Proteolysis and Volatile Components of Reduced-Fat Cheeses Made from Ultrafiltered Milk and Different Starters Supplemented With

Proteolysis and volatile components of reduced-fat cheeses made from ultrafiltered milk and different starters supplemented with lactobacilli and Lac-Prt-lactococci J Rodriguez, T Requena, E Valero, Non Renseigné, R Lopez-Fandino, M Juarez

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J Rodriguez, T Requena, E Valero, Non Renseigné, R Lopez-Fandino, et al.. Proteolysis and volatile components of reduced-fat cheeses made from ultrafiltered milk and different starters supplemented with lactobacilli and Lac-Prt-lactococci. Le Lait, INRA Editions, 1997, 77 (6), pp.717-728. hal- 00929558

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Original article

Proteolysis and volatile components of reduced-fat cheeses made from ultrafiltered milk and different starters supplemented with lactobacilli and Lac-Prt- lactococci

J Rodriguez l, T Requena 1*,E Valero 2, 1 Martfnez-Castro 2, R Lépez-Fandifio 3, M Juarez 1

1 lnstituto dei Frio (CSlC), Ciudad Universitaria, 28040 Madrid; 2 lnstituto de Quimica Orgânica General (CSlC); 3 lnstituto de Fermentaciones lndustriales (CSlC), Juan de la Cierva 3, 28006 Madrid, Spain

(Received 20 January 1997; accepted 12 May 1997)

Summary - Proteolysis and volatile components of serni-hard cheeses manufactured from a mixture of cow's, ewe's and goat's milk, with reduced fat content and concentrated by ultrafiltration, were folJowed during ripening. A starter culture (IFPL), consisting of several strains of Lactococcus, Lac- tobacillus and Leuconostoc, selected previously for its high proteolytic activity, and the same starter with the addition of a concentrate of Lactococcus lactis subsp lactis TI, lacking the ability to utilize lactose and casein (Lac Prr), were tested with the aim of accelerating cheese ripening as compared with a commercial starter (FD). Casein hydrolysis was not affected by the type of starter culture employed, whereas appreciable differences were found in the non-volatile water-soluble nitrogen fraction. The peptide pattern allowed a clear distinction among cheeses made with different bacterial cultures. IFPL cheeses exhibited, after 1 month of ripening, a higher content of total free amino acids than FD cheeses after 3 months of ripening. Furthermore, the addition of L lactis TI to the IFPL culture noticeably increased the release of total free ami no acids during ripening, as well as the content of total volatile compounds. reduced-fat cheese / proteolysis / starter / volatile component / LacrPrt" lactococci

Résumé - Protéolyse et composés volatils de fromages à teneur réduite en matière grasse pré- parés avec du lait ultrafiltré et différents levains additionnés de lactobacilles et de lactocoques Lac-Prt-. La protéolyse et les composés volatils ont été suivis pendant l'affinage de fromages à teneur reduite en matière grasse, préparés à partir d'un mélange de lait (vache, brebis, chèvre) ultra- filtré. Pour accélérer l'affinage, un levain (IFPL) contenant plusieurs souches de Lactococcus, Lac- tobacillus et Leuconostoc (préalablement sélectionnés pour leur activité protéolytique élevée), et le

* Correspondence and reprints 718 J Rodrfguez et al même levain additionné d'une souche de Lactoccocus lactis subsp lactis Tl (LacPrc), ont été com- parés avec un levain commercial (FD). Le type de levain employé n'a pas montré d'effet sur l'hydro- lyse de la caséine, mais semble entraîner des différences dans la fraction azotée hydrosoluble non volatile. Les profils peptidiques des fromages préparés avec les trois levains étaient différents et très caractéristiques. La teneur en acides aminés des fromages préparés avec le levain IFPL après seule- ment un mois d'affinage était plus élevée que celle des fromages préparés avec le levain FD après 3 mois. L'addition de la souche L lactis TI augmentait la production des acides aminés pendant l'affinage. Les fromages préparés avec cette souche avaient aussi la plus haute teneur en composés volatils (alcools, cétones, esters et dérivés soufrés). fromage à matière grasse réduite/ protéolyse/ levain/ composé volatil/ lactocoque LacrPrt"

