Characterization of live biotherapeutic product (LBP) batches typically includes a measurement of viability, such as colony forming units (CFU). However, strain-specific CFU enumeration assays can be complicated by the presence of multiple organisms in a single product with similar growth requirements. To overcome specific challenges associated with obtaining strain-specific CFU values from multi-strain mixtures, we developed a method combining mass spectrometry-based colony identification with a traditional CFU assay. This method was assessed using defined consortia made from up to eight bacterial strains. Among four replicate batches of an eight-strain mixture, observed values differed from expected values by less than 0.4 log10 CFU among all strains measured (range of differences, -0.318 to + 0.267). The average difference between observed and expected values was + 0.0308 log10 CFU, with 95% limits of agreement from -0.347 to 0.408 (Bland-Altman analysis). To estimate precision, a single batch of eight-strain mixture was assayed in triplicate by three different users, for a total of nine measurements. Pooled standard deviation values ranged from 0.067 to 0.195 log10 CFU for the eight strains measured, and user averages did not differ significantly. Leveraging emerging mass-spectrometry-based colony identification tools, a novel method for simultaneous enumeration and identification of viable bacteria from mixed-strain consortia was developed and tested. This study demonstrates the potential for this approach to generate accurate and consistent measurements of up to eight bacterial strains simultaneously and may provide a flexible platform for future refinements and modifications. KEY POINTS: • Enumeration of live biotherapeutics is essential for product quality and safety. • Conventional CFU counting may not differentiate between strains in microbial products. • This approach was developed for direct enumeration of mixed bacterial strains simultaneously.

This study described the quality and microbial influence on ozone water (OW) and ultra-high pressure (UHP) processing alone or in combination with refrigerated catfish fillets. The analysis parameters included total volatile base nitrogen (TVBN), thiobarbituric acid reactive substances (TBARs), chromaticity, microbial enumeration, 16S rRNA gene sequencing, electronic nose (E-nose), and sensory score. The study found that compared with the control (CK), ozone water combined with ultra-high pressure (OCU) delayed the accumulation of TVBN and TBARs. The results of sensory evaluation illustrated that OCU obtained a satisfactory overall sensory acceptability. The counting results suggested that compared to CK, OCU significantly (p < 0.05) delayed the stack of TVC, Enterobacteriaceae, Pseudomonas, lactic acid bacteria (LAB), and hydrogen sulfide-producing bacteria (HSPB) during the storage of catfish fillets. The sequencing results reflected that the dominant were Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria at the phylum level, and the dominant were Acinetobacter, Pseudomonas, Lelliottia, Serratia, Shewanella, Yersinia, and Aeromonas at the genus level. The dominant was Acinetobacter in initial storage, while Pseudomonas and Shewanella were in anaphase storage. Based on the TVC and TVBN, the shelf life of catfish fillets was extended by at least 3 days compared to the control. In short, the combination of ozone water and ultra-high-pressure processing is a favorable strategy to control microbial quality and delay lipid oxidation during catfish storage.

In a proof-of-concept study, an acidic (pH 5.8) biochar was created using a low pyrolysis temperature (350 °C) and steam activation (800 °C) to potentially improve the soil physicochemical status of an eroded calcareous soil. Biochar was added at 0%, 1%, 2%, and 10% (by wt.) and soils were destructively sampled at 1, 2, 3, 4, 5, and 6 month intervals. Soil was analyzed for gravimetric water content, pH, NO3-N, plant-available Fe, Zn, Mn, Cu, and P, organic C, CO2 respiration, and microbial enumeration via extractable DNA and 16S rRNA gene copies. Gravimetric soil water content increased with biochar application regardless of rate, as compared to the control. Soil pH decreased between 0.2 and 0.4 units, while plant-available Zn, Mn, and P increased with increasing biochar application rate. Micronutrient availability decreased over time likely due to insoluble mineral species precipitation. Increasing biochar application raised the soil organic C content and remained elevated over time. Increasing biochar application rate also increased respired CO2, yet the CO2 released decreased over time. Soil NO3-N concentrations significantly decreased with increasing biochar application rate likely due to microbial immobilization or denitrification. Depending on application rate, biochar produced a 1.4 to 2.1-fold increase in soil DNA extracted and 1.4- to 2.4-fold increase in 16S rRNA gene abundance over control soils, suggesting microbial stimulation and a subsequent burst of activity upon biochar addition. Our results showed that there is promise in designing a biochar to improve the quality and water relations of eroded calcareous soils.