Natural diversity of aminotransferases and dehydrogenase activity in a large collection of Lactococcus lactis strains J.B. Brandsma a, * , E. Floris b , A.R.D. Dijkstra b , L. Rijnen c , J.A. Wouters b , W.C. Meijer a a CSK food enrichment, P.O. Box 225, 8901 BA Leeuwarden, The Netherlands b NIZO food research, P.O. Box 20, 6710 BA Ede, The Netherlands c 620, Chemin des Hauts de Nı ˆmes, Villa Jardin 11, 30900 Nı ˆmes, France article info Article history: Received 31 December 2007 Received in revised form 10 June 2008 Accepted 11 June 2008 abstract The natural diversity of Lactococcus lactis was studied with respect to the flavour-generating enzymes leucine (Leu-AT) and aromatic (AraT) aminotransferase, and hydroxyisocaproic acid dehydrogenase (HicDH). The screening of 175 L. lactis strains required the development of enzyme assays to enable the use of high throughput screening facilities. Natural diversity was expressed as the difference between 10% of the strains with the highest and lowest enzyme activity. The natural diversity found for Leu-AT, AraT and HicDH activity was 13-, 49- and 30-fold, respectively. Leu-AT activity showed a weak correlation with the AraT and HicDH activity, whereas there was no correlation between AraT and HicDH activity. In the group of strains with the highest Leu-AT activity, 5- and 6-fold differences of AraT and HicDH activities were found, respectively. These results demonstrated that it is possible to select strains with specific combinations of important ripening enzymes that could enhance cheese flavour formation. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Soluble proteins released from casein by the action of chymosin and plasmin are degraded by lactic acid bacteria (LAB) and play a major role in the flavour development of matured cheese. With a complex set of proteolytic and peptidolytic enzymes, LAB are able to degrade the released proteins in several steps from oligopeptides via di- and tripeptides to, finally, amino acids (Baankreis, 1992; Kunji, Mierau, Hagting, Poolman, & Konings, 1996). Amino acids are converted by aminotransferases to a-ketoacids that can be degraded by enzymatic or non-enzymatic reactions to flavour- generating compounds (Alting, Engels, van Schalkwijk, & Exterkate, 1995; Engels et al., 2000; Rijnen et al., 2003; Smit et al., 2004; Yvon, Thirouin, Rijnen, Fromentier, & Gripon, 1997). The typical mature flavour of Gouda and Cheddar cheeses depends on the degradation of branched chain amino acids (BcAA) and methionine (Engels et al., 2000; Weimer, Seefeldt, & Dias, 1999). The a-ketoacids derived from aromatic amino acids (ArAA) are converted to floral generating flavours (Rijnen et al., 1999) and are generally considered as off-flavours in these types of cheeses (Broadbent et al., 2004). As well as the formation of flavour- generating compounds, a-ketoacids can also be converted to compounds that do not contribute to flavour (Yvon & Rijnen, 2001). The enzymatic conversion of a-ketoacids originating from BcAA and methionine towards hydroxy acids is especially considered to be unwanted. An important enzyme in this respect is hydrox- yisocaproic acid dehydrogenase (HicDH), which converts a-ketoa- cids from BcAA, methionine and also ArAA into hydroxy acids (Hummel, Schutte, & Kula, 1985). Broadbent et al. (2004) suggested the use of strains with high HicDH activity to prevent the formation of floral off-flavours in Cheddar. Unfortunately, in cheese experi- ments, it was demonstrated that the overall flavour development was less appreciated, probably due to the reduction of a-ketoacids originating from BcAA and methionine. It was demonstrated by Rijnen et al. (2003) that BcAA, methionine and ArAA degradation can also be influenced in cheese by Lactococcus strains with different relative activities of branched chain (BcaT) and aromatic aminotransferase (AraT). This suggests that the aroma profile and aroma intensity of cheese can be manipulated by selecting Lacto- coccus strains with specific combinations of BcaT, AraT and HicDH activities. Therefore, such work emphasizes the relevance of studying the biodiversity and correlation of these enzymes in L. lactis strains. The natural diversity of proteolytic enzyme activities in Lacto- coccus strains has been studied mainly in a limited number of strains at the level of proteinases and peptidases. Coolbear, Pillidge, and Crow (1994) found that the total proteinase activity in L. lactis subsp. cremoris and subsp. lactis varies with a factor of 8- and 15- fold, respectively. In the same set of Lactococcus strains, the diver- sity of six peptidases was also determined. The variation in levels of * Corresponding author. Tel.: þ31 582844242; fax: þ31 582844210. E-mail address: h.brandsma@cskfood.com (J.B. Brandsma). Contents lists available at ScienceDirect International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj 0958-6946/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2008.06.004 International Dairy Journal 18 (2008) 1103–1108