Ripening of extra-hard cheese made with mesophilic DL-starter

International Dairy Journal 20 (2010) 844e851

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Ripening of extra-hard cheese made with mesophilic DL-starter U. Rehn a, M.A. Petersen a, K. Hallin Saedén b, Y. Ardö a, * a b

Department of Food Science, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark Norrmejerier, Mejerivägen 2, SE-906 22 Umeå, Sweden

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 September 2009 Received in revised form 19 May 2010 Accepted 1 June 2010

Extra hard cheese is commonly made with thermophilic starters using high temperatures to stimulate expulsion of whey. In this work, microflora, proteolysis and volatiles were investigated in an extra-hard cheese made with mesophilic DL-starter, produced using challenging cooking temperatures for the starter bacteria over several hours. Cheese from six commercially produced vats was investigated over 56 weeks. The number of starter bacteria decreased after three weeks of ripening. Casein breakdown was characterised by chymosin and plasmin activity on as1- and b-caseins, respectively. Peptide profiles showed accumulation of Lactococcus derived peptides from as1-CN f1e23, and the peptide b-CN 29e93 as a result of joint plasmin and chymosin activity and absence of highly proteolytic thermophilic Lactobacillus, commonly present in extra-hard cheese. The composition of amino acids depended mainly on starter during the first 26 weeks of ripening. The content of volatiles depended both on ripening time and the starter used. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Hard and extra-hard cheese (49e56% and <51% moisture in non-fat substance, MNFS, respectively, Codex Alimentarius, 2008) is mainly produced using thermophilic starters and high cooking temperatures, whereas mesophilic DL-starters are commonly used in northern Europe for several semi-hard cheese varieties (54e69% MNFS) produced with lower cooking temperatures, such as Danbo, Jarlsberg, Herrgård, Leerdammer and Gouda cheese (Ardö, 1993, 2004; van den Berg, Meijer, Düsterhöft, & Smit, 2004). The Swedish full fat and hard to extra-hard cheese Västerbottensost (50% MNFS) is produced with mesophilic DL-starter and cooked at high temperatures for several hours (Ardö, 1993), which creates stressful conditions for the starter bacteria. Västerbottensost has been produced in the county of Västerbotten in Sweden since the 19th century. This technology may require interesting ripening prerequisites, which were investigated in this work. Mesophilic DL-starters are composed of Lactococcus lactis subsp. lactis (Lc. lactis), Lc. lactis subsp. cremoris (Lc. cremoris), Lc. lactis subsp. lactis biovar diacetylactis (Lc. diacetylactis) and different subspecies of Leuconostoc mesenteroides (Leuconostoc). Mesophilic DL-starters commonly comprise an undefined mixture of strains of the species mentioned. Important properties of the starter that influence cheese ripening are autolysis, the activity and specificity

* Corresponding author. Tel.: þ45 3533 33193; fax: þ45 3533 3190. E-mail address: [email protected] (Y. Ardö). 0958-6946/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2010.06.001

of their proteolytic enzymes, including enzymes involved in peptide and amino acid release and further metabolism to flavour compounds. Autolysis of starter bacteria may be induced by deteriorated growing conditions, such as lack of suitable nutrients, salt content or heating during cheese manufacture (Bie & Sjöström, 1975a, 1975b; Boutrou et al., 1998; Hannon et al., 2003; Lortal & Chapot-Chartier, 2005). The released intracellular material, enzymes and nutrients, have been shown to have a major impact on cheese ripening. The agents involved in initial casein (CN) degradation in semihard cheese are the coagulant and the milk enzyme plasmin, which are active mainly on as1-casein and b-casein, respectively. The coagulant is, however, more or less inactivated in hard cheese varieties cooked at high temperatures of about 50e55
C (Ardö, 2001). High plasmin activity has been observed in the semi-hard cheese Herrgård as a result of accumulation of b-casein derived peptides, especially b-CN f29e93 (Ardö, Lilbæk, Kristiansen, Zakora, & Otte, 2007). The cell-bound proteinase (CEP, lactocepin), of the starter Lactococcus sp. hydrolyse the peptides produced by the initial degradation of the caseins, as reviewed by Sousa, Ardö, and McSweeney (2001). The proteolytic activity and specificity of lactocepin have been discussed in relation to production of small peptides derived from the chymosin produced fragment as1CN f1e23, such as f1e9, f1e13, f1e16 and f1e17 (Broadbent et al., 2002). Besides lactocepin, the proteolytic system of lactococci is composed of transport systems for amino acids and small peptides, as well as several intracellular peptidases. Amino acids are released from the peptides by microbial activity and may be converted into

U. Rehn et al. / International Dairy Journal 20 (2010) 844e851

volatile compounds with subsequent impact on cheese flavour (Ardö, 2006). The aim of this work was to investigate ripening of extra-hard cheese made with mesophilic DL-starter using high cooking temperatures at the limit for survival of the starter over a period of several hours. The microbiology, proteolysis and volatile compounds over 56 weeks of ripening of commercially produced Västerbottensost were investigated. 2. Materials and methods

845

30
C for 72 h and plates with MRS agar and vancomycin were incubated anaerobically at 25
C for 96 h. 2.4. Capillary electrophoresis of casein components The casein components were analysed by dispersing cheese samples in a citrate solution according to Ardö and Polychroniadou (1999). The citrate dispersion was then diluted (volume ratio 1:1) in a urea sample buffer and analysed using capillary electrophoresis (CE) and centrifuged as described by Jensen, Vogensen, and Ardö (2009).

