|Title||Formation of Amino Acid Derived Cheese Flavour Compounds|
|Source||Wageningen University. Promotor(en): J.T.M. Wouters; Gerrit Smit, co-promotor(en): W.J.M. Engels. - Wageningen : S.n. - ISBN 9789058089960 - 127|
Product Design and Quality Management Group
|Publication type||Dissertation, externally prepared|
|Keyword(s)||geurstoffen en smaakstoffen - aldehyden - kazen - melkzuurbacteriën - aminozuren - aroma - fermentatieproducten - flavour compounds - aldehydes - cheeses - lactic acid bacteria - amino acids - aroma - fermentation products|
|Categories||Dairy Science / Flavours|
|Abstract||Lactic acid bacteria (LAB), among them Lactococcus lactis, are often used for the fermentation of milk into various products, such as cheeses. For their growth and maintenance LAB metabolise milk sugar, protein and fat into various low molecular compounds, which sometimes have strong flavour characteristics. This thesis focuses on the production of one class of these compounds as a model system: aldehydes, in particular the key-flavour compounds 3-methylbutanal and 2-methyl propanal, which are enzymatically derived from the amino acids leucine and valine. Comparing several strains belonging not only to the genus Lactococcus , but also to the genera Lactobacillus , Streptococcus , Leuconostoc , Bifidobacterium , Propionibacterium , Brevibacterium , Corynebacterium and Arthrobacter with regard to various enzyme activities involved in the conversion pathway of leucine to 3-methylbutanal, revealed a large variation between and within species. In particular,a-keto acid decarboxylase activity, leading to 3-methylbutanal was identified as a rate-limiting step in this pathway.
Identification of thea-keto acid decarboxylase gene was obtained by screening of mutant libraries of the decarboxylase-positive strain L. lactis B1157 with a newly developed method for high throughput analysis (HTS) of volatiles, present in the headspace of miniaturised fermentations. The gene had a very high homology with a (most probably) truncated gene in the genome of L. lactis IL1403 annotated as ipd . The molecular weight of the TPP dependant enzyme was 60.9 kDa, its estimated pI is 5.03. The enzyme activity was hardly affected by high salinity and activity was found to be optimal at pH=6.3. Moreover, of all potential substrates tested, the activity towards branched-chaina-keto acids was the highest, for which reason it is proposed to be annotated as BcKAD (branched-chain keto acid decarboxylase).
The newly developed method for analysis of volatiles was also applied for monitoring the non-enzymatic conversion of the leucine-derived keto acid to the branched chain aldehyde 2-methylpropanal. 2-Methylpropanal is the aldehyde, which was believed to be a product of the enzymatic conversion of valine. The non-enzymatic reaction proceeded in the presence of manganese ions and oxygen, the products were the aldehyde and either carbon oxides or oxalate. The conversion was maximal at pH 5.5 and pH>8. A range ofa-keto acids were converted under the sameconditions,and keto acids with an electron-pulling side chain had the highest conversion rates.
The results described in this thesis add to the understanding of the control of flavour formation in fermented products like cheese. The thesis focuses mainly on the existence and relevance of some of the amino acid converting pathways of certain bacterial strains. In addition to this, further research on regulation and/or complementation of these pathways in/by several strains is recommended. The HTS-method also enables the selection of strains from large culture collections for production of certain volatiles and for application of this property in cheese making.