaining Spore Viability During Storage and Unit-Dose Manufacture

Storage of the API in unopened sealed pouches below 25°C and 60% RH is required. If the powder is exposed to relative humidity above 70% for 24 h, water activity can rise above 0.45 and initiate spore germination, with viable count losses of 0.5–2.0 log₁₀ depending on carrier matrix. The API is incompatible with strong acids below pH 3.0, ethanol concentrations above 30% v/v, and oxidising disinfectants such as hypochlorite at 200 ppm. Simultaneous formulation with antibiotics should be avoided unless compatibility with spore germination is demonstrated by challenge test; spore forms are more tolerant than vegetative cells to many aminoglycosides in disk diffusion screening, but germination in vivo may be suppressed. After wet granulation, final granule moisture should be dried to ≤ 5.0% before encapsulation to prevent capsule shell deformation and viable-count drift. Batch-to-batch variation in viable count is controlled within ±0.3 log₁₀ by standardising fermentation harvest against a reference spore suspension.