2.1. Experimental design Microbial flora, proteolysis and development of volatile aroma compounds in extra-hard cheese (VästerbottensostÒ) were analysed for six commercially produced vats of cheese (V1eV6) from a single dairy plant. The six vats of cheese were produced within seven weeks during winter. Cheeses were ripened at the producer’s facilities and samples were obtained at the ages of 24 h, 3, 12, 26 and 56 weeks. Cheese making comprised long cooking time periods, for several hours, above 40
C. Coagulant (75/25 of chymosin and bovine pepsin, 180 IMCU, obtained from Kemikalia, Skurup, Sweden) was added to milk at a concentration of 0.3 mL L1 of milk. Cheeses were produced in cylinders (w16 cm height) of 18 kg, brine salted to a content of around 1.2% in the cheese, waxed, and ripened for different periods at specific temperatures between 10 and 16
C. The sensory properties of the cheeses were evaluated by the producer after 56 weeks of ripening and all cheeses were graded as high quality Västerbottensost. The effect of using two different mesophilic DL-starters (DL1 and DL2; Chr. Hansen, Hørsholm, Denmark) was also investigated. Vats V2, V3 and V4 were made with DL1 and vats V1, V5 and V6 were made with DL2. 2.2. Gross composition Moisture content of the cheeses was determined according to the IDF standard method (IDF, 2004). The pH was measured with a pH electrode inside a tightly packed grated cheese sample added a suitable amount deionised water. Moisture content, fat in dry matter (FDM) and pH after 24 h were measured at the dairy using a FoodScanÔ (Foss, Hillerød, Denmark) and a pH electrode, respectively, whereas moisture content and pH at 3, 12, 26 and 56 weeks were measured at the University of Copenhagen. 2.3. Quantification of lactic acid bacteria The preparation of cheese samples was carried out by aseptic sampling of 10 g of cheese (taken from the interior of the cheese). Each sample was mixed with 90 mL 2% (w/v) sterile sodium citrate (Merck, Darmstadt, Germany) solution and mixed in a Seward Stomacher 400 (Struers A/S, Ballerup, Denmark) for 2 min (normal speed). Cheese samples were spread on De Man, Rogosa and Sharpe (MRS) agar (Merck) in which pH was adjusted to 6.5 and 5.4 (after sterilisation) using 1 M NaOH and 1 M HCl, respectively, and incubated in anaerobic jars (AnaeroGenÔ, Oxoid, Basingstoke, England) for 72 h. MRS agar plates at pH 5.4 were incubated at 37
C to give an estimate of non-starter lactic acid bacteria (NSLAB), whereas plates with MRS at pH 6.5 were incubated at 30
C to give an estimate of the total amount of lactic acid bacteria (LAB). To enumerate the starter Lactococcus sp. and Leuconostoc sp. after 24 h, cheese samples from batch V3, V4, V5 and V6 were spread on M17 (Merck) agar with 0.5% lactose (w/v) for Lactococcus sp. and MRS agar (Merck) with 0.2 mg mL1 vancomycin (Calbiochem, Merck) for Leuconostoc sp. (Mathot, Kihal, Prevost, & Divies, 1994). The M17 plates with lactose were incubated aerobically at

2.5. Peptide analysis by reversed phase high performance liquid chromatography A pH 4.6 soluble fraction was made from a citrate dispersion of cheese as described by Ardö and Polychroniadou (1999). To remove casein precipitate and fat, samples were centrifuged (1800 g at 4
C for 30 min). The supernatant was filtered through a 0.20 mm filter (Sartourius MiniSart, Hannover, Germany) and stored at 20
C until analysed. The peptide analysis was carried out with reversed phase high performance liquid chromatography (RP-HPLC) as described by Ardö and Gripon (1995). 2.6. Peptide identification by liquid chromatographyemass spectroscopy The LCeMS analysis was carried out using an Agilent 1100 LCeMSD Trap (Agilent Technologies A/S, Nærum, Denmark) operated with LS/MSD Trap Control data analysis software version 4.01. A Zorbax C18 column (2.1  150 mm, 5 mm, Agilent Technologies A/S) was operated at 40
C. Of the sample prepared for RP-HPLC (Section 2.5), 15e50 mL was injected. The LCeMS procedure was carried out as described in Ardö et al. (2007), with the modification of recording of mass spectra using a standard range from 100 to 2200 m/z at the normal scan resolution and the target mass set to 1521 m/z. Data were processed by Bruker Daltonics Data Analysis software (Bruker, Billerica, MA, USA) version 2.1. The GPMAW software version 3.04 (ÓLighthouse data, Odense, Denmark) was used to assign peptide masses to particular sequences of the caseins. 2.7. Amino acid composition Analysis of the amino acid composition was carried out by RPHPLC according to Bütikofer and Ardö (1999). The 20 amino acids of casein were analysed, as well as the breakdown products citrulline (Cit) and ornithine (Orn) from Arg and g-amino butyric acid (GABA) and a-amino butyric acid (AABA) from Glu. Pre-column derivatisation with o-phthaldialdehyde (OPA, Agilent, Birkerød, Denmark) and fluoroenylmethyl chloroformate (FMOC, Agilent) was used. The amino acid concentration was calculated as mmol kg1 of cheese using standard curves. Individual amino acids were expressed as mmol kg1 of cheese and as relative content of the total amount of amino acids (mol%). 2.8. Volatile compounds Analysis of volatile compounds was carried out by dynamic headspace sampling and gas chromatographyemass spectroscopy (D-HS GCeMS). Frozen cheese samples were thawed in a refrigerator overnight. Tap water (100 mL) and 1 mL of 4-methyl-1-pentanol (50 mg L1) as an internal standard were added to cheese cubes (80 g, w10  10 mm2). The mixture was homogenised at 13,500 rpm with an Ultra Turrax T25 for 2 min. Dynamic headspace