INTRODUCTION toc, which has been proved to be suitable for the manufacture of semi-hard goat's milk Demand for low-fat cheeses has increased cheese (Requena et al, 1992). A Lactococcus during the past few years, as a consequence strain from this starter was plasmid cured of the consumers' requirements for low calo- in order to reduce its proteolytic activity rie and low cholesterol foods. Unfortuna- (Pre) and capacity to metabolize lactose tely , the reduction in the fat content impairs (Lac"), keeping its peptidolytic activity the sensorial properties of the cheese, and (Requena and McKay, 1993). This deriva- therefore, different strategies have been used tive lactococcal strain (Tl), along with the in order to improve texture and flavour parental strain (IFPL 359), was found to (Drake and Swanson, 1995). The use of accelerate proteolysis in low-fat cheese siur- ultrafiltration (UF) concentrates gives higher ries (Rodriguez et al, 1996a). Furthermore, yields and softer curds, however, there are reduced-fat semi-hard cheeses made with a problems to he solved arising from the slow- mixture of cow 's , ewes and goat's milk down of ripening and flavour development, concentrated by UF, and with the addition of which is probably due to increased levels IFPL starter supplemented with Tl , reached of proteinase and peptidase inhibitors and the ability of whey proteins to link sorne the desired characteristics of a mature cheese aroma compounds (De Koning et al, 1981; in a shorter period of time, as compared with Lelièvre and Lawrence, 1988; Bech, 1993). cheeses made with a commercial starter In addition, chee se ripening and flavour (Rodrfguez et al, 1996b). Proteolysis was development can be accelerated by addition enhanced in the IFPL cheeses, which achie- of extra enzymes, heat-shocked starters or ved a right balance of flavour and aroma. modifiedbacteria to the cheese milk, or Because small peptides, free amino acids, by the use of elevated ripening tempera- free fatty acids, volatile components and tures and combinations of the se strategies their by-products are thought to contribute to (Coulson et al, 1992; El Soda, 1993). Bac- cheese flavour, we have carried out a detai- terial enzymes can also be added by using led study on the evolution of proteolysis, bacteria with high autolytic properties (Crow lipolysis and formation of volatile corn po- etai, 1995). nents during ripening of those experimental We have developed a specifie starter cul- cheeses. In this paper these results are rela- ture (IFPL) consisting of several strains of ted to the previously published data on Lactococcus, Lactobacillus and Leuconos- enhancement of flavour development. Starter effect on UF cheese ripening 719

MATERIALS AND METHODS Peptide analysis by HPLC

Cheese samples Peptide analyses were carried out as described by Gonzâlez de Llano et al (1995). Cheese (10 g) was mixed with 50 mL deionized water and Cheeses were manufactured in the pilot plant of homogenized using an Ultraturrax (5 min at 40 "C), the Laboratoire de Recherches de Technologie The homogenate was then held for 10 min at Laitière (Inra, Rennes, France), from approxi- 40 "C and filtered through glass wool. After- mately 190 kg of heated (92 "C, 4 s) serni-skim- wards, it was filtered through a Durapore 0.45 med (23 g/kg fat) mixed milk (cow's/ewe's/goat's: jlm filter (Millipore Corp, Bedford, USA) and 55115/30). Milk was ultrafiltered at 50 "C to six- 50 jlL was injected on to the column. fold concentration as described by Goudédranche et al (1980). The concentrate (25 kg) was sepa- A Beckman System Gold HPLC was used rated in three batches and the various freeze- together with a System Gold Software data acqui- dried starter cultures were added to give counts sition system (Beckman Instruments Inc, San of 1010 colony forming units (cfu)lkg retentate. Ramon, USA). Separations were performed on a Batch FD contained a commercial starter culture CI8 Nucleosil 5 jlm, 300 A column (250 x 4.6 (Flora Danica MSP, Chr Hansen, Denmark), mm) (Macherey Nagel Düren, Düren, Germany) batch IFPL contained strains from the culture at 40 "C. Solvent A was 1 mL trifluoroacetic collection of the Instituto dei Frio (Departamento acid (TFA; Merck, Darmstadt, Germany)1L MilIi- de Productos Lâcteos, Madrid, Spain), and batch Q water (Millipore) and solvent B was 1 mL IFPL (T 1) contained the IFPL starter culture and a concentrate of L lactis subsp lactis TI (Lac- TFAIL in acetonitrile (HPLC grade from Schar- Pre) (which increased total counts in the retentate lau, Barcelona, Spain) and water (60:40 v/v). up to 1011 cfulkg). Cheese manufacturing, ripe- Separations were performed folIowing the ning conditions and sampling were described method described by Gonzâlez de Llano et al previously (Rodrïguez et al, 1996b). Mean values (1995). A flow rate of 1 mL/min was employed of total solids (TS) and salt on moisture of with detection at 220 nm. cheeses were 41.4% and 2.1 %, respectively, and the average contents of fat and protein were The aromatic amino acids tyrosine, phenyla- (%TS) 36.0 and 53.3, respectively. The mois- lanine and tryptophan, as weil as whey proteins ture in non-fat solid content of the cheeses was (œ-lactalbumin and B-Iactoglobulin A and B) 68%. Microbiological and physico-chemical cha- (Sigma, St Louis, USA) were run on the HPLC to racteristics of cheeses have been reported as weil identify their retention times. (Rodrfguez et al, 1996b ).

