FFoorrmmaattiioonn ooff AAmmiinnoo AAcciidd DDeerriivveedd CChheeeessee FFllaavvoouurr CCoommppoouunnddss Bart A. Smit Promotoren: Prof. dr. J.T.M. Wouters Hoogleraar Zuivelkunde, Wageningen Universiteit Prof. dr. G. Smit Hoogleraar in “Molecular Flavour Sciences”, Wageningen Universiteit Co-Promotor: Dr. W.J.M. Engels Projectleider, NIZO food research, Ede. Promotie-commissie: Prof. dr. O.P. Kuipers Rijksuniversiteit Groningen Prof. dr. W.M. de Vos Wageningen Universiteit Prof. dr. ir. M.A.J.S. van Boekel Wageningen Universiteit Dr. J. Sikkema Friesland Coberco Dairy Foods, Deventer. FFoorrmmaattiioonn ooff AAmmiinnoo AAcciidd DDeerriivveedd CChheeeessee FFllaavvoouurr CCoommppoouunnddss Bart A. Smit Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, prof. dr. ir L. Speelman, in het openbaar te verdedigen op vrijdag 23 april 2004 des namiddags te vier uur in de Aula. Smit, B.A. Flavour Formation from Amino Acids in Fermented Dairy products. Thesis Wageningen University, The Netherlands, 2004 - with summary in Dutch. ISBN 90-5808-996-7 Key words: Lactic acid bacteria, Lactococcus lactis, amino acid, leucine, flavour, aroma, aldehyde, 3- methylbutanal, 2-methylpropanal, dairy fermentation, cheese. CCoonntteennttss Chapter 1. General introduction................................................................................7 Chapter 2. Diversity of leucine catabolism in various micro-organisms involved in cheese ripening and identification of the rate controlling step in 3-methylbutanal formation .....................................33 Chapter 3. Development of a high throughput screening method to test flavour-forming capabilities of anaerobic micro-organisms ................47 Chapter 4. Non-enzymatic conversion of α-keto acids in relation to flavour formation in fermented foods................................................................61 Chapter 5. Identification, cloning and characterisation of a branched-chain α-keto acid decarboxylase from Lactococcus lactis involved in flavour formation ..................................................................................75 Chapter 6. Discussion and concluding remarks......................................................89 Summary..................................................................................................................101 Samenvatting...........................................................................................................105 References ...............................................................................................................111 Nawoord..................................................................................................................123 List of publications..................................................................................................125 Curriculum vitae......................................................................................................127 CChhaapptteerr 11 General introduction Formation of Amino Acid Derived Cheese Flavours 7 Chapter 1. 1.1. Abstract Fermentation of food can contribute greatly to its nutritional value, perception and texture besides to longer and safer use. Especially nutritious foods like milk are often fermented to a wide variety of products by various micro-organisms. Lactic acid bacteria like Lactococcus lactis are often used for the fermentation of milk into various cheeses. Most lactic acid bacteria convert lactose into lactic acid, thereby lowering the pH. This decrease in pH, together with the addition of salt and the removal of water results in a stable product. Besides production of acid, micro-organisms like lactic acid bacteria often produce other metabolites from milk sugar, protein and fat. The reactions primary take place for own maintenance and growth, but the resulting low molecular compounds have sometimes strong flavour characteristics. The most dominant flavour compounds (key-flavours) can be isolated and identified. Their subsequent recombination usually results in a good impression of the original product-flavour. Many key flavours in semi-hard cheeses like Gouda, are derived from protein degradation. The biochemical conversion from protein to flavour compounds starts with proteolysis and peptidolysis, which results in free amino acids. These can be converted in several ways, leading to a large variety of (flavour) compounds. This thesis focuses on the production of one class of these compounds as model system: aldehydes, in particular the key-flavour 3-methylbutanal, which are derived from the amino acid leucine. Only few lactic acid bacteria are known to produce considerable amounts of this aldehyde. The aim of this research is to gain understanding on the formation pathway of this kind of flavour compounds, in order to improve the control of flavour development during fermentation. Targeted strain selection is one of these tools, which might open opportunities for application in flavour diversification and/or acceleration of the cheese ripening process. Key-words Fermentation, lactic acid bacteria, Lactococcus, flavour formation, protein, amino acids. 