Abstract
In the US, sodium nitrate is used as a preservative and curing agent in processed meats and is therefore a regulated ingredient. Nitrate reducing bacteria (NRB) can convert vegetable nitrate into nitrite allowing green/clean label status in the US as per the USDA-FSIS definition of ‘natural nitrite’. The current ‘in-liquid’ test tube assay for detecting nitrite is not suitable for screening mixtures of bacteria nor is commercial nitrate broth suitable for growth of many Gram (+) bacteria. M17 broth was therefore used to develop M17-nitrate broth to be inclusive of Gram (+) bacteria. An ‘on-agar’ colony-screening assay was developed to detect the conversion of nitrate to nitrite on agar plates and could detect one NRB+ colony among ~300–500 colonies on a single plate. Samples that might have NRB were spread-plated on M17 agar plates, sandwiched with nitrate agar, and after incubation followed with sequential agar overlays containing the reagents used in the nitrate reduction assay; the appearance of red color zones above colonies indicated the presence of nitrite. NRB derived from various samples were confirmed for nitrate conversion and both nitrate and nitrite were quantified by C8 reversed-phase (RP) ion-pairing high performance liquid chromatography (HPLC) analysis (1 ppm limit of detection). Staphylococcus carnosus, a strain commonly used for nitrate reduction, was able to convert 1100 ppm M17-nitrate broth to 917 ppm nitrite. Staphylococcus caprae and Panteoa agglomerans, NRB isolated using the M17-nitrate agar assay, were also able to ferment the same broth to 916 ppm and 867 ppm nitrite, respectively. This is the first report of an on-agar colony screening assay for the detection and isolation of nitrite reducing bacteria allowing NRB to be readily isolated. This may allow for the identification of new bacteria that may have a more efficient process to generate nitrite, and possibly concomitant with production of additional natural antimicrobials, as vegetable nitrite becomes more widely used to prevent spore germination.
Highlights
In the US, sodium nitrate is used as a preservative and curing agent in processed meats and is a regulated ingredient
Nitrate reducing bacteria (NRB) were spread-plated on M17 agar plates, sandwiched with nitrate agar, and after incubation followed with sequential agar overlays containing the reagents used in the nitrate reduction assay; the appearance of red color zones above colonies indicated the presence of nitrite
NRB derived from various samples were confirmed for nitrate conversion and both nitrate and nitrite were quantified by C8 reversed-phase (RP) ion-pairing high performance liquid chromatography (HPLC) analysis
Summary
Active cultures were grown in tryptic soy broth (TSB, BD Bacto, Franklin Lakes, NJ, USA) in 9-mL tubes at 37 ◦ C. Bacteria were cultured overnight in nitrate broth (HiMedia Labs, Mumbai, India) at 37 ◦ C and tested for the presence of nitrite. If there is no color change, either nitrate remains unreduced and is determined by the addition of zinc powder which is a strong reducer that will reduce nitrate to nitrite to turn the culture red, confirming there was unreduced nitrate in the tube. If there is still no color change with the addition of zinc powder, the only explanation is that nitrate was reduced to nitrite, and further reduced to other nitrogen compounds (Figure 1): a. The nitrate reduction assay using individual broth cultures was slow and laborious The nitrate reduction assay using individual broth cultures was slow and laborious for screening potential NRB from food/animal samples. A plain agar layer containing addition of nitrate reagent A [19,20] and thin agar overlays for overlaysoffor recovery of injured bacteria recovery of injured bacteria [21,22]
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