Exploiting natural microbial diversity for development of flavour starters

W.L.P. Bredie and M.A. Petersen (Editors) Flavour Science: Recent Advances and Trends 9 2006 ElsevierB.V. All rights reserved.

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Exploiting natural microbial diversity for development of flavour starters Johan E.T. van Hylckama Vlieg, Annereinou Dijkstra, Bart. A. Smit, Wim J.M. Engels, Liesbeth Rijnen, Marjo J.C. Starrenburg, Gerrit Smit and Jeroen A. Wouters NIZO food research, P.O. Box 2 O, 6 710 BA Ede, The Netherlands

ABSTRACT The recent elucidation of many of the biochemical pathways involved in flavour formation in fermented food products has given an impetus to the food and ingredients industry to reshape their strain development programs. In this paper we highlight some of the latest developments and illustrate the prospects for starter culture development by exploitation of the diversity in flavour forming capacity among natural isolates. 1. I N T R O D U C T I O N Nowadays, fermentation is not only applied as a means of food preservation but special attention is paid to flavour development. Starter cultures, especially Lactic acid bacteria (LAB), play a crucial role in flavour formation and in recent years the understanding of the physiological characteristics that determine the production of flavour compounds has increased significantly. This is exemplified by the elucidation of the metabolic pathways involved in flavour formation in dairy products. The pathway of casein breakdown leading to the development of various key flavour and off-flavour compounds has drawn particular attention. Initially, caseins are converted to large peptides by rennet and microbial proteases. Most LAB produce an extensive set of peptidases that further degrade these peptides to smaller oligopeptides and amino acids that have desired (for example 'sweet' or 'brothy') taste or an undesired (for example 'bitter') taste. Finally, volatile flavour compounds are produced from amino acids by various enzymatic and non-enzymatic conversions. Several excellent reviews are available that summarise recent advances in research on the microbiology, biochemistry and molecular biology of flavour formation by lactic acid bacteria [ 1-3]. An important finding has been that a large diversity in desired enzyme activities occurs among natural strains. Consequently, high-performing starter cultures can be developed

62 by careful selection and combination of strains with desired activities. Recent technological breakthroughs in the field of automated screening and genomics allow the efficient exploitation of this large biodiversity. In such screening programs, miniaturised fermentations are carried out in 96-well format using robotics for liquid handling, keyenzyme activity measurement with colorimetric substrates, and analysis of flavour compounds with GC-TOF or HPLC-MS. Subsequently, strains exhibiting the desired activities are tested in product model systems and pilot product trials leading to the rapid identification of high-performing strains. The availability of automated screening platforms has given an incentive to the starter and food industries to reshape their strain development programs by targeting the key enzymes, genes and metabolites for these traits. In the current paper we will illustrate the power of this approach by highlighting an example of the development of starters that exhibit desired peptidase activities required for the removal of bitter off-flavours. 2. M A T E R I A L S A N D M E T H O D S

Enzyme activity tests were performed on 2 ml GMl7-grown cultures in 96-well 2 ml plates in quadruplet. Overnight-grown cultures were centrifuged and washed with 2 ml 50 mM sodium phosphate buffer pH 7.2. Subsequently, cells were disrupted using a mini bead-beater 96 Cell Disrupter (Merlin Diagnostic Systems, The Netherlands) with about 300 ~tl of 0.1 mm Zirconia / Silica (Merlin Diagnostic Systems BV, The Netherlands) and 1 ml 50 mM sodium phosphate buffer pH 7.2. The resulting suspension was centrifuged (10 min, 8300 g at 4 ~ and the supernatant containing the cell free extract was used to determine enzyme activities. All peptidase activities were determined in 96-well format at 30 ~ by online monitoring of the release of pnitroanilide from the following substrates: PepN, lys-p-nitroanilide; PepXP, H-Ala-Prop-nitroanilide; PepA, H-Glu-p-nitroanilide 3. RESULTS As described above, peptidases are key enzymes in the production of flavour compounds from casein in dairy fermentation. In order to assess the diversity of peptidase activities, we have quantified the activity of three peptidases, aminopeptidase N (PepN), X-Pro dipeptidyl-peptidase (PepXP) and glutamyl-aminopeptidase (PepA) in a strain collection of Lactococci. The collection contained three groups of strains, a group of dairy isolates belonging to the subspecies cremoris, a group of dairy isolates belonging to the subspecies lactis, and a group of wild strains belonging to the subspecies lactis. A miniaturised and automated screening procedure performed in a 96well plate format was used to grow the bacteria and quantify the peptidase activities in crude extracts (Figure 1). The results show that peptidase activities are highly strain dependent and that the average PepN activity in cremoris strains is approximately threefold higher than the average activity in dairy isolates of the subspecies lactis. Within the latter subspecies the activity in dairy isolates is two-fold higher than the activity in nondairy isolates. A similar pattern is observed with PepXP activities. PepA activities did

63 not correlate with subspecies or isolation source. The higher levels of peptidase activities in dairy isolates may reflect their adaptation to the dairy environment where these enzymes may provide effective access to the amino acids in dairy proteins. L. l a c t i s cremoris

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Figure 1. Diversity of aminopeptidase N (PepN), X-Pro dipeptidyl-peptidase (PepXP) and peptidaseXP and Glutamyl-aminopeptidase (PepA) peptidase activity among L. lactis subsp cremoris and L. lactis subsp, lactis grown in LM 17 medium. The power of the approach described for industrial strain development is exemplified by the development of a debittering starter culture. A bitter off-flavour may occur in many food fermentations and is often caused by unbalanced proteolysis resulting in the accumulation of certain hydrophobic peptides with a strong bitter taste. It has been shown that PepN produced by L. lactis is capable of degrading a bitter peptide that accumulates in bitter cheese [4]. Extensive screening programs have helped to identify strains that produce high levels of PepN. Strains that combine the high PepN activity with other beneficial characteristics have been shown to eliminate the bitter taste of certain cheese products. Some of these strains are currently marketed as commercial starter cultures.

64 4. C O N C L U S I O N S AND F U T U R E O U T L O O K In recent years the screening for flavour generating cultures has developed from a trialand error process, limited by lack of knowledge on the available cultures, to a process that efficiently screens strains for the desired combination of flavour forming enzyme activities. By operating different analytical techniques in an automated screening platform, the functionalities and flavour compounds that can be screened for are almost unlimited. It is now feasible for example to screen large numbers of strains for the production of a specific flavour compound [5] or true flavour profiles. Moreover, performing such screenings in matrices closely mimicking the product increases the predictive value of the strain selection process, thereby providing an effective means of valorising natural LAB diversity for starter culture development. References

1. J.E. Christensen, E.G Dudley, J.A Pederson and J.L. Steele, Antonie Van Leeuwenhoek Int. J. Gen. Moler Microbiol., 76 (1999) 217. 2. G. Smit, J.E.T. van Hylckama Vlieg, B.A. Smit, E.H.E. Ayad and W.J.M. Engels, Aust. J. Dairy Technol., 57 (2002) 61. 3. M. Yvon and L. Rijnen, Int. Dairy J., 11 (2001) 185. 4. P.S. Tan., T.A. van Kessel, F.L. van de Veerdonk, P.F. Zuurendonk, A.P. Bruins and W.N. Koning, Appl. Environ. Microbiol., 59 (1993) 1430. 5. B.A. Smit, W.J.M. Engels, J.T.M. Wouters and G. Smit, Appl. Microbiol. Biotechnol., 64 (2004) 396.