8 Smit, B.A. Introduction 1.2. Fermented dairy products Milk and dairy products are an important part of a well balanced diet all over the world. Milk contains many nutrients, for nursing and sustaining a newborn animal. The high nutritious value of milk makes it sensitive to spoilage by bacteria when keeping it for longer times. In some cases (controlled) spoilage, fermentation, leads to a more stable product, often due to the production of conserving acids by lactic acid bacteria. More generally fermentation can be defined as the change of the properties of a raw material by micro-organisms or enzymes to improve its safety, shelf life, nutritious value or perception (Steinkraus, '94). Major factors to achieve a well- controlled fermentation process are the use of only selected, well-characterised micro-organisms, the pre-treatment of raw material, and the control of process parameters such as temperature, pH, mixing intensity etc. Examples of the very diverse group of fermented dairy products are cheese, butter, buttermilk, kefir, yoghurt and quarg (labneh). Other products derived from dairy fermentations are lactic acid and whey, which are applied as food and non-food ingredients. The long history of the first fermented dairy products with an improved shelf life has presumably started together with the domestification of animals. These fermented milks most probably resembled products like yoghurt. Although the appearance of these products did not change very much over time, the focus on yoghurt fermentations has changed from preservation to improvement of characteristics like texture, flavour and health promoting activity. The latter focus started around the 1900’s, when Metschnikoff studied the health effects of fermented milks, in relation the longer life expectance of people on the Balkan (Metschnikoff, '08). Nowadays several types of yoghurts have been developed with an improved health imago, structure and flavour. By removing a part of the water phase, the dry matter content and the appearance of the fermented milk changed largely to a more or less cheese-like product. The earliest records of cheese making are probably from the ancient Egypt and from around 300 BC also more textured cheese-like products, like Domiati had become established (Robinson, '95). Due to regional characteristics, like climate, distinctive varieties of cheese emerged. For example, it appears that there is a distinct relationship between climate and the bacteria associated with the fermented product of that region. In northern Europe, mainly mesophilic bacteria have been used, and in warmer climates the major part of the flora consisted of thermophiles. Later, in the Middle Ages, human activities played a dominant role in the development of cheese varieties. Transporting cheese from production area to large conurbations and trading it there demanded cheese with a longer shelf life. Therefore harder and drier cheeses became common, although soft cheeses retained their position in some areas. The advent of refrigeration and fast, reliable transport changed this pattern again, and especially in the USA soft cheeses claim a major part of the Formation of Amino Acid Derived Cheese Flavour Compounds 9 Chapter 1. market. Nowadays, a large range of cheese types is available and, from an economical point of view, cheese is the most important fermented milk product. In Table 1.1 some popular cheeses are categorised based on hardness and use of an additional culture (Ridgway, '99). The most common categorisation of cheese is based on their water content, which can be noticed by the hardness of the cheese. The main source of bacteria in these modern cheese varieties is the primary starter, which is a mixture of selected lactic acid bacteria (LAB). In addition to this starter, some other micro-organisms (secondary starter) might be present in the cheese, and these might add largely to the product characteristics of the cheese. The introduction of new microbial strains in cheese making is a powerful tool to change product characteristics. In Gouda cheeses produced with non-dairy LAB (wild strains) large differences in flavour formation were observed (Ayad ea., '00). The background of the formation of these differences however is still unclear. Table 1.1 Some major types of cheese, categorised on hardness and secondairy starter used. (Edited after Ridgway, '99) Category Secondary starter Examples Country Extra Hard - Parmesan Italy Grana Padano Italy Hard - Cheddar England Manchego Spain Edam The Netherlands Semi-Hard - Gouda The Netherlands Port Salut France Eye formers Emmental Switzerland Maasdam The Netherlands Gruyère Switzerland Surface bacteria Limburger Germany (smear) Tilsit Germany Kernhemmer The Netherlands Soft Cottage cheese USA Fromage frais France Mozerella Italy Feta Greece Mould-veined Danish blue Denmark (blue) Gorgonzola Italy Roquefort France Surface mould Brie France (white) Camembert France 10 Smit, B.A.