according to the present invention , accelerated cheese ripening can be obtained by the use of carboxy - peptidases . the carboxy - peptidase preparation should be free from endo - protease activity , and should be able to at least release amino acids that are important for cheese flavour formation , such as leucine , phenylalanine , valine and methionine . the carboxypeptidase is added at activity levels between 1 and 2500 cpg / g substrate ( e . g . cheese milk ), preferably 1 - 250 cpg / g substrate or more preferably 1 - 25 cpg / g substrate . cpg - units are defined in example 1 . protease activity is measured by the hydrolysis of a casein ( 6 g / l assay solution ) at ph 6 . 0 , 4 ° c . for 1 hour . 1 pu is the amount of enzyme that produces , in one minute , a ( tca - soluble ) hydrolysate , which ( 280 nm ) absorbance is equal to a tyrosin solution of 1 μm ). the carboxypeptidase preparation is defined as free of endo - protease activity when the ratio of endo - protease activity ( pu )/ carboxypeptidase activity ( cpg ) in the preparation is less than 0 . 01 , preferably less than 0 . 001 and most preferably less than 0 . 0005 . the carboxy - peptidase is preferably a broad spectrum carboxy - peptidase that is able to release to majority of the natural amino acids from peptides or proteins . broad spectrum carboxypeptidase is defined as an enzyme that is able to release at least 80 % of the natural amino acids in amounts detectable by the method as described in example 3 of this application . preferably the carboxypeptidase preparation contains a carboxy - peptidase in which 90 % of the carboxypeptidase activity is caused by a single enzyme , measured as described in example 1 , but combinations of carboxypeptidases are also allowed . we have surprisingly found that the use of a purified carboxy - peptidase cpd i ( pepg ) from an aspergillus strain is well able to by its own accelerate cheese ripening . examples of suitable aspergilli are a . niger , a . oryzae and a . sojae . preferably cpd 1 from a . niger is used . the enzyme has been described ( dal degan , ribadeau - dumas & amp ; breddam , appl . environ . microbiol ( 1992 ) 58 , 2144 - 2152 ) and sequenced ( svendsen & amp ; dal degan , bioch . biophys . acta ( 1998 ) 1387 , 369 - 377 ). the carboxy - peptidase can also be used to accelerate cheese flavour development in emcs . other preferred applications of the carboxy - peptidase are in the field of flavour development in fermented foods like fermented sausages and beers in which , similar to the situation in cheese , availability of free amino acids like valine , leucine , isoleucine and phenylalanine as well as sulfur containing amino acids like methionine are known to be of particular importance . fig1 shows the activity profile in relation to the ph . the amino acid sequence of carboxypeptidase i ( pepg ) of a . niger is described ( svendsen & amp ; dal degan , bioch . biophys . acta ( 1998 ) 1387 , 369 - 377 ). degenerate pcr primers were designed to clone the pepg gene from a genomic library from aspergillus niger n400 ( cbs 120 . 49 ), using methods known to the skilled person in the art . the gene was fused to the 3 ′ end of the glucoamylase promotor . analogous examples of fusions of structural genes to the glucoamylase promotor have been described ( ep - a - 0420358 , ep - a - 0463706 and wo99 / 38956 ). first the pepg structural gene was pcr amplified from a genomic fragment containing the gene and purified . second , the promotor region of the glaa gene was pcr amplified using , at the 3 ′ end , a primer that overlaps the 5 ′ end of the pepg structural gene . third , the two pcr fragments were fused via fusion pcr with an oligonucleotide primer 5 ′ of the glaa promotor , and an oligonucleotide overlapping the stopcodon of pepg in the reverse direction . fourth , the resulting fusion fragment was cloned in the a niger expression vector pgbtop7 ( wo99 / 38956 ), resulting in a fusion plasmid containing the glaa promotor , pepg structural gene and the glaa terminator . this plasmid was digested with hindiii and co - transformed with pgbbaas - 1 digested with xho i to aspergillus niger iso502 , essentially as described in wo 99 / 38956 . transformants selected for growth on acetamide plates were analysed using colony pcr to check for the presence of pepg expression cassette , using known techniques . the gene sequence was determined , and the genomic dna sequence , coding sequence and corresponding amino acid sequence are given as seq id no . : 1 , 2 and 3 respectively . a . niger pepg transformants were cultivated in shake flask using methods as described previously ( wo 99 / 38956 ). after growth for 6 days at 34 ° c ., supernatants were analysed on activity . activity of pepg was determined by adding 10 μl of the culture supernatant to a 990 μl of a solution containing 45 mm na - acetate ( ph 4 . 5 ), 0 . 95 mm edta and 0 . 