Measurement of pH micro-heterogeneity in natural cheese matrices by fluorescence lifetime imaging

Abstract

Cheese, a product of microbial fermentation may be defined as a protein matrix entrapping fat, moisture, minerals and solutes as well as dispersed bacterial colonies. The growth and physiology of bacterial cells in these colonies may be influenced by the microenvironment around the colony, or alternatively the cells within the colony may modify the microenvironment (e.g., pH, redox potential) due to their metabolic activity. While cheese pH may be measured at macro level there remains a significant knowledge gap relating to the degree of micro-heterogeneity of pH within the cheese matrix and its relationship with microbial, enzymatic and physiochemical parameters and ultimately with cheese quality, consistency and ripening patterns. The pH of cheese samples was monitored both at macroscopic scale and at microscopic scale, using a non-destructive microscopic technique employing C-SNARF-4 and Oregon Green 488 fluorescent probes. The objectives of this work were to evaluate the suitability of these dyes for microscale pH measurements in natural cheese matrices and to enhance the sensitivity and extend the useful pH range of these probes using fluorescence lifetime imaging (FLIM). In particular, fluorescence lifetime of Oregon Green 488 proved to be sensitive probe to map pH micro heterogeneity within cheese matrices. Good agreement was observed between macroscopic scale pH measurement by FLIM and by traditional pH methods, but in addition considerable localized microheterogeneity in pH was evident within the curd matrix with pH range between 4.0 and 5.5. This technique provides significant potential to further investigate the relationship between cheese matrix physico-chemistry and bacterial metabolism during cheese manufacture and ripening.

Application of advanced light microscopic techniques to gain deeper insights into cheese matrix physico-chemistry

“…Moreover, the fluorescence lifetime of many fluorescent dyes is a sensitive indicator of local chemical properties, such as ion concentration, pH, oxygen concentration or presence of other fluorescent molecules (Lakowicz 2006). Consequently, FLIM can not only be used to distinguish different fluorophores on the basis of their characteristic lifetimes (rather than their spectral properties) but also to distinguish among different environments based on changes in lifetime of the same fluorophore, for example local pH within the cheese matrix (Burdikova et al 2015). Fluorescence lifetime microscopy can also provide additional valuable information in fat composition studies when combined with various fluorescent dyes (e.g.…”

Bacterial Colonies in Solid Media and Foods: A Review on Their Growth and Interactions with the Micro-Environment

“…These consistent results demonstrate that the diffusion of lactic acid was not the limiting factor for growth neither in gelatine nor in a model cheese containing colonies which radius was smaller than 150 μm. Furthermore, in ripened commercial Cheddar cheeses, pH microgradients have been observed at the microscopic scale of a few μm using the fluorescence life-time (FLIM), but not especially around colonies (Burdikova et al, 2015 ). The accumulation of lactic acid around the colonies has been suggested as the main explanation for the lower growth rate in renneted milk gels when compared with that in liquid milk (Stulova et al, 2015 ).…”

“…The accumulation of lactic acid around the colonies has been suggested as the main explanation for the lower growth rate in renneted milk gels when compared with that in liquid milk (Stulova et al, 2015 ). The simplified composition (no fat, no NaCl) and the homogeneous structure of the model cheese (Jeanson et al, 2013 ) may explain the non-accumulation of lactic acid around small colonies whilst in commercially available cheeses (Burdikova et al, 2015 ), lactic acid concentration may vary at the microscopic scale because of a more heterogeneous microstructure.…”

Probiotics– the journey continues

“…Sensitive fluorescence lifetime imaging techniques have been used to examine the cheese matrix at a microscopic scale (Burdikova et al . ), revealing localised variations in pH levels within the cheese.…”