U. Rehn et al. / International Dairy Journal 20 (2010) 844e851

sampling was carried out in flasks with the cheese slurry placed in a water bath (30
C) with continuous stirring (200 rpm) for 60 min. Volatile compounds were purged from the cheese slurry with an N2 flow (200 mL min1) onto a Tenax trap (250 mg Tenax-TA, mesh size ¼ 60/80, Buchem B.V., Apeldoorn, The Netherlands). Volatiles were desorbed from the trap and analysed by GCeMS as described by Juric, Bertelsen, Mortensen, and Petersen (2003). Data analysis was carried out with MSD Chemstation software (G1701DA ver. D.00.00.38, Agilent Technologies A/S). Identification of spectra peaks was carried out by probability-based matching with mass spectra in the G1035A Wiley library (HewlettePackard, Palo Alto, CA, USA). All integrated areas were divided by the area of the internal standard (4-methyl-1-pentanol) and are presented as relative responses.

a 10

log cfu g-1 Lactic acid bacteria cheese

846

9 8 7 6 5 4 3 2 1 0

5

10

15

20

25

30

35

40

45

50

55

60

40

45

50

55

60

Ripening time (weeks)

2.9. Data analysis Statistical analyses (one-way ANOVA) were carried out with R version 2.8.1 (R Foundation for Statistical Computing, Vienna, Austria). 3. Results 3.1. Gross composition The moisture content decreased from approximately 36 to 32% (w/w) during the cheese ripening period (Table 1). The variation in moisture content between the cheeses after 24 h was higher than for 3 weeks ripening and thereafter. The pH increased from around 5.3 after 24 h to around 5.6 after 56 weeks of ripening. The average of the fat in dry matter (FDM) of the cheeses was 51.9% with a standard deviation of 0.7%. 3.2. Microbial composition Starter bacteria counts were higher than NSLAB counts in the cheeses up to 3 weeks of ripening (Fig. 1). Higher cell numbers on MRS at pH 6.5 were observed for cheeses made with DL1 compared with DL2 at 24 h and 3 weeks of ripening. Between 3 and 12 weeks, bacteria counts on MRS agar pH 6.5 decreased towards the number of counts on MRS agar pH 5.4, which indicates a decrease in the cultivable starter bacteria. The bacteria counts on the two MRS media were similar after 26 weeks of ripening. At 24 h and 3 weeks of ripening, a large variation in the number of LAB on MRS pH 6.5 was seen between the cheeses. The highest numbers at 24 h and after 3 weeks were observed for V2 (log 7.7 and 8.0 cfu g1, respectively) and the lowest for V1 (log 6.1 and 6.3 cfu g1,

log cfu g-1 Lactic acid bacteria cheese

b

10 9 8 7 6 5 4 3 2 1 0

5

10

15

20

25

30

35

Ripening time (weeks) Fig. 1. Total number of lactic acid bacteria, log cfu g1 cheese, on MRS at pH 6.5 and 30
C (a) and log cfu g1 cheese non-starter lactic acid bacteria on MRS at pH 5.4 and 37
C (b) for six vats of cheese: V1 (-), V2 (,), V3 (:), V4 (6), V5 (A) and V6 (>). V2, V3 and V4 were made with DL1 and V1, V5 and V6 were made with DL2.

respectively). The estimated enumeration of Lactococcus sp. and Leuconostoc sp. after 24 h (on M17 with lactose and MRS agar with vancomycin, respectively) showed a higher amount of both Lactococcus sp. and Leuconostoc sp. in V3 and V4 as compared to V5 and V6 (Table 2). 3.3. Casein degradation Cheeses from the six vats showed similar casein breakdown during ripening, and results for V2 are shown as an example (Fig. 2). Chymosin action on k-casein resulted in the appearance of para-kcasein. After 3 weeks of ripening, the initial breakdown of

Table 1 Gross composition of the cheeses. Values of moisture content represent means of triplicates and values for pH are means of duplicates. Cheesea

FDMb (%)

Ripening time (weeks) Moisture (%)

pH

0c

3

12

26

56

0c

3

12

26

56

0c

V1 V2 V3 V4 V5 V6

33.2 37.4 37.1 36.2 35.6 35.8

33.3 34.9 35.6 34.7 34.4 35.0

32.9 33.7 32.8 32.7 33.4 33.4

32.5 33.5 32.8 32.8 32.7 32.7

32.2 31.6 32.2 32.0 32.1 31.9

5.27 5.28 5.36 5.39 5.30 5.32

5.38 5.40 5.36 5.39 5.30 5.33

5.40 5.40 5.43 5.40 5.39 5.41

5.44 5.47 5.58 5.60 5.45 5.49

5.63 5.60 5.70 5.71 5.54 5.60

53.2 51.3 52.4 51.5 51.6 51.5

Mean SD

36.1 1.5

34.7 0.8

33.2 0.4

32.8 0.3

32.0 0.2

5.32 0.05

5.36 0.04

5.41 0.01

5.51 0.07

5.63 0.07

51.9 0.7

a b c

V1eV6 refer to six vats. Fat in dry matter. Sample taken after 24 h (before brining).