Amino acid content Polyacrylamide gel electrophoresis of the casein fraction Free amino acids were extracted from cheese according to the method of Resmini et al (1993). Cheese (5 g) was mixed with 10 mL deionized Sarnples (1.5 g) of cheese were blended with 40 mL water and homogenized using an Ultraturrax citratelHCI buffer, pH 2.2 (composed of 19.61 g (5 min at 40 "C). Whole casein was separated tri-sodium citrate dihydrate, 0.1 g sodium azide by precipitation with 100 mL acetic acid/L to and 200 mg EDT A in 1 L deionized water) and pH 4.6. The casein precipitate was washed repea- filtered through a Whatman no 40 filter paper. tedly with deionized water and centrifuged (3000 g, The filtrate was mixed with a sulphosalicylic 20 min, 5 "C) after each wash, before it was acid solution in citrate buffer (final concentra- freeze-dried, The Iyophilized casein fractions tion of 30 g/L), filtered, heated at 84 "C for were analyzed by means of urea-PAGE accor- 10 min, to denature whey proteins, centrifuged ding to the method of Ramos et al (1977). Elec- (5000 rpm, 10 min) and filtered (0.2 uiti), The trophoresis was carried out on slabs 0.7 mm thick filtrate (50 jlL) was analyzed using a Biochron and the bands were stained with Coomassie blue 20 amino acid analyzer (Pharmacia LKB, Uppsala, R250 (Winter et al, 1977). Sweden). 720 J Rodrfguez et al

Analysis of volatile components equipped with a PTV inje ctor, with helium as carrier gas (split ratio 1:20). Oyen temperature Cheese (1 g) was suspended in 3 mL of Milli-Q was held at 70 "C for 3 min, raised up to 190 at water (previously boiled for 20 min) using an 13 "Czrnin, with a final hold of 25 min. Other Ultraturrax, and the pH adjusted to 7.5 by addi- details were as described by Juârez et al (1992). tion of NaOH 0.1 N as recommended by Bosset and Gauch (1993). 2-Hexanone was added as internai standard. Analyses were carried out in tri- RESUL TS AND DISCUSSION plicate. Volatile compounds from cheese were isolated with a HP-7695 Purge & Trap concentrator cou pied to a HP 6890 gas chromatograph Casein degradation (both from Hewlett-Packard, Avondale, USA) following the procedure described by Bosset No differences were found among the three and Gauch (1993) with minor modifications. Chromatographie column was from fused silica batches with respect to the primary casein (50 m x 0.22 mm x 0.25 pm) coated with BP-20 breakdown. In ail cases, us-caseins to the (SGE, Ringwood, Australia). Nitrogen was used three species were hydrolysed faster than as carrier gas with a flow rate of 1 mL/min in B-caseins. After 15 days of ripening, the split mode (split ratio 1:20). Column temperature typical breakdown pattern consisted of was held at 35 "C for 15 min and then program- degradation products of high electrophore- med at 5 "Czrnin up to 180 "C for 5 min. The system efficiency (concentrator + column) was tic mobility, probably derived from the measured in isothermal conditions and cons ide- action of rennet on us-caseins, together with red satisfactory (H = 0.39 mm at k = 2.4). Peak bands of low mobility and y-caseins resul- areas were calculated using the HP-ChemSta- ting from the hydrolysis of B-caseins. This tion software. did not change significantly during the ripening period studied. Figure 1 shows, as an example, the electrophoretic pattern of GC-MS analysis caseins from cheeses made with IFPL starter with the addition of L lactis subsp lactis Volatiles were isolated following a dynamic Tl during ripening. headspace procedure based in stripping with nitrogen and trapping on Tenax, followed by thermal desorption, detailed elsewhere (Valero et al, 1997). GC-MS analysis was carried out using Peptides present in the water a capillary column identical to that described soluble fraction above with helium as carrier gas and a GC-8000 gas chromatograph with a MD-800 quadrupole mass detector working in El mode at 70 eV Figure 2 shows the reverse phase-HPLC (Fisons Instruments, Milan, Italy) separation of peptides in the water soluble fractions of the cheeses at 90 days of ripening. In agreement with the increased reten- Free fatty acid (FF A) analysis tion of whey proteins in the curd of the UF chee ses , a considerable amount of œ-lactal- Lipid extraction was carried out on an acidified bumin and B-Iactoglobulin, as weil as sorne cheese slurry using ethyl ether (Martfn-Hernân- large water-soluble degradation products, dez et al, 1988). Methylation of the FFA frac- were eluted after 80 min (results not shown). tion was performed with 20% of tetramethy- The RP-HPLC profiles allowed a clear dis- lammonium hydroxide in methanol (Martfnez- tinction between batch FD, made with the Castro et al, 1986). FFA were analyzed on a fused commercial starter, and the batches made silica capillary column (50 m x 022 mm x 0.22 pm) coated with Silar 5CP (Chrompack, Middelburg, with the IFPL starter. This indicates that the Netherlands) using a Perkin-Elmer 8420 unlike the primary proteolysis, peptidoly- apparatus (Perkin-Elmer, Beaconsfield, UK) sis in the cheeses was greatly influenced by Starter effect on UF cheese ripening 721

