Food Chemistry 111 (2008) 606–615

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Changes of free amino acid content of Teleme cheese made
with different types of milk and culture
Eleni C. Pappa a,*, Kyriaki Sotirakoglou b
National Agricultural Research Foundation, Dairy Research Institute, 452 21 Katsikas, Ioannina, Greece
Department of Mathematics, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece

a r t i c l e i n f o a b s t r a c t

Article history: The evolution of concentration of free amino acids in Teleme cheese made from sheep, goat or cow milk,
Received 24 October 2007 using a thermophilic, mesophilic or a mixture of a thermophilic, a mesophilic culture throughout ripening
Received in revised form 8 February 2008 was studied. The total free amino acid (TFAA) content increased at all stages of ripening, regardless of the
Accepted 10 April 2008
milk and culture used. In general, the TFAA content was higher in cheeses made from cow’s milk than that
of the cheeses made from ewe’s milk; cheese from goat’s milk ranged over intermediate levels. Also,
higher concentrations of TFAA were found in cheeses made with the thermophilic than with the meso-
philic culture. Cheeses made with the mixture of thermophilic–mesophilic culture ranged over interme-
White-brined cheese
diate levels. The results of this study have shown that Leu, Glu, Phe, Val and Lys were the major FAA of
Amino acids Teleme cheese at all stages of ripening, regardless of the type of milk and culture used.
Starter culture Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction the volatile fraction of cheese (Salles et al., 2000) because they
are precursors of many volatile aroma compounds (e.g. amines,
Proteolysis is the most complex and, in many varieties, the most carboxylic acids, thiols, esters, alcohols, aldehydes and thioesters).
important biochemical event during ripening. The pattern of prote- White-brined cheese, like other types of ripened cheese, needs
olysis can be summarized as follows: the caseins are hydrolyzed maturation to develop the required properties. White-brined
initially by residual coagulant activity retained in the curd and cheeses are in great demand in warm climates and their preserva-
by plasmin and perhaps by other indigenous proteolytic enzymes tion in brine is necessary. Teleme cheese is a soft white- brined
to a range of intermediate-sized peptides, which are hydrolyzed cheese that can be produced using caprine, ovine or bovine milk
by proteinases and peptidases from the starter lactic acid bacteria, alone or any mixture of them (Greek Codex Alimentarius, 1998).
non-starter lactic acid bacteria (NS-LAB) and secondary microflora In Teleme cheese production, traditional yogurt is used as starter
to shorter peptides and amino acids (Upadhyay, McSweeney, by the traditional cheese-makers, while mixtures of commercial
Magboul, & Fox, 2004). Every type of cheese has its own character- starters, including mesophilic starters, are used in industrial
istic free amino acid (FAA) pattern, resulting from the enzymatic production (Anifantakis, 1991). The kind of milk used in cheese
degradation of peptides by various enzymes and also from amino production affects the activity of the starter culture and has a
acid interconversion and degradation (Polo, Ramos, & Sanchez, direct effect on the free amino acid profile of the cheese.
1985). The concentrations of the different amino acids in a cheese There are few publications available on amino acid profile of
are related to the manufacturing technology (type of curd, addition Teleme cheese. Mallatou, Pappa, and Boumba (2004) studied the
of starters, ripening conditions), duration of ripening and the ex- profile of amino acids of Teleme cheese made from sheep’s, goat’s
tent and type of proteolysis (Christensen, Johnson, & Steele, or mixture of sheep’s-goat’s (1:1) milks with mixed thermophilic–
1995). Certain free amino acids (FAA) are extremely important in mesophilic cultures. Buriana and Farag (1983) determined the ami-
flavour and taste development, e.g. Arg is related to bitterness, no acid profile of Teleme cheese made from cow, sheep or mixtures
while Pro, Ser and Asn are related to sweetness (Izco & Torre, (1:2) of these milks with Lactococcus lactis subsp. lactis and Lb.
