Spoilage potential, biofilm formation and blue pigment production by Pseudomonas paracarnis

Dairy product contamination with psychrotrophic microorganisms is a concern for the dairy industry. Pseudomonas spp. have been frequently associated with blue pigmentation on the surface of fresh cheeses in recent years, but the structure of this pigment has not yet been elucidated. Furthermore, the production of lipase and protease by the Pseudomonas genus has been studied for many years due to the importance of these enzymatic activities in food spoilage. In addition to the production of hydrolytic enzymes and pigments, this genus is also recognized for its capability of biofilm formation, which represents a great risk for the permanence of Pseudomonas in the industrial environment. The main goal of this work was to evaluate the spoilage potential, biofilm formation capacity, and blue pigment production of Pseudomonas isolated from spoiled cheese. The production of blue pigment by strains belonging to Pseudomonas carnis, Pseudomonas paracarnis, and Pseudomonas fluorescens species was screened in an in vitro approach. Metabolites produced by P. paracarnis A006 were identified using gas chromatography followed by mass spectrometry (GC- MS) after its solubilization and extraction. The influence of different cheese manufacturing parameters on the production of pigments in a cheese-mimicking matrix (mini-cheese) was assessed using Response Surface Methodology (RSM) for Box- Behnken design (BBD). The colorimetric analyses of mini-cheese were carried out to obtain color variations and validation of the RSM approach. The deteriorating potential of the pigmented (P. paracarnis - A006) and non-pigmented (P. fluorescens ATCC 13525) strains was evaluated in vitro and in situ (mini-cheese). Proteolytic and lipolytic activities were quantified using azocasein and p-nitrophenyl palmitate, respectively, as substrates. Its ability of biofilm formation was assessed by applying the crystal violet method. P. paracarnis A006 was selected as the best producer of blue pigment among the evaluated strains, but it was not possible to identify its pigment chemical structure using the GC-MS approach. However, another 114 metabolites were identified. RSM highlighted the use of starter culture containing Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis and Streptococcus thermophilus, in the cheese-making process, inhibits the multiplication of Pseudomonas. The inoculation of these lactic acid bacteria led to the inhibition of Pseudomonas growth, as well as the acidification of the cheeses reduced the production of blue pigment. The mathematical model defined by RSM determines that the absence of salt, a pH of 6.28 and an inoculum of 1.2 % of starter culture minimize the blue pigment production. The availability of nutrients, time and temperature of incubation interfere with proteolytic and lipolytic activity. P. paracarnis A006 and P. fluorescens ATCC 13525 showed proteolytic above 2.0 ∆A/mL.h, which demonstrate that both strains have a high proteolytic potential. Lipolytic activity of tested strains, like its proteolytic activity, is a strain-dependent characteristic and strongly affected by temperature and incubation time. The results of this work also revealed lower biofilm formation capacity over time at 25°C in both nutritional conditions, except for P. paracarnis A006 cultured in MMP. Eight out of nine genes located in the aprX-lipA operon, which encode genes related to the proteolytic and lipolytic activity, were identified in the genome of P. paracarnis A006 and P. fluorescens ATCC 13525. Therefore, defining cheese-making parameters is an interesting strategy to minimize the technological problems caused by Pseudomonas spp. Regarding this context, the RSM approach proved to be efficient. However, the chemical structure of the blue pigment produced by Pseudomonas spp. must be elucidated to have more information about the factors that can be controlled to minimize its production. Keywords: Response surface methodology. Mini-cheeses. Hydrolytic activity.