l 2 3 4 5 It is assumed that large or hydrophobie peptides should exhibit higher retenti on on a reverse phase eolumn, and it has been sug- gested that peptides eluting after Trp (reten- y-CN E tion time: 35 min) eonstitute the hydrophobie peptide portion (Lau et al, 1991; Gonzàlez de Llano et al, 1995). The pre- B-CN -, sence of hydrophobie peptides in eheese has been assoeiated with bitter taste, and their ewes~ & goats' [ degradation upon ripening to yield hydro- a.-CN phi lie peptides is thought to eontribute to cows' eheese flavour (Cliffe et al, 1989; Engels a.1-CN and Visser, 1994). The total amount of peptide material was higher in bateh FD than in the other two batehes, although the IFPL eheeses eontained more peptides in the fraction with the latest retention time. Never- theless, it should be noticed that no bitter flavour was deteeted by the taste panel in the eheeses made with the IFPL starter Fig 1. Urea-PAGE pattern of the casein fraction (Rodriguez et al, 1996b). of the cheeses made with the mixed starter of local strains (lFPL) supplemented with Lacto- coccus lactis subsp lactis TI, LacPrr , after 15 (1),30 (2),45 (3),60 (4) and 90 (5) days of ripe- Amino acid content ning. Distribution (urée-PAGE) de la fraction caséi- Table 1shows the results of free amino aeid nique des fromages préparés avec le levain IFPL content in UF-eheeses made with either PD contenant plusieurs souches, additionné d'une or IFPL starters during ripening. After 1 souche de Lactococcus lactis subsp lactis Tl month of ripening, IFPL chee ses eontained LacPrt ,après 15 (l), 30 (2), 45 (3), 60 (4) et 90 (5) jours d'affinage. higher content of free amino aeids than FD eheeses after 3 months of ripening. The total level of free amino aeids in FD eheese at the type of starter used. These differences this time was 56% of that found in IFPL eould be rnainly due to strain variations in eheese, and only 41 % of that found in IFPL arninopeptidase aetivities (El Abboudi et al, (Tl). Strains of the IFPL starter had been 1991; Requena et al, 1993; Parra et al, 1996) seleeted on the basis of their high proteoly- and to differenees in sensitivity for lysis tic aetivities (Requena et al, 1993) and could (Chapot -Chartier et al, 1994; Wilkinson et al, be responsible for the aeeeleration of the 1994). Arninopeptidase N aetivity of L lac- free amino aeid release in UF-eheeses and fis has been shown to play a raie in the for the lower amount of peptide material hydrolysis of low rnoleeular mass bitter pep- (fig 2). Increase in total amino acids when tides that are formed during the breakdown strains of mesophilie lactobaeilli are inclu- of easein by rennet or starter proteinase ded as cheese starter adjuncts has been (Baankreis, 1992; Tan et al, 1993). No sub- reported (Lemieux et al, 1989; Lee et al, stantial differenees were found between the 1990). The mutant strain Tl (Lac- Pre), in peptide patterns of the two batehes made addition to retaining ail peptidase activity with the IFPL starter throughout the ripe- from the parental strain L lactis subsp lactis ning period. IFPL 359 (Requena and McKay, 1993), has 722 J Rodrfguez et al