2 mm fa - phe - ala ( obtained from bachem ). the change in optical density at 337 nm was followed . the decrease in optical density is a measure for the pepg activity . one enzyme unit ( 1 cpg ) is defined as the amount of enzyme needed to decrease the optical density at 337 nm by 1 absorbance unit per minute under the test conditions . the transformant showing the highest cpg value per ml was selected for pepg expression . pepg was purified from the culture broth of aspergillus niger expressing the enzyme according to the method described ( dal degan , ribadeau - dumas & amp ; breddam , appl . environ . microbiol ( 1992 ) 58 , 2144 - 2152 ) with the exception that the cabs - sepharose step was omitted . the activity of the final substantially purified enzyme was established to be 150 cpg / ml , using the activity measurement as described in example 1 . endo - protease activity was below detection limits (& lt ; 0 . 6 pu / ml ). the production and purification of pepg were repeated yielding a final preparation containing 650 cpg / ml carboxy - peptidase activity and 2 . 25 pu / ml endo - protease activity . the ratio pu / cpg for the latter preparation was 0 . 003 . the substrate specificity of the purified pepg was determined using substrates z - ala - x , in which z is benzyloxycarbonyl and x is any of the amino acids ( one letter code ) a , d , e , f , g , h , i , k , l , m , n , p , q , r , s , t , v , w , y . all substrates were obtained from bachem , except when x = q or t , which substrates were obtained from pepscan ( the netherlands ) the enzyme specificity was determined at ph 4 . 0 and 40 ° c . in solutions that contained 3 mm of the peptide substrates . the reaction was started by addition of the 5 μl enzyme solution ( 440 units / ml ) to 95 μl of the reaction mixtures . samples were taken for each substrate immediately at t = 0 minutes and spotted on tlc - plates ( merck hptlc [ plates 20 × 10 silica gel 60 ), another sample was taken after 45 minutes incubation and also spotted on the same tlc plate . as a control , the substrate solutions without the enzyme were spotted on the same tlc plate . the plate was stained for free amino groups by spraying with a ready to use ninhydrin spray ( acros ). enzyme activity was rated from −( no activity ), +/− ( low activity ) to +( little activity ) to +++++ ( very high activity ). very high activity (+++++) on a particular substrate is scored when all the substrate has already been converted at the t = 0 sample . results are as follows : ref : data extracted from dal degan , ribadeau - dumas & amp ; breddam , appl . environ . microbiol ( 1992 ) 58 , 2144 - 2152 . the table shows that the cloned and purified pepg is similar to the one described by dal degan in 1992 , but there are some unexpected differences . the enzyme preferentially liberates the hydrophobic amino acids f , i , l , m and v . the preference of the cloned gene is , however , different from the one described by dal degan et al , which has highest preference for i whereas the cloned enzyme shows highest activity on l , m and v . also , the purified enzyme is rather active on k , more active than e . g . on a and d , which is different from the data described by dal degan et al . clearly , the carboxy - peptidase is has a very broad substrate specificity and is able to handle all amino acids except possibly c , which was not tested . miniature cheeses were produced as described by shakeel - ur - rehman et al . ( protocol for the manufacture of miniature cheeses in lait , 78 ( 1998 ), 607 - 620 ). raw cows milk was pasteurized by heating for 30 minutes at 63 ° c . the pasteurized milk was transferred to wide mouth plastic centrifuge bottles ( 200 ml per bottle ) and cooled to 31 ° c . subsequently , 0 . 72 ml of starter culture ds 5lt1 ( dsm gist b . v ., delft , the netherlands ) was added to each of the 200 ml of pasteurised milk in the centrifuge bottles and the milk was ripened for 20 minutes . than , cacl 2 ( 132 μl of a 1 mol . l − 1 solution per 200 ml ripened milk ) was added , followed by addition of the coagulant ( 0 . 04 imcu per ml ). in case the experimental involved the use of pepg , this enzyme was added together with the coagulant . the milk solutions were held for 40 - 50 minutes at 31 ° c . until a coagulum was formed . the coagulum was cut manually by cutters of stretched wire , spaced 1 cm apart on a frame . healing was allowed for 2 minutes followed by gently stirring for 10 minutes . after that , the temperature was increased gradually to 39 ° c . over 30 minutes under continuous stirring of the curd / whey mixture . upon reaching a ph of 6 . 2 the curd / whey mixtures were centrifuged at room temperature for 60 minutes at 1 , 700 g . the whey was drained and the curds were held in a water bath at 36 ° c . the cheeses were inverted every 15 minutes until the ph had decreased to 5 . 2 - 5 . 3 and were then centrifuged at room temperature at 1 , 700 g for 20 minutes . after manufacture the cheese were ripened at 12 ° c . and sensory analysis was performed after 3 and 6 weeks of ripening y a minimum panel of 3 people . several dosages of pepg to the cheese milk were used : 0 (= control ), 5 , 50 and 500 cpg / 200 ml cheese milk . the addition of pepg clearly led to an overall increase in flavour intensity , leading to a more matured taste as compared to the control cheese . this was the case at all levels of pepg addition , even though the lowest level of addition ( 5 cpg / 200 ml ) required 6 weeks ripening to give a clear effect . at the other doses ( 50 and 500 cpg / ml ) the effect on taste was already obvious after 3 weeks of ripening . the results clearly showed that pepg accelerates cheese flavour development , and that the effect was dose dependent , and that no off - tastes had developed . gouda cheeses were prepared in 200 l vats using the starter culture delvotec ® dx31d ( obtained from dsm ) and maxiren600 ( obtained from dsm ; 55 imcu / i milk ; dsm ) as the coagulant , using a standard gouda cheese manufacture protocol known to the person skilled in the art . raw milk was standardised to a casein to fat ratio of approximately 0 . 