U. Rehn et al. / International Dairy Journal 20 (2010) 844e851

The result of plasmin activity was observed after 24 h as the occurrence of peaks in the retention time interval 75e80 min containing the peptides b-CN f1e105 and f1e107 (A1/A2 genetic variants; peptides 25/29 and 20/26, respectively). Between 24 h and 26 weeks, these peptides were further degraded to b-CN f29e107, b-CN f29e105 (peptides 18/24 and 21/28) by the cleavage of Lys28eLys29 by plasmin (Ardö et al., 2007) and b-CN f29e93 (peptide 19/27) probably as a result of chymosin activity on Leu192eTyr193. Microbial activity on the plasmin derived peptide b-CN f1e28 (peptide 14) resulted in formation of b-CN f12e28, f1e28, f8e28 and f7e28 (peptides 9e12). The peptide as2-CN f5e21 (peak 7) may result from plasmin activity on the bond Lys21eGln22 (Le Bars & Gripon, 1993).

Table 2 Results from estimated enumeration of Lactococcus on M17 agar and of Leuconostoc on MRS agar with vancomycin after 24 h (before brining). Cheesea

M17 (log cfu g1)

MRS with vancomycin (log cfu g1)

V3 V4 V5 V6

7.7 7.4 6.7 6.6

6.6 7.3 3.6 5.6

a

847

V3eV6 refer to four vats.

as1-casein 8P and 9P by chymosin was observed as an increase in as1-CN f24e199, referred to as as1-I-casein 8P and 9P. The peaks corresponding to as1-casein 8P and 9P disappeared between 12 and 26 weeks. During further ripening, the as1-I-caseins were degraded

3.5. Amino acid release and metabolism

to eventually disappear completely. The breakdown of b-casein, by the action of plasmin, could be observed by the appearance of g-caseins at 24 h (Fig. 2). A decrease in the peaks corresponding to the g-caseins between 26 and 56 weeks showed that further degradation was mainly initiated by plasmin, because no peaks other than g-caseins were seen (Ardö, 2001). The casein fraction of the cheese ripened for longer periods mainly consisted of g3-casein.

V2 cheese had the highest total amino acid content all through ripening and V5 cheese had the lowest (Fig. 4). The quantitatively dominating amino acids in the ripened cheeses were Glu, Lys, Leu, Val, and Pro (average of all cheeses were 14, 12, 12, 8, and 7 mol%, respectively, of the total content of amino acids). The content of Pro, which may introduce a sweet flavour note, in the 56-weeks ripened cheese varied between 19.3 mmol kg1 cheese and 30.7 mmol kg1 cheese (V2 and V5 respectively; results not shown). Significant differences between cheeses made with the two different starters (DL1 and DL2) were observed for the relative content of ten amino acids after 3 weeks of ripening, seven after 12 and six after 26 weeks of ripening, whereas after 56 weeks of ripening, only the relative amount of Cit was significantly different between cheeses made with different starters, which during ripening was always significantly higher in cheeses made with DL2 (Table 4). The results demonstrate less impact of starter during later stages of ripening. Of the amino acids which produce important cheese flavour compounds by subsequent catabolism, the relative content of Ile and Met were significantly lower in cheese made with DL2, whereas the relative contents of Leu, Phe and Tyr were significantly lower in cheeses made with DL1. The amino acid Glu, which is produced during amino acid catabolism, had a significantly higher relative content in cheeses made with DL1 (Table 4).

3.4. Peptide formation Main peptides found in the pH 4.6 soluble fraction of the cheeses were tentatively identified using LCeMS (numbered peaks in Fig. 3 and Table 3). The amino acids Tyr, Phe and Trp appeared on the HPLC-chromatogram at the retention times 15, 20 and 31 min, respectively, and they increased during ripening. The activity of chymosin was shown by formation of the peptides as1-CN f1e23 (peak 15), k-CN f106e169 (peak 16) and b-CN f193e209 (peak 17). Furthermore, the accumulating peptides as1-CN (f1e8, 1e9, 1e7, 1e6, 1e13 and 1e14) corresponding to peptides 1, 3 and 5, 6, respectively (Table 3 and Fig. 3), may originate from the starter Lactococcus sp. protease activity on the chymosin produced peptide as1-CN f1e23 (Ardö, 2001; Broadbent et al., 2002).

300

-CN

-CN A

-CN

200

26 weeks -CN A Para- -CN

150

-I-CN 8P

-CN A

12 weeks 100

-CN 8P -I-CN 9P -CN

50

-CN A

Absorbance at 214nm

-CN A

56 weeks

250

3 weeks

-CN A -CN 9P -CN

24 h

0 20

22

24

26

28

30

32

34

36

38

40

42

44

46

48

50

Migration time (min) Fig. 2. Capillary electrophoresis curves presenting casein breakdown in cheese V2 over 56 weeks of ripening. (CN: casein). Para-k-CN refers to k-CN f1e105, g1-CN refers to b-CN f29e109, g2 refers to b-CN f106e209, g3-CN refers to b-CN f107e209, b-CN A1 and A2 refers to the two genetic variants of b-CN, as1-I-CN 8P refers to as1-CN f24e199 and as1-I-CN 9P refers to as1-CN f24e199 9P. Labelling of peaks is according to Ardö (2001).