0.6

0.4

o N N -< 0.2

0.0

0 20 40 60 BO

0.6

0.4

0 N N

-< 0.2

0.0

0 20 40 60 80

0.6

0.4

0 N N

-< 0.2

0.0

o 20 40 60 BO Time (min)

Fig 2. HPLC profiles of the water soluble nitrogen fraction of cheeses made with the commercial starter (FD) (a), the mixed starter of local strains (IFPL) (b), and the IFPL starter supplemented with Lactococcus lactis subsp lactis Tl, Lac-Pre (c), after 90 days of ripening. Profils HPLC de lafraction azotée soluble dans l'eau des fromages préparés avec du levain commercial (FD), le levain IFPL contenant plusieurs souches, et le même additionné d'une souche de Lactococcus lactis subsp lactis Tl (Lac Prtï, après 90 jours d'affinage. Starter effect on UF cheese ripening 723

00 00 q "1; o \0

00 oq ('Cl

"0 00 00 00 o oq q q «) ('Cl o ('Cl

"0 00 or> ~

"0 o 0\

"0 or> or> o oq oq ('Cl or> or> \0 o

o 00 oq C"! -o

\0 \0

~0\ o

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~\0 -: o

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"0 o o or> lrl C"! $ oq o o o ('Cl

Cl) <:: Cl) ou; CL) <:: 'C e .5 Cl) »0; CIl E-< > 724 1 Rodriguez et al shown ability to autolyse and to increase six-fold increase in proline and four-fold in peptidase activity in a cheese-like medium lysine (table 1). Substrates including lysine (Rodrfguez et al, 1996a). Elevated levels of and proline residues have been found to be amino acids at the beginning of the ripening preferably hydrolysed by the intracellular period in cheese correspond with earlier enzymic fraction of L lactis subsp laetis Iysis of starter bacteria (Birkeland et al, IFPL 359 (Requena et al, 1993). These 1992; Wilkinson et al, 1994; Christensen et results indicate that it is possible to change al, 1995). the composition of free amino acids in cheeses by modifying the peptidolytic pro- Although the concentrations of free perties of starter bacteria. The use in chee- amino acids increased with the age of the semaking of a strain that overproduces a cheese, the relative proportion of each amino certain peptidase activity has a similar effect acid remained essentially constant. Amino (Christensen et al, 1995). acids in higher proportion in aIl chee ses were Glu (16%), Leu (14%), Val (12%) and Ser (9%), and contributed about 50% to the total free amino acids. Main differences be- Volatile components tween batches were due to variations in proline and lysine proportion. The total content The volatile components identified in the of these amino acids at the end of the ripe- cheeses are listed in table II. Figure 3 shows ning period was particularly higher in IFPL the chromatograms of volatile components (Tl) cheeses than in FD cheeses, showing a found in the three cheese batches at 90 days

Table II. Volatile components identified in ripened reduced-fat ultrafiltered cheese by GC (tentative) and GC/MS. Composés volatils formés dans des fromages affinés à teneur réduite en matière grasse. identifiés par GC et GC/MS.

Peak RT Compound GC GC/MS

1 4.1 Diacetyl + 2 4.5 Acetaldehyde + 4 5.5 Acetone + 6 6.8 Ethyl acetate + + 7 7.3 Methanol + 3-methyl butanal + 8 8.1 Dimethyl sulphide + 9 8.5 Ethanol + 9 8.6 2-pentanone + + 10 10.4 Methyl butyrate + + Il 11.8 n-propanol + 13 13.5 Ethyl butyrate + + 15 16.9 Dimethyl disulphide + + 19 20.3 Methyl hexanoate + + 21 23.2 2-heptanone + + 2-methyl butanol + 22 25.9 Ethyl hexanoate + + 22 25.9 3-methyl butanol + + 25 27.2 2-heptanol + + 28 29.7 Methyl octanoate + + 30 31.8 2-nonanone + + Starter effect on UF chee se ripening 725