9 and pasteurised for 15 seconds at 72 ° c . pepg was added immediately prior to addition of maxiren at a level of 25 cpg / l . a control cheese was prepared from the same milk batch using the same manufacturing process but without addition of pepg . after coagulation the curd was cut and stirred for 20 minutes at a low speed . following this half the whey was replaced with warm water ( 40 % of initial volume ) to increase the temperature from 31 ° c . to 36 ° c . after this the curd / whey mixture was stirred , at an increasing speed , for another 30 - 40 minutes until the curd was firm enough for drainage . the curd was hooped and left for half an hour before being placed into 5 kg moulds . the cheeses were pressed , using increasing pressure , for around 4 hours after which the cheeses were left to rest overnight . the following morning they were placed into brine for 24 hours . after allowing the cheeses to dry , a protective coating was placed on the cheese and the cheeses started their ripening period . during this period they were kept at 15 ° c ., relative humidity of approximately 80 %. the cheeses were regularly turned and coated during the ripening period . cheeses were organoleptically assessed after 6 weeks and 3 months of ripening by using a trained panel consisting of a minimum number of 8 people . after 6 weeks , the cheese containing pepg received significantly higher scores for sweetness as compared to the control cheese . after 3 months of ripening , the cheese containing pepg was clearly different from the control cheese , showing significantly higher flavour ( and also clearly different creaminess . the cheese flavour of the cheese containing pepg was pleasant and more mature , as compared to the control cheese . the experiment clearly showed that pepg accelerates the cheese flavour development and that no off - tastes developed . the enzyme reaction was determined in buffers of various ph . the buffer at ph 2 , 3 and 4 contained 0 . 1m sodium phosphate , 0 . 05m citric acid and 0 . 05m acetic acid ; the buffer at ph 4 , 5 and 6 contained 0 . 05 m sodium phosphate , 0 . 05m acetic acid , and 0 . 05m tris . the ph was adjusted to the correct value using 4m hcl or 4m naoh . substrate solution contained 8 mm fa - phe - ala in methanol . the assay solution contained 965 μl buffer , 25 μl substrate solution and 10 μl purified enzyme . reactions were performed at 25 ° c ., and change of absorption at 337 nm was followed for 10 minutes . the relative activity was calculated fomrt he change in absorbance . the results are the average of two separate measurements , except for the measurement at ph is 4 , which was performed in duplo in two different buffers . the results are given in fig1 . the profile shows of fig1 that the enzyme has a ph optimum of ph 4 . the optimum is clearly higher than ph optimum of 3 . 1 - 3 . 4 for the carboxy peptidase described by dal degan ( dal degan , ribadeau - dumas & amp ; breddam , appl . environ . microbiol ( 1992 ) 58 , 2144 - 2152 and references sited therein ). a cheese paste was prepared from a mixture of 90 % 5 weeks old gouda cheese and 10 % old gouda cheese , basically as described by smit et al . (( 1995 ). ch - easy model : a cheese - based model to study cheese ripening . in p . etievant , bioflavours . ( pp . 185 - 190 ). young gouda cheese ( approximately 6 weeks old ) was purchased from a local supermarket , and was finely grated . milliq - water was added to the grated cheese to reach a final water content of approximately 50 % and , after mixing , the cheese paste was divided in portions of 200 grams in separate containers . the mixture was heated during 5 minutes at 80 ° c . to eliminate microbial growth . one of the containers was analyzed to verify elimination of microbial growth by plate count analysis for bacteria , yeasts and moulds . the other containers were stored at 4 ° c . for further use . when absence of microbial growth was confirmed by the plate count analysis , the prepared cheese pastes in the remaining containers were used . prior to making additions , the cheese paste was heated to 55 ° c . and gently cooled to 30 ° c . pepg solutions were prepared in milliq water containing 1 . 6 , 0 . 16 and 0 . 016 cpg / ml . subsequently , 2 ml of each pepg solution was added to individual containers containing 200 g cheese paste , and the mixture was mixed by stirring ; the control paste contained no pepg but only milliq the containers were than stored at 17 ° c . after 4 weeks the flavour of the pastes was organoleptically assessed by a sensory panel . increasing pepg concentrations resulted in clearly increasing intensity of flavour of the cheese pastes . the pepg is apparently useful in emc processes to generate cheese flavour .
the present invention relates to a process for the flavour development in a fermented food whereby a carboxypeptidase is used .