848

U. Rehn et al. / International Dairy Journal 20 (2010) 844e851

a

-lactalbumin -lactoglobulin

0.6

V1

Phe

Absorbance at 210 nm

0.5

V2 20,25,26,29

14

0.4 5

16 15

13

6 5 5

0.1

5

16 15

13

6

0.2

13

14

16 15

13

14

16 15

6

1

V3

20,25,26,29

14

0.3

6

V4 20,26,29

V5 20,25,26,29 17

V6

0.0 10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

Retention time (min) -lactalbumin -lactoglobulin

b 3.0

Phe

5

1,2

2.5 Absorbance at 210 nm

6 8 7 9 10 12

Trp 3 4

Tyr

5 1,2 3

2.0

1,2 3 1.5

1,2 3

0.5

1,2 3 0.0 10

15

20

25

13

9 10 5

X

6

11,12 10

4

16

14

9 10

11

11 13 10

4

X

X

6 7 9

4 40

45

11 13 50

16

14 55

Y X: 19,24 Y: 27,28,29

Y X: 19,21,24 Y: 26,27

17 18

14

5

35

X: 19,21,24 Y: 27,28

18

16

14

5 6

X: 19,24 Y: 27,28

60

65

V3 V4 V5

Y X: 19,20,22,23 Y: 26,27

17 18 70

V2

Y

X

6

30

Y

17 18

5 4

X: 19,24 Y: 26,27,29

18

16

14

Y

V1

X

11,12

6

4

1.0

1,2 3

X 18

16

14

75

V6

80

85

90

Rotation time (min)

c

Phe

3.0

1,2 Tyr

19

Trp 3

56 4

2.5

1,2

5 3

Absorbance at 210 nm

9

11

17 18

9

11

17 18

-lactalbumin -lactoglobulin

V1

27 19

6

2.0

27

butanol, with the GCeMS analysis method used (presented as 2/3methyl-butanal and 2/3-methyl-1-butanol, respectively). Coelution was also observed for 2-pentanone and 2,3-butanedione (diacetyl). The content of primary alcohols and 3-methyl-3-buten-1-ol significantly increased between 26 and 56 weeks of ripening, while the content of secondary alcohols generally decreased (not significantly) or remained rather constant (Table 5). The contents of all identified secondary alcohols, however, were significantly higher in cheeses made with DL2 than with DL1, both after 26 and 56 weeks of ripening. The contents of 2-butanol, 2-methyl-1-propanol and 2/ 3-methyl-1-butanol were also significantly higher in cheeses made with DL2 after 56 weeks. The contents of the aldehydes 2/3methylbutanal, however, were significantly higher in cheeses made with DL1. All identified ketones were also significantly higher in cheeses made with DL1 after both 26 and 56 weeks, with the exception of 2-butanone after 56 weeks of ripening. The ketones generally increased during ripening between 26 and 56 weeks, however not significantly. The contents of phenol, 2-nonanal and 2-methyl furan significantly increased between 26 and 56 weeks with no differences found between the cheeses made with different starters. The ethyl esters of acetic, butyric and hexanoic acid dominated the gas chromatograms for all cheeses, and six other esters were identified (Table 5). Their contents did not change significantly between 26 and 56 weeks of ripening, except for ethyl decanoate that decreased, and methyl hexanoate that was only found in the 56 weeks old cheeses (Table 5). Three different sulphur containing compounds were identified: dimethyl-sulfide (DMS), dimethyl-disulfide (DMDS) and dimethyltrisulfide (DMTS). The contents of DMTS decreased significantly between 26 and 56 weeks of ripening (Table 5). No differences in contents of sulphur compounds could be explained by the type of starter.

V2

4. Discussion V3

1.5

V4

1.0

V5

0.5

V6

0.0 10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

Rotation time (min)

Fig. 3. Peptide production after 24 h (a), 26 weeks (b) and 56 weeks (c). Numbered peptides were analysed by LCeMS and are presented in Table 3. Note the different scale on y-axis for (a).

AABA was not detected in any of the cheeses and GABA was only detected in low amounts, which showed the absence of microflora that converts Glu into GABA and AABA (results not shown). Trp was only found at low levels (<0.4 mol%) in the ripened cheeses that were under the detection limit for the younger cheeses (results not shown). Arg was only detected in low amounts in V2 at 3 weeks and in V2 and V3 at 12 weeks. The results for Ala at 26 weeks may contain traces of Arg due to co-elution (results not shown). 3.6. Volatile compounds Volatile compounds identified after 26 and 56 weeks of ripening included alcohols, aldehydes, ketones, esters and sulphur containing compounds (Table 5). Unfortunately, it was not possible to separate 2-methyl-butanal and 3-methyl-butanal, as well as the corresponding alcohols 2-methyl-1-butanol and 3-methyl-1-