~ 3 13 15 " A .. 9 21 3Q .. 4 19 .. 6 10 11 .. 7 18 17\ .. 2 16 20~ 28 1 ~23J , ! L ... -J ~ ~ v.1,ll .. , -

4 9 10 13 15

11 8

·25, 1 16 2 19

8 3 Fig 3. Chromatograms of 6 volatile components found 2~ 23 7 28· in cheeses prepared with the 20 24 commercial starter (FD) (A), 15 21 the mixed starter of local 5 strains (IFPL) (B) and the IFPL starter supplemented with Lactococcus lactis subsp lactis TI, Lac-Prt- (C), after 90 days of ripening. Peak numbers correspond to table II. Peaks 3,5, ~ ·1 13 15 19 22 , 12,14,16,17,18,20,23, " 4 ~I9 C 24,26,27,29, not identified. .. 25 Chromatogrammes des com- posés volatils trouvés dans 2 desfromages préparés avec .. 7 du levain commercial (FD) 17 (A), le levain IFPL conte- .. 11 20 nant plusieurs souches (B), 10 12 et le même additionné d'une .. souche de Lactococcus lactis . 15 subsp lactis Tl (Lac- Prr ï (C), après 90 jours d'affi- .. 3 5 nage. Les pics sont numéro- 2i d262830 tés comme dans le tableau 14 ll. Pics 3, 5,12,14,16,17, \v. it Il ~j >ul~ " 1--- 18,20,23,24,26,27,29: non identifiés. ' 726 J Rodrfguez et al of ripening. Sorne of the components were 4.5 to 7.8% for a standard solution and from identified from their mass spectra and GC 6.5 to 10.9% for a sample solution. Values retention while other were tentatively assi- which have been reported as satisfactory gned by comparison with retention times of are: 3.1% (Yang and Min, 1994), 1.6-8% standards and published elution orders (Bosset (Collin et al, 1993),7.8-18% (Wood et al, and Gausch, 1993). Ketones, ethyl esters 1994). Total volatile quantities (expressed as and methyl esters, aldehydes, alcohols, the sum of 30 quantified peaks) increased dimethyl sulphide and dimethyl disulphide during ripening in the three batches as pre- were found in ripe cheeses from the three sented in figure 4. The highest total vola- studied batches. These type of compounds tile content was found in batch IFPL (Tl), have been found in other Spanish semi-hard which also had the best score in sensorial cheeses (Martfnez -Castro et al, 1991; Bosset analysis (Rodrfguez et al, 1996b). The higher and Gauch, 1993; Rodrfguez et al, 1996a). level of volatile components found in Free fatty acids did not appear in the chro- batches IFPL and IFPL(TI) could be related matograms since sample solutions were at to their increased proteolysis level, as shown pH 7.5. Differences among volatile com- by the free amino acid content (table 1). ponents of cheeses from different batches at the same ripening time were mainly quan- titative. Free fatty acids (FF A) In order to compare results, peak areas in every chromatogram were related to that of Total FFA in the ripe cheeses from batches internai standard 2-hexanone, and expres- IFPL and IFPL(T 1) were 1345 mg/kg and sed as uglg cheese. Reproducibility of the 1324 mg/kg, respectively, and 1114 mg/kg volatile determination was estimated from in batch FD. The low levels of total FF A variation coefficients which ranged from can be explained by the reduced fat content

~ 4 ~ ë5 > 3 ëiio f- 2.

o-l-----r-~-..,.----'---___r---..,.------r--- 30 60 90 Days of ripening

Fig 4. Total volatile components measured (;tg/g) in cheeses prepared with the commercial starter (FD) (0), the mixed starter of local strains (IFPL) (0) and the IFPL starter supplemented with Lactococcus lactis subsp lactis Tl, LacPrr (11),at 30,60 and 90 days of ripening. Composés volatils totaux (}lg/g) dans des fromages préparés avec du levain commercial (FD) (0), le levain IFPL contenant plusieurs souches (0), et le même additionné d'une souche de Lactococcus lactis subsp lactis Tl (Lac Prtï (.1), après JO, 60 et 90jours d'affinage. Starter effect on UF cheese ripening 727

