Paenibacillus spp. BD3526, a bacterium exhibiting a protein hydrolysis circle surrounded with an obvious precipitation zone on skim milk agar, was isolated from raw yak (Bos grunniens) milk collected in Tibet, China. Phylogenetic analysis based on 16S rRNA and whole genome sequence comparison indicated the isolate belong to the genus Paenibacillus. The strain BD3526 demonstrated strong ability to produce protease with milk clotting activity (MCA) in wheat bran broth. The protease with MCA was predominantly accumulated during the late-exponential phase of growth. The proteolytic activity (PA) of the BD3526 protease was 1.33-fold higher than that of the commercial R. miehei coagulant. A maximum MCA (6470 ± 281 SU mL⁻¹) of the strain BD3526 was reached under optimal cultivation conditions. The protease with MCA was precipitated from the cultivated supernatant of wheat bran broth with ammonium sulfate and purified by anion-exchange chromatography. The molecular weight of the protease with MCA was determined as 35 kDa by sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) and gelatin zymography. The cleavage site of the BD3526 protease with MCA in κ-casein was located at the Met₁₀₆–Ala₁₀₇ bond, as determined by mass spectrometry analysis.

Strain JR3^T was isolated from Chinese pickled potherb mustard (Brassica juncea Coss.) purchased from a local market in Shanghai, China. A polyphasic approach including 16S rRNA gene sequence analysis, average nucleotide identity (ANI) analysis, digital DNA–DNA hybridization (dDDH), determination of G+C content and various phenotypic analyses was employed to characterize strain JR3^T. The bacterium was rod-shaped, Gram-stain-positive, terminal round endospore-forming and catalase-positive. The strain could grow at a wide range of temperatures (20–45 °C) and pH values (6.0–8.0). Optimal growth of strain JR3^T occurred at 35–40 °C and a pH value of 7.0. The strain exhibited growth at salt (NaCl) concentrations of up to 3% (w/v). The G+C content of the genomic DNA was 44.0 mol%. The major fatty acids were C16 : 0, C19 : 0 c9, 10, summed feature 10 (C18 : 1 c11/t9/t6) and C18 : 1 c9. 16S rRNA gene sequencing revealed that strain JR3^T represents a member of the genus Lacrimispora, and it has higher sequence similarity with Lacrimispora amygdalina BR-10^T (=DSM 12857^T) (98.72%), Lacrimispora saccharolyticum WM1^T (98.29%) and Lacrimispora xylanolytica sy1 (98.22%). The dDDH value for strain JR3^T and phylogenetically related species within the genus Lacrimispora ranged from 17.7% to 29.9%. The ANI of strain JR3^T with its closely related taxa was far lower than the threshold (95%−96%) used for species differentiation. Results of phylogenetic, physiological and phenotypic characterization confirmed that strain JR3^T represented a novel species within the genus Lacrimispora, for which the name Lacrimispora sinapis sp. nov. is proposed. The type strain is JR3^T=CCTCC AB 2024044^T=LMG 33655^T.

Monascus species are widely used in traditional fermented foods for pigment and flavor formation, but citrinin contamination remains a major safety concern that limits broader food applications. Therefore, this study aimed to evaluate the citrinin risk of a furu-derived Monascus ruber strain, BC20, by integrating phenotypic screening across food-relevant matrices with genome-resolved analysis. After 14 days of cultivation across eight matrices, including fungal media as well as dairy-, cereal-, and bran-based substrates, citrinin was not detected by immunoaffinity cleanup combined with HPLC–FLD (LOD, 4 μg/kg; LOQ, 12 μg/kg). To investigate the genetic basis of this phenotype, we generated a chromosome-scale genome assembly for BC20 and conducted comparative analyses across a total of 19 Monascus genomes. ANI analysis and phylogenomic inference consistently placed BC20 within the ruber–pilosus clade. Comparative synteny analysis showed that the citrinin-associated locus in BC20 no longer retained an intact cluster configuration but instead exhibited a remnant-locus architecture, and similar patterns were also observed in several related genomes from the same clade. By contrast, the monacolin K (mk) locus remained syntenically conserved in BC20, supporting locus-specific structural disturbance rather than assembly-derived pseudo-absence. Additionally, its antifungal susceptibility was determined. Overall, BC20 represents a M. ruber candidate strain with undetectable citrinin, and this study provides a practical analytical framework for citrinin risk screening in food-related Monascus isolates.