2000). Although the direct contribution of FAA to the typical casei. Polychroniadou and Vlachos (1979) investigated the amino
cheese flavours are somewhat limited, they are associated with acid profile of Teleme cheese made from a mixture of goat’s-
sheep’s (1:9) milk with thermophilic culture, whereas, Alichanidis,
* Corresponding author. Tel.: +30 26510 94785; fax: +30 26510 92523. Polychroniadou, Tzanetakis, and Vafopoulou (1981) studied the
E-mail address: (E.C. Pappa). amino acid profile of Teleme cheese from deep-frozen curd made

0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.

the column was equili- 64.64 and 0. Samples were applied using a rheodyne injector (model 7725i). (Waters. 1998). IL.90  30 cm) was used. Louis. whereas. sonicated in a water bath for 20 min and centrifuged at Laboratorium.3. On the day of cheese-making. 6 and 8 are convex. A 2. mine: acidic and neutral amino acids: basic amino acids 1:1:1. was manufactured using (a) sheep’s milk of the ‘‘Boutsiko” breed The method of Bidlingmeyer. USA) and were filtered through a lots of cheese were obtained. trol station. These results could serve as a model for fu. was added as the internal Teleme cheese with three starter culture variables: (i) a freeze. on a HV hydrophilic filter (Millipore) with 0. Identification of the cheese amino acids was achieved by compar- 2. culture (M). standardized milk was pasteurized at 63 °C for 30 min and then Standard mixtures of basic. at 33 °C. bulgaricus and 1 M sodium acetate 1:2:2. The working amino acid standard was prepared as described in Teleme cheese was manufactured as described previously (Pap. Teleme cheeses 0–3% eluent B over 13. Each kind of milk was used in making taining 0. as de- from the Agricultural Station of Ioannina (b) goat’s milk from a scribed in the PICO Tag amino acid analysis Operator’s Manual local native goat population and (c) cow’s milk from a herd of (1984) was applied. 13. Methionine sulfone was added as an internal standard and P43% fat in dry matter (Greek Codex Alimentarius. Phenylisothiocyanate (PITC) (sequential of milk CaCl2 solution (40%. v/v/v) were added to the dried samples. USA). A Waters reverse phase column (3. 3000g for 10 min. brated with solvent A for 20 min. w/v) was added. The samples were incu- the microorganisms Lactococcus lactis subsp. respectively and 6. respectively. Pappa. respectively. Run time was 67 min with flow rate 1 ml min 1. Tag system. are no reports of the effect of different cultures on the amino acid as well as on the day they were transferred to the cold room. The objective of this work was to study the effect of three com. Horsholm.45 lm pore size before day after day basis. to obtain a first quality cheese that is 656% moisture v/v/v). Free amino acids were extracted from cheese Friesian cows. The (standard solution: internal standard 1:1. FRC-60 (Hansen’s Laboratorium. an ice bath and then centrifuged at 20. respectively and afterwards cheeses were trans. Lb. K. near the Dairy Research Institute where the as follows: 1 g of cheese was weighed and 10 ml of 0. E. S. a mesophilic or a mixture of a thermophilic. FYS-11 (Rhone-Poulenc blender. until both pH and moisture values reached the limits of and 15% methanol. 60. transferred to the cold room at 22.55 with acetic acid con- After manufacture.mesophilic culture. cremoris. Rennet powder HA-LA grade) and thiethylamine were from Pierce (Rockford. which were vacuum-dried again. 17 and 15 dients. at a quantity of 0. & Zerfiridis. . with 40% trichloroacetic acid. then 3–6% eluent B made from sheep’s milk using thermophilic. and curve 11 maintains starting conditions days. cheeses were ripened in the warm room taining 2. To assist curdling (St. Denmark) was added at a The solvents were of HPLC grade. then followed by a purge at 100% elu- goat’s milk. The mixture was left for 10 min in (ii) a freeze-dried concentrated mixture of thermophilic–meso. 500 ll of supernatant were diluted (1:1. thermophilic–meso. respectively. Materials and methods nium 32 software package. itored at 254 nm. Briefly. 