Cheddar cheese cooking temperature induces differential lactococcal cell permeabilization and autolytic responses as detected by flow cytometry: implications for intracellular enzyme accessibility

Abstract

Aims: To determine the influence of cheese cooking temperature on autolysis and permeabilization of two lactococcal starter strains in broth and in Cheddar cheese juice during ripening. Methods and Results: Flow cytometry (FCM) was used to identify and enumerate intact and permeabilized cells in broth and in Cheddar cheese juice. Levels of intracellular enzyme activities were quantified concurrently. Permeabilized cell numbers increased for both strains in broth following a temperature shift from 32 to 38°C and was accompanied by an increase in the level of accessible intracellular enzyme activities. The relative proportions of intact and permeabilized cell populations, as detected by FCM in cheese juice, changed during 42-day ripening. Permeabilized cell populations increased during ripening for both strains; however, an increase in accessible intracellular enzyme activity was observed only for the highly autolytic strain Lactococcus lactis AM2. Conclusions: Differences in the autolytic and permeabilization response induced by cooking temperature in two lactococcal strains affects intracellular enzyme accessibility in Cheddar cheese. Significance and Impact of the Study: This study highlights the importance of the autolytic and permeabilization properties of lactic acid bacteria starter strains and their impact on cheese ripening.

The growth, physiology and toxigenic potential of Bacillus cereus in cooked rice during storage temperature abuse

“…FCM has previously been employed to study the physiology of microbes in a variety of foodstuffs including milk (Gunasekera, Veal, & Attfield, 2003;Holm, Mathiasen, & Jespersen, 2004), drinking water (Pianetti et al, 2005), cheese (Sheehan, O'Loughlin, O'Cuinn, FitzGerald, & Wilkinson, 2005) and ground beef (Tortorello, Stewart, & Raybourne, 1998). Previously, the authors reported the use of FCM to demonstrate a heterogenous response by B. cereus endospores to simulated cooking regimes and refrigerated storage (Cronin & Wilkinson, 2008a data and provided a deeper insight into the physiological status of populations of B. cereus stored at various temperatures over the course of the 6 days incubation.…”

Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review

“…However, in many of the published studies few direct comparisons have been carried out between data obtained by FCM enumeration and that obtained by traditional plate counting. Sheehan et al (2005) noted strain related differences in the evolution of live, dead and permeabilised starter LAB strains in Cheddar cheese during ripening. FCM methodology involved the use of a SYTO9/PI stain combination with comparison of data from gates previously generated using a number of control populations.…”

“…This estimation of viability is based on the assumption that intact (PI negative) cells are always live/viable while PI positive cells are always dead/ damaged. However, most FCM profiles will yield a double positive population (PI and SYTO 9 positive) which may actually make up a very significant proportion of cells in sample e.g cheese during ripening (Sheehan et al, 2005;Yanachkina et al, 2016). The latter cells may have differing metabolic activities and may well reflect a VBNC state.…”

“…The main advantages of FCM for microbiology are: rapid assay times and data generation (1-2 min), high numbers of cells that can be analysed per sample (10,000 and upwards), minimal sample volume (from 5 μl), potential high throughput, multiplicity of stains available to examine various aspects of cell viability, structure and/or metabolism (multiparametric), and less labour and space required compared with conventional plating techniques (Wilkinson, 2016). Flow cytometry is used within microbiology in an expanding range of applications including; medical diagnostics (Aebisher, Bartusik, & Tabarkiewicz, 2017;Diaz, Herrero, Garcia, & Quiros, 2010), water treatment and supply quality monitoring (De Roy, Clement, Thas, Wang, & Boon, 2012;Egli & Kotzsch, 2015;Hammes et al, 2008), brewing fermentation monitoring (Achilles, Harms, & Müller, 2006;Bühligen et al, 2014;Overton, 2015), pharmaceutical, environmental testing (Herrero & Diaz, 2015), the dairy industry (Doolan, Nongonierma, Kilcawley, & Wilkinson, 2014;Pane, 2013;Sheehan, O'Loughlin, O'Cuinn, FitzGerald, & Wilkinson, 2005;Yanachkina, McCarthy, Guinee, & Wilkinson, 2016) and for manufacturers of probiotic cultures and products (Davis, 2014;Raymond & Champagne, 2015). The general industrial applications of FCM include rapid detection of pathogens, estimation of cell damage following processing steps such as heating, spray drying and freeze drying, and monitoring of bioprocesses (Diaz et al, 2010;Herrero & Diaz, 2015).…”

Flow cytometry applications in the food industry

“…FCM has been proven useful for measuring the lysis of cheese bacteria. Using the LIVE/DEAD BacLight bacterial viability kit, the lysis of Lactococcus lactis was monitored by counting the number of intact and permeable cells at diVerent time points [20,79]. FCM showed the presence of permeabilized cells at day 1 of cheese ripening.…”