The casein and peptide profiles of the extra-hard cheeses were characterised by chymosin and plasmin activity on as1- and b-casein, respectively, as well as accumulation of microbial breakdown products of peptides produced by these activities. Almost all as1-casein were degraded into as1-I-casein within 12 weeks of ripening. This has also been observed in the semi-hard cheese Herrgård after the same ripening time (Antonsson, Molin, & Ardö, 2003). In hard cheese varieties made with thermophilic starter and higher cooking temperatures, like Parmigiano Reggiano, as1casein may still remain in the ripened cheese due to inactivation of chymosin during cheese making (Gobbetti, 2004). The b-caseins were fully hydrolysed after 26 weeks of ripening and after 56 weeks the casein fraction of the cheese contained mainly g3-casein, confirming that plasmin activity was the main factor. The accumulation of the peptides b-CN f29e93 A1 and A2 was concluded to be characteristic for a cheese with significant plasmin and chymosin activity and limited proteolytic activity of the mesophilic starter, as compared with thermophilic starters, to further hydrolyse these peptides, which also has been observed in Herrgård cheese (Ardö et al., 2007). The free amino acids may contribute to the background flavour of cheese, and they are of high interest also due to their potential of being converted into a variety of aroma compounds. The total amount of amino acids in these cheeses after 12 and 26 weeks was found to be similar to the amount in the semi-hard cheese, Herrgård, (measured after 14 and 33 weeks, respectively) (Antonsson et al., 2003). A high content of free amino acids is characteristic of extra-hard cheese varieties ripened for a long time, in general

U. Rehn et al. / International Dairy Journal 20 (2010) 844e851

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Table 3 Suggested peptides (based on LCeMS results) found in the cheeses during ripening. Peptidea

RT (min)

Molecular mass (Da)

LCeMS

HPLCa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

2.8e3.3 2.9e3.7 3.4e4.1 7.4e7.5 11.1e11.5 11.4e11.8 13.4 13.0e13.4 14.8e15.0 16.0e16.8 17.5e17.6 18.1e18.2 16.2 21.2e22.0 26.2e26.3 26.6e26.8 29.8e29.9 32.3e32.5 33.1e33.2 32.7e33.1 33.1 33.1 33.1

29.0 29.0 29.7 34.0 38.6 39.8 41.8 42.7 44.8 45.9 48.7 49.0 50.7 56.6 64.7 65.0 72.2 75.7 76.7 76.7 76.7 76.7 76.7

24 25 26 27 28 29

32.8e33.2 33.4 33.2e33.9 33.8e33.9 33.9e34.0 33.9e34.0

76.7 76.7 76.7e78.0 78.0 78.0 78.0

Experimental

Suggested peptide

Referenceb

as1-CN f1-8 as1-CN f1e9c as1-CN f1-7 as1-CN f1-6 as1-CN f1-13 as1-CN f1-14 as2-CN f5-21 as2-CN f5-20 b-CN f12-28 b-CN f11-28 b-CN f8-28 b-CN f7-28 as1-CN f1-16 b-CN f1-28 as1-CN f1-23 k-CN f106-169 b-CN f193e209d b-CN f29e107 A1 b-CN f29e93 A1 b-CN f1e107 A1 b-CN f29e105 A1 b-CN f30e93 A1 b-CN f29e96 A2/ b-CN f30e97 A2 b-CN f29e107 A2 b-CN f1e105 A1 b-CN f1e107 A2 b-CN f29e93 A2 b-CN f29e105 A2 b-CN f1e105 A2

1 1,2 1 1 1,2 1,2 1,3,9 3 1,3 1,3 1,3 1 1,2 1,5,8 1,2,5,7,8 5 1,2,5,6,8 4,5,8 4 4,5,8 4,5,8 4 4

Theoretical

1140e1141 1012 875e876 748 1536 1665e1666 2274 2144 2214 2342e2344 2595e2596 2708e2710 1877e1878 3478e3480 2764e2765 6778e6780 1881e1882 9022e9024 7529e7532 12484 8757e8758 7403e7404 7730

1140 1012 875 746 1536 1665 2273 2145 2212 2341 2594 2707 1877 3478 2764 6776 1880 9023 7529 12483 8757 7402 7733

8981e8983 12218e12219 12442e12445 7490e7491 8717 12177e12179

8982 12217 12443 7490 8717 12177

4,9 4,6,8 4,9 4 4,5,8 4,5

a

Peptide numbers and HPLC retention times (RT) refer to Fig. 3. Peptide identification supported with results from analysis of cheese: 1) Gagnaire, Molle, Herrouin, and Leonil (2001), 2) Broadbent et al. (2002), 3) Lund and Ardö (2004), 4) Ardö et al. (2007), 5) Larsson, Zakora, Dejmek, and Ardö, (2006) and supported with results from experiments carried out in solution: 6) Visser and Slangen (1977), 7) McSweeney, Olson, Fox, Healy, and Højrup (1993), 8) Exterkate, Lagerwerf, Haverkamp, and van Schalkwijk (1997), 9: Le Bars and Gripon (1993). c Appears as a shoulder on peptide 1. d Mass spectra gave indications of other peptides present in the same peak. b

Amino acid (mol% of total content)

Ripening time (weeks) 3

12

26

56

Relative content in DL1 > DL2 Glu Ser Thr Ile Lys Met

* * * * ** *

** ns ** ns * **

ns ns ns * * ns

ns ns ns ns ns ns

Relative content in DL1
* ns * * *

* ** ns ns *

* ns * * *

* ns ns ns ns

500 cheese)

Table 4 Significant differences of the relative content of the amino acids (mol%) between cheeses made with starter cultures DL1 (vats V2, V3, V4) and DL2 (vats V1, V5, V6), respectively (significance: *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant).