25 ll of philic culture (MX).C. standard. Drainage was under ical grade. holding 24. Kandarakis. con- cheese-makings took place. 20 ll of a redrying solution (triethylamine: methanol: Lactococcus lactis subsp. consisting of the microorganisms Strepto. goat’s or cow’s tives by RP-HPLC using the PICO-Tag amino acid analysis system milk. the milk was standardized to casein to fat (1984). Eluent A comprised at 19 °C. This was repeated five times. Milford. equipped with a 50 ll sample loop. Experimental design volume of 20 ll of the amino acids was injected into the column. over 10.73 for the sheep.1. MO.75. France). w/w) for 16 h according to the Operator’s Manual (1984).000g for 10 min. using thermophilic. Samples were suspended in 100 ll of Pico Tag sample milk was used in making the three variables of cultures and nine diluent (Waters. v/v) coccus thermophilus and Lactobacillus delbrueckii subsp. Curves 5.5% acetonitrile. delbrueckii subsp.0 g per 100 kg milk. 16 and 12 days. except that glutamine (2. a solvent pump (mod- el 600E). cheeses made from at 34% eluent B for 12 min. mixed or mes. then 6–9% eluent B over 6 min using philic or mesophilic culture were transferred to the cold room at curve 5. MA. too (gluta- ratios of 0. Denmark). Before each injection. a helium degasser and a tunable absorbance detector (model 486). To our knowledge.4 mM methionine sulfone. MA. methionine sulfone and glutamine were purchased from Sigma tioned was added to it.. Gradient elution was as follows: ferred to a cold room (5 °C) for up to 6 months. mixed or mesophilic culture. The mixture was homogenized using an Ultra-Turrax dried yogurt thermophilic culture (T). After cooling. during ripening. Separations were conducted at 45 °C and the absorbance was mon- The experimental design was that described elsewhere (Pappa. 20 ll of derivatizing solution ganismds was 1:1) and (iii) a freeze-dried concentrated mesophilic (methanol: water: triethylamine: phenylisothiocyanate 7:1:1:1. v/v). 2006a).2. Milford. Samples were analyzed on a WATERS ture research in this field. namely a thermophilic. 2. each kind of uum-dried. the deproteinized supernatant were vacuum-dried in the PICO- consisting of the microorganisms Lactococcus lactis subsp. detail in the PICO Tag amino acid analysis Operator’s Manual pa et al. one of the cultures already men. 40% water (19 °C).5 min using curve 11. until the next step. Eluent B was 45% acetonitrile. Curves 5. injection. Sotirakoglou / Food Chemistry 111 (2008) 606–615 607 with the same type of milk and culture. and Tarvin (1984). 70 mM sodium acetate adjusted to pH 6. 0. 120 and 180. goat and cow milk. profile of Teleme cheese. Cohen. there Cheeses were sampled for analysis on days 1. R-703 (Hansen’s Laboratorium. respectively.5 mM) was added. linear and concave gra- ophilic culture were transferred to the cold room at 23. Denmark) consisting of v/v/v/v) were added to the dried samples. The chromatograms were processed using the Millen- 2. USA). on free amino The FAA were identified as phenylthiocarbamyl (PTC)-deriva- acid release of Teleme cheese made from sheep’s. acidic and neutral amino acids and cooled at 37 °C. (Hansen’s Laboratorium.5 min and re-equlibration to 100% eluent A.5 min using curve 8. 11 days. Analysis of free amino acids (FAA) and chemical analysis mercial concentrated starters. were ent B for 4. Anifantakis. That is. then 9–34% eluent B over 20 min using curve 6. 2006a). Cheese-making and sampling ing retention times with pure standards. cremoris.1 M HCl. lactis. lactis and Lactococcus bated for 10 min at room temperature and then they were vac- lactis subsp. The solvent system consisted of two eluents made pressure and salting took place in brine (14% NaCl. All other reagents were analyt- quantity of 3. 8 and 11 are gradients programmed by the Waters HPLC con- cheeses made from cow’s milk using thermophilic.6 and 656%. HPLC consisting of a controller (model 600).5% (v/v). bulgaricus. (the ratio of thermophilic to mesophilic microor. thermophilus. Teleme cheese HPLC analyses were done in duplicate. USA).

