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Microbial Stress Responses and Gene Expression during Aging of Cation-substituted full fat Cheddar cheese
Sodium replacement is a potential alternative to direct sodium reduction in foods wherein an equally salty cheese will attract consumers without the risk of high sodium intake. However bacteria in Cheddar cheese responsible for cheese flavor development may experience a different form of stress and respond differently when sodium is, for example, substituted by potassium. We are yet to understand how salt change is likely to alter flavor formation in Cheddar cheese. We proposed to study whether bacteria continue to experience salt stress even with sodium reduction or with use of different salt mixtures with less sodium in full fat Cheddar cheese containing different fat levels. We investigated how starter bacteria respond to stress from different salt combinations during ageing of Cheddar cheese. In order to study starter bacterial stress, gene expression studies were done by qPCR targeting six known salt stress-related genes (3 induced, 3 repressed by NaCl stress, based on literature) for starter lactococcal subspecies in cheeses with different salt combinations. Five cation combinations with different levels of sodium replacement – 100% Na (control with 2.2% total salt), 75:25 and 50:50 Na:K, 50:40:10 of Na:K:Ca, and low sodium (0.7% total salt) – were chosen for the analyses. Starter lactococci survived well in response to stress, with initial variations post salting due to cations diminished over aging (P<0.05). Initial responses of the dual enolase genes of lactococci matched known responses from other studies, with enoA being induced more by high salt and enoB repressed by high salt (P<0.05), respectively. Expression of dnaK, which encodes a molecular chaperone and is known to be induced immediately with acid and salt stresses, steadily plateaued and remained high (> 10^6 copies) throughout 6 mo of aging. Initial stress response to other cations varied between the 6 genes, with only the cheese substituted with 50:40:10 of Na:K:Ca exhibiting lower transcript induction (P<0.05) of all stress response genes. This indicates that calcium even at low levels plays a role in mitigating lactococcal stress. Notably, RNA extracted for gene expression directly from cheeses was devoid of any cross-contaminating genomic DNA, which confirmed that only transcripts were detected. The presence of detectable mRNA even after 6 mo of Cheddar cheese aging confirms that lactococci are still physiologically active independent of their exhibiting growth on media plates. This study presents a novel perspective on cation-controlled gene expression through cheese aging.
Keywords: lactococci, cation substitution, gene expression