Conversion of Amino Acids to Short and Branched-Chain-Fatty Acids by Started and Adjunct Bacteria

Principal Investigator:

Dr. Bart C. Weimer, Utah State University

Co-Investigators:

none

Project Summary:

Short-chain fatty acids have a role in Cheddar cheese flavor development. The mechanisms responsible for their production in cheese are not established. Microbial lipases are involved, however, lactic acid bacteria typically possess extremely weak lipolytic ability and do not produce improved flavor, even with use of strains that over express the lipase (Holland et al., 1996). Alternatively, short chain fatty acids in Cheddar cheese may arise from microbial catabolism of branched chain amino acids. The aim of this study was to determine the ability of volatile fatty acid (VFA) production in lactococci associated with cheese processing in absence of carbohydrate and in carbohydrate starvation.

All bacteria tested produced VFA in laboratory conditions with diversity in genera and species in complex laboratory media. Strains of lactococci, lactobacilli, and brevibacteria convert amino acids to various VFA. Brevibacteria produced 10-100 times more VFA than the other bacteria tested. Studies linking carbohydrate starvation and amino acid metabolism demonstrated that some strains appeared to die by growth studies. However, brevibacteria and lactococci maintained the ability to produce increasing amounts of VFA in carbohydrate starvation conditions in a chemically defined media (CDM), despite an apparent decrease in viable cells. Isovaleric acid alone was produced by strains in CDM at pH 7, while no fatty acids were detected in CDM at cheese-like conditions. These studies indicate amino acids are converted to VFA by cheese related bacteria. Further, they indicate that the amount of sugar present regulates these metabolic processes.

NMR studies with radiolabeled amino acids demonstrated the interconversion occurs in these bacteria. Brevibacteria produced significantly more FFA from branched chain amino acids than lactococci. Lactococci produced FFA only after the onset of carbohydrate starvation.

Deletion mutants from J. Steele (Wisconsin) and M. Yvon (France) that lacked specific aminotransferase enzymes retained the ability to produce fatty acids from amino acids, but the type of VFA produced was different compared to the wild type. This indicates that multiple metabolic pathways exist for production of VFA in lactococci. This will allow a hypothesis for the catabolic pathway in cheese. Screening for VFAs' production from various amino acid precursors showed that genera involved are not diverse within the species but different between genera.

Another attempt to exclude the aminotransferase reaction was made by using precursor a-keto acids as substrates for in vivo catabolism to VFAs. The keto acids degraded in solution during incubation to VFAs. However, lactococci and lactobacilli produced VFAs above the observed background degradation. They also produced different products from what would be expected from a reaction subsequent to an aminotransferase in the catabolic pathway. This makes the role of an aminotransferase in VFA production more complicated than it would seem to be.

Lactococcus lactis ssp. lactis ML3 was inoculated into CDM (modified from Gao et al., 1997) at pH 7.2 and 5.2, containing 0.2% lactose. The cells metabolized the small amount of lactose to become carbohydrate starved for 6 months. ML3 entered the non-culturable state within 10 days of inoculation into CDM, but remained metabolically active and contained ATP. They eventually produced branched-chain fatty acids such as isovaleric and isobutyric acid. The lack of milk fat in CDM and associated fatty acid production during starvation implicates branched-chain amino acid catabolism during starvation as a plausible explanation for branched-chain fatty acid production. Monitoring the activities of aminotransferases in cell-free extracts lead to no patterns correlated patterns with branched-chain fatty acid production. Current work is underway to determine patterns of amino acid utilization and genes expressed associated with the catabolic pathways.

Use of keto acids (added as a reactant in an enzyme assay) was found in all strains tested. Lactococci are being used to determine the exact pathway for this conversion with NMR.

Publications:

Balasubramanian Ganesan, B.C. Weimer, K. Seefeldt, R. Koka, and B. Dias. 2002. Fatty acid production by lactococci, lactobacilli and brevibacteria. Submitted.

Theses:

none

Published Abstract:

Weimer, B. C., B. Ganesan, and K. Seefeldt. 2000. Volatile fatty acid production by starter and adjunct bacteria. International Dairy Federation Biennial Cheese Flavor Conference, Banff, Alberta.

Presentations:

Balasubramanian Ganesan and Bart Weimer. 2002. Fatty acid production by lactococci during carbohydrate starvation in a chemically defined medium. American Society for Microbiology, Salt Lake City, UT.