Design of Ready-to-Use “Ball-in-Ball” Staphylococcus aureus Microsphere Based on Novel Cryoprotectant and Drop Freeze-Drying Technology: Effective Preservation and Application

<i>Staphylococcus aureus</i> (<i>S. aureus</i>) poses a significant threat to public health and safety, and enhancing the monitoring of <i>S. aureus</i> in food is essential to curb and prevent foodborne transmission. In order to obtain strains for more convenient and rapid use in quality control or quantitative analysis, this study designed a ready-to-use "ball-in-ball" microsphere based on a novel cryoprotectant combined with drop freeze-drying technology. When using a cryoprotectant that contains 1.5% bovine serum albumin, 4.5% trehalose, 8.2% polyethylene glycol 8000, and 4.1% D-mannitol, the survival rate of <i>S. aureus</i> can reach 98.2 ± 2.6%. This cryoprotectant effectively prevents <i>S. aureus</i> from shrinking, deforming, and damaging cell walls. Additionally, it shows desirable protective efficiency for other Gram-positive bacteria. The molding of microspheres is efficient and cost-effective, demonstrating good uniformity and stability without the need for pre-freezing. The moisture content and the count of <i>S. aureus</i> showed no significant changes over 90 days at -20 °C. In the simulated contaminated sample, the recovery rate of <i>S. aureus</i> in milk and green tea was 83.1-93.7%. This study could provide a practical approach to improve the monitoring efficiency of <i>S. aureus</i> and shows potential as a generalized strategy for preparing ready-to-use strains related to food safety.