a significantly lower relative content of Leu in the same cheeses, indicating that Leu was catabolised. The amount of methyl ketones generally increased during ripening, while secondary alcohols generally decreased, however, the differences between cheeses made with different starters were higher. Ketones have earlier been shown to make up an important group of volatile compounds in many hard cheese varieties such as Grana Padano, the second largest group of volatiles, and Parmigiano Reggiano, the largest group of volatiles (Barbieri et al., 1994; Moio & Addeo, 1998). Furthermore, 2-pentanone and 2-heptanone were

450

Total amino acid content (mmol kg

(Gobbetti, 2004), however, proteolytic activity in cheese with thermophilic starter bacteria is broader compared to the cheeses in this study. In several extra-hard cheese varieties, like Parmigiano Reggiano, Fossa and Mahón, the quantitatively dominating amino acids are, as in the cheeses studied here, found to be Glu, Val, Leu and Lys (Battistotti & Corradini, 1993; Frau, Massanet, Rosselló, Simal, & Cañellas, 1997; Gobbetti et al., 1999). The significantly higher content of 2/3-methyl-butanal in cheeses made with the starter DL1 as compared to DL2 could be correlated with

350

400

300 250 200 150 100 50 0 0

5

10

15

20

25

30

35

40

45

50

55

60

Ripening time (weeks)

Fig. 4. Total free amino acid content (mmol kg1 cheese) in V1 (-), V2 (,), V3 (:), V4 (6), V5 (A) and V6 (>).

850

U. Rehn et al. / International Dairy Journal 20 (2010) 844e851

Table 5 Volatile compounds identified after 26 and 56 weeks of ripening presented as an average of the integrated areas (relative to the internal standard) of cheeses made with the same starter (DL1 or DL2).a RTb (min)

26 weeks

Sign.d

56 weeks

DL1

DL2

Sign.c

DL1

DL2

Sign.c

10.4 18.8 3.9 5.3 9.1 17.8 8.4 7.5 13.6 15.1 32.7

ALCOHOLS 1-Butanol 1-Hexanol 2-Propanol 2-Butanol 2-Pentanol 2-Heptanol 3-Pentanol 2-Methyl-1-propanol 2/3-Methyl-1-butanole 3-Methyl-3-buten-1-ol Phenol

0.022 0.024 0.26 0.27 0.12 0.077 0.0081 0.043 0.048 0.0089 0.016

0.016 0.022 0.42 0.60 0.22 0.18 0.013 0.055 0.065 0.010 0.015

** ns *** ** ** *** ns ns ns ns ns

0.041 0.035 0.15 0.16 0.080 0.059 0.0054 0.046 0.053 0.031 0.027

0.032 0.047 0.34 0.42 0.26 0.22 0.011 0.065 0.12 0.043 0.027

ns ns * * * * ** * ** ns ns

*** *** ns ns ns ns ns ns ns *** **

2.6 3.1 4.2 12.1 20.2 16.0

KETONES 2-Propanone 2-Butanone 2-Pentanone/diacetyle 2-Heptanone 2-Nonanone 3-Hydroxy-2-butanone

0.38 0.75 1.25 0.32 0.023 0.85

0.072 0.14 0.13 0.047 0.0081 0.032

*** * ** *** *** *

0.56 0.71 1.42 0.59 0.042 1.20

0.25 0.33 0.51 0.25 0.023 0.13

* ns * * * ***

ns ns ns ns * ns

3.3 23.5 20.3

ALDEHYDES 2/3-Methylbutanale Benzaldehyde Nonanal

0.30 0.017 0.018

0.095 0.012 0.017

* ns ns

0.41 0.018 0.0098

0.24 0.016 0.015

* ns ns

ns ns *

3.0 3.9 5.6 9.7 14.6 21.5 26.3 18.8 12.4

ESTERS Ethyl acetate Ethyl propanoate Ethyl butanoate Ethyl pentanoate Ethyl hexanoate Ethyl octanoate Ethyl decanoate 2-Hydroxyethyl propanoate Methyl hexanoate

4.21 0.076 3.57 0.012 0.42 0.062 0.13 0.072 n.d.

3.75 0.044 2.51 0.0082 0.32 0.053 0.11 0.041 n.d.

ns ns ns ns ns ns ns ns ns

3.09 0.074 3.55 0.014 0.55 0.073 0.031 0.074 0.0043

3.48 0.062 3.16 0.013 0.50 0.071 0.029 0.059 0.0043

ns ns ns ns ns ns ns ns ns

ns ns ns ns ns ns *** ns ***

2.3 6.5 19.6

S-COMPOUNDS Dimethyl-sulfide (DMS) Dimethyl-disulfide (DMDS) Dimethyl-trisulfide (DMTS)

0.021 0.033 0.017

0.021 0.044 0.017

ns ns ns

0.018 0.061 0.0044

0.020 0.061 0.0046

ns ns ns

ns ns ***

5.1 10.1 2.9

OTHERS Alpha-pinene Delta-3-carene 2-Methyl-furan

0.043 0.019 0.011

0.043 0.017 0.0067

ns ns ns

0.033 0.016 0.017

0.035 0.016 0.016

ns ns ns

ns ns **

n.d.: not detected. a Significant differences between cheeses with different starters as well as between 26 and 56 weeks are indicated (*P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant). b Retention time. c One-way ANOVA between cheeses with different starters. d One-way ANOVA between 26 and 56 weeks of ripening. e 2-Pentanone and diacetyl (2,3-butanedione) co-eluted in the same peak; 2/3-methyl-1-butanol and 2/3-methylbutanal and could not be separated by mass spectra.

shown to be the two most abundant methyl ketones in aged Manchego cheese (Villasenõr, Valero, Sanz, & Martinez Castro, 2000). It could be concluded that the choice of starter significantly influenced the ratio between methyl ketones and secondary alcohols all through ripening. No large differences were seen between the cheeses concerning the contents of the identified esters and sulphur containing compounds. Further investigation should reveal the importance of ethyl esters of acetic, butyric and hexanoic acid, for the flavour of extra-hard cheese made with mesophilic DLstarter. The total amount of starter bacteria after 24 h was lower in the studied cheeses as compared with Herrgård cheese after 1 day: log 6.1e7.7 and log 8e9 cfu g1 cheese in Västerbottensost and Herrgård, respectively (Ardö, Thage, & Madsen, 2002). This may be explained by the use of relatively high temperatures for long time periods during cheese making.

Higher cell numbers on MRS pH 6.5 (as a measure of total amount of LAB) after 24 h and after 3 weeks were observed in cheeses made with DL1 as compared with DL2. The number of starter bacteria early after production may be crucial for the ripening pattern in several ways. They contribute to a large amount of a range of enzymes of great importance for metabolism of cheese constituents, especially after damage of the cell membranes that finally may lead to their lysis. These enzymes contribute to amino acid release and conversion to compounds with flavour impact (Ardö, 2006). 5. Conclusions The number of starter bacteria started to decline after 3 weeks of ripening and the total number of lactic acid bacteria varied in the interval log 6e7 from 12 weeks and throughout ripening. Proteolysis of the extra-hard cheese with mesophilic DL-starter was

U. Rehn et al. / International Dairy Journal 20 (2010) 844e851

characterised by both plasmin and chymosin activities, which is untypical for hard cheese varieties with higher cooking temperatures, for which chymosin is more or less inactivated. Peptides from starter activity on the chymosin derived peptide as1-CN f1e23 and plasmin derived peptide b-CN f1e107, also characterised the peptide profile. The peptide b-CN f29e93 accumulated characteristically during ripening, likely as a result of joint plasmin and chymosin activity and the absence of proteolytic thermophilic bacteria to mediate further degradation. The type of mesophilic DL-starter influenced the composition of free amino acids and the content of volatile compounds in the ripened cheeses. Secondary alcohols generally decreased during ripening, whereas methyl ketones generally increased, and significant differences were found between cheeses made with different DL-starters. Acknowledgements Finn Kvist Vogensen and Bashir Aideh are gratefully acknowledged for fruitful discussions. Mona Østergaard is acknowledged for excellent technical help with the analysis of caseins, peptides and amino acids. We also thank Mehdi Darestani Farahani for kindly running the GCeMS analyses. References Antonsson, M., Molin, G., & Ardö, Y. (2003). Proteolysis of the semi-hard cheese Herrgård made at different dairies. Exploratory study. Milchwissenschaft, 58, 145e148. Ardö, Y. (1993). Swedish cheese varieties. In P. F. Fox (Ed.), Cheese: Chemistry, physics and microbiology, Vol. 2 (2nd ed., pp. 254e256) London, UK: Chapman and Hall. Ardö, Y. (2001). Cheese ripening. General mechanisms and specific cheese varieties. Bulletin of the International Dairy Federation, 369, 7e12. Ardö, Y., et al. (2004). Semihard Scandinavian cheese made with mesophilic DL-starter. In Y.H. Hui, L. Meunier-Goddik, Å.S. Hansen, J. Josephsen, W.-K. Nip, P.S. Stanfield et al. (Eds.), Handbook of fermented food and beverages (pp. 277e290). New York, NY, USA: Marcel Dekker Inc. Ardö, Y. (2006). Flavour formation by amino acid catabolism. Biotechnology Advances, 24, 238e242. Ardö, Y., & Gripon, J.-C. (1995). Comparative study of peptidolysis in some semihard round-eyed cheese varieties with different fat content. Journal of Dairy Research, 62, 543e547. Ardö, Y., Lilbæk, H., Kristiansen, K. R., Zakora, M., & Otte, J. (2007). Identification of large phosphopeptides from b-casein that characteristically accumulate during ripening of the semi-hard cheese Herrgård. International Dairy Journal, 17, 513e524. Ardö, Y., & Polychroniadou, A. (Eds.). (1999). Laboratory manual for analysis of cheese. Luxembourg: European communities, Office for Official Publications of the European Communities. Ardö, Y., Thage, B. V., & Madsen, J. S. (2002). Dynamics of free amino acid composition in cheese ripening. Australian Journal of Dairy Technology, 57, 109e115. Barbieri, G., Bolzoni, L., Careri, M., Mangia, A., Parolai, G., Spagnoli, S., et al. (1994). Study of the volatile fraction of Parmesan cheese. Journal of Agricultural and Food Chemistry, 42, 1170e1176. Battistotti, B., & Corradini, C. (1993). Italian cheese. In P. F. Fox (Ed.), Cheese: Chemistry, physics and microbiology, Vol. 2 (3rd ed., pp. 221e244) London, UK: Chapman and Hall. van den Berg, G., Meijer, W. C., Düsterhöft, E.-M., & Smit, G. (2004). Gouda and related cheeses. In P. F. Fox, P. L. H. McSweeney, T. Cogan, & T. P. Guinee (Eds.), Cheese: Chemistry, physics and microbiology, Vol. 2 (3rd ed., pp. 103e140). San Diego, CA, USA: Elsevier Academic Press.

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