of cheeses and the scaree !ipolytic activity of BossetJO, Gauch R (1993) Comparison of the volatile the starter cultures. Moderate lipolysis has flavour compounds of six European' AOC' cheeses by using a new dynamic headspace GC-MS been reported in semi-hard cheeses ripened method.lnt Dairy 1 3,359-377 without molds (De la Fuente et al, 1993), Chapot-Chartier MP, Deniel C, Rousseau M, Vassal and there is commonly very !ittle lipolysis in L, Gripon JC (1994) Autolysis of two strains of low-fat cheeses (Dimos, 1992; Ardë, 1994). Lactococcus lactis during cheese ripening.lnt Dairy 14,251-269 In conclusion, present results show that IFPL Christensen JE, Johnson ME, Steele JL (1995) Pro- starter contributes to the aceeleration of fla- duction of Cheddar chee se using a Lactococcus vour development in UF-cheeses mainly lactis ssp cremoris SKII derivative with enhan- through changes in both the water-soluble ced aminopeptidase activity. lnt Dairy 1 5, 367-379 nitrogen fraction and the volatile compo- Cliffe AJ, Reveil D, Law BA (1989) A method for the reverse phase HPLC of peptides from Cheddar nents. This supports previous findings on cheese. Food Chem 34,147-160 the contribution of low molecular-mass Collin S, Osman M, Delcambre S, EI-Zayat A, Dufour compounds, like small peptides, ami no J (1993) Investigation of volatile flavor compounds acids, FFA or their breakdown products to in fresh and ripened Domiati cheeses. 1 Agric Food Chem 41,1659-1663 the flavour of different types of chee se Coulson J, Pawlett D, Wivell R (1992) Accelerated (Engels and Visser, 1994). The addition to ripening of Cheddar cheese. Bulllnt Dairy Fed the IFPL starter of L lactis subsp lactis Tl 269,29-35 (Lac Prr) greatly increased the contents of Crow VL, Coolbear T, Gopal PK, Martley FG, McKay free amino acids and volatile components LL, Riepe H (1995) The role of autolysis of lactic in the cheeses during ripening, which cor- acid bacteria in the ripening of cheese.lnt Dairy 1 5, 855-875 related with the best final scores on both De la Fuente MA, Fontecha MA, Juarez M (1993) aroma and flavour (Rodrfguez et al, 1996b). Fatty acid composition of the triglyceride and free fatty acid fractions in 'different cow's, ewe's and goat's milk cheeses. Z Lebensm Unters Forsch 196, 155-158 ACKNOWLEDGMENTS De Koning PJ, De Boer R, Both P, Nooy PFC (1981) Comparison of proteolysis in a low-fat semi-hard The authors would like to acknowledge finan- type of cheese manufactured by standard and by cial support for this study from the Comision ultrafiltration techniques. Neth Milk Dairy 1 35, Interministerial de Ciencia y Tecnologfa (Project 35-46 ALI 95-0046-C02-0 1). Dr J Rodriguez is the Dimos A (1992) A comparative study by GC/MS of recipient of a scholarship from the Spanish Coun- the flavour volatiles produced during the matura- cil for Scientific Research (CSIC). tion of full-fat and low-fat Cheddar cheese. Master Thesis, Latrobe University, Bundoora, Victoria, Australia Drake MA, Swanson BG (1995) Reduced- and low- fat cheese technology: A review. Trends Food Sei Techno16,366-369 El Abboudi M, El Soda M, Pandian S, Barreau M, Tre- REFERENCES panier G, Simard RE (1991) Peptidase activities in debittering and nondebittering strains of lactoba- Ardô Y (1994) Proteolysis andflavour development cilli.lnt Dairy 1 1,55-64 in reducedfat cheese. Doctoral Thesis, Lund Uni- El Soda M (1993) Accelerated maturation of cheese.lnt versity, Sweden Dairy 1 3, 531-544 Baankreis R (1992) The raie of lactococcal peptidases Engels WJM, Visser S (1994) Isolation and compara- in cheese ripening. Doctoral Thesis, University of tive characterization of components that contribute Amsterdam, the Netherlands to the flavour of different types of cheese. Neth Bech AM (1993) Characterising ripening in UF-cheese. MilkDairy148,127-140 lnt Dairy 13,329-342 Gonzalez de Llano D, Polo C, Ramos M (1995) Study Birkeland SE, Abrahamsen RK, Langsrud T (1992) of proteolysis in artisanal cheeses: high perfor- Accelerated cheese ripening: use of Lac- mutants of mance liquid chromatography of peptides. 1 Dairy lactococci. 1 Dairy Res 59, 389-400 Sci78,1018-1024 728 J Rodrfguez et al

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