Isolation and Characterization of Nitrate Reducing Bacteria for Conversion of Vegetable-Derived Nitrate to ‘Natural Nitrite’

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.

DNA Barcoding and Water Quality Analysis of Nitrifying Bacteria in Lebak Lebung Swamp, South Sumatera

“…The presence of ammonium-oxidizing bacteria is indicated by a change in the color of the media from red to yellow due to changes in the pH of the media due to the oxidation of ammonium to nitrite, while the presence of nitrate-producing bacteria is indicated by changes in the media. color from clear to cloudy [28,29]. Next, the bacterial colonies that grow on certain media in petri dishes are taken one by one and planted back in the dish to be purified by incubating at room temperature (27)(28)℃ in anaerobes conditions.…”

“…color from clear to cloudy [28,29]. Next, the bacterial colonies that grow on certain media in petri dishes are taken one by one and planted back in the dish to be purified by incubating at room temperature (27)(28)℃ in anaerobes conditions.…”

Screening for extracellular synthesis of silver nanoparticles by bacteria isolated from Al-Halfaya oil field reservoirs in Missan province, Iraq

“…To demonstrate the existence of the enzyme in different bacteria, the nitrate reductase test was performed according to Bhusal and Muriana (2021).…”

Comparison of Sodium Nitrite and ‘Natural’ Nitrite on the Inhibition of Spore Germination and Outgrowth of Clostridium sporogenes in Low- and High-Fat Frankfurters

“…One alternative that has been offered is 'natural nitrite' (i.e., microbially fermented vegetable-derived nitrate into vegetable nitrite). This was first initiated in the US in response to a product category labelled 'natural' foods whereby artificial ingredients are not allowed (e.g., sodium nitrite) [8,9]. The consumption of vegetables has been considered 'healthy', yet they contain high levels of nitrate that are converted to nitrite in the intestinal tract and are considered the source of >95% of consumer exposure to nitrite [8,10,11].…”

“…In a prior paper we identified nitrate-reducing bacteria that were capable of fermenting vegetable-derived nitrate to natural vegetable nitrite and discussed the regulatory status of natural nitrite in the US, which is different than in the EU [9]. In this study, the objectives were to characterize three strains of Clostridium sporogenes as surrogate microorganisms for C. botulinum/C.…”