B_Ala AAB 4 Component 2 (10.68% of the total variability and was defined by AAB. Gly and Lys. PCA of the 27 free amino acids resulted in eight principal components with eigen. 1.608 E. USA and GraphPad Prism version 4.42%) 2 Asp AAB ability. at different ages. . first three principal components were the most important. Cheeses made from Amino acids were identified according to their retention times sheep’s or cow’s milk had the opposite characteristics and were by comparison with a standard mixture solution chromatogram. MD. The first principal Cys Thr Gaba Tyr component explained 30. 2. a comparison of the values of each Cit Met parameter of Teleme cheese manufactured with one type of milk -4 Gaba (sheep’s. Consequently.. One-way analysis of variance was used for the -9 -6 -3 0 3 6 comparison of the data of each parameter of Teleme cheese made Component 1 (30. near Pro and Ala and therefore 3. Leu Arg Lys Ile Val Orn indicating that they were positively correlated with each other. at each -6 age. Cheeses made from goat’s milk were clus- tered on the negative side of PC3. standard solutions of pure amino acids had eigenvalues greater than 2.8. b-Ala (posi- milk and the stage of ripening. The third principal component explained another 10.85% of the total variability and fied peaks.0.75% of the total variability. Sotirakoglou / Food Chemistry 111 (2008) 606–615 The dry matter (DM) of the cheese samples was determined Biplot according to the IDF standard (IDF. having the highest contents of TFAA. Principal component analysis (PCA) was applied to pooled data in order to reduce the dimensionality of the data and to detect the most important causes of variability. and the results were ex- 6 pressed on a dry matter basis. therefore they were negatively correlated to Statistical analysis was performed using statistical packages this. 0 GlyPhe Orn mixed thermophilic–mesophilic or mesophilic) and the kind of Gln Arg Tyr milk (sheep’s. free amino acids were represented as a function Cit of both first and second principal components. ing opposite to Gaba. 1). together with the intact FAA. 2 AAB and b-Ala were for selecting discriminant variables. The least significant differences were obtained using the LSD test Fig. 2). SPSS Inc. while -6 -4 -2 0 2 4 6 cheeses 120 to 180 days old were placed on the negative side of Component 2 (10. All the cheeses that were located on the left of Fig. a number of pal component could be a representative of the kind of milk. In Fig.75%) with one type of milk and one type of culture. these cheeses had the lowest content of Ser. cipal component could be a representative of the type of culture. These amino acids were Gln Phe His placed closed together on the negative side of the horizontal axis. CA. the third princi- During the extraction and derivatization process. Asn. The eight selected components accounted for 79.68%) 2. sented the opposite characteristics. USA. San Diego. than cheeses made with the other cultures. suggesting that they were well represented from the first PC.14% of the total vari. a common statistical cut-off point. Chicago. from 1 -4 to 60 days old. When necessary.4. The second principal component explained the second and third principal components.68%) PC1 (Fig.2. which could be considered as Ala Pro representative of the ripening time. Principal component analysis. goat’s or cow’s) as factors and ripening time as Asn Leu -2 split–splot factor. placed on the positive side of PC3. On the other hand. Wilk’s lambda (k) criterion was used tive values) and Gaba (negative value). Linear regression analyses placed close together on the positive side of PC2. generally. Plot of the free amino acids as a function of Asn. However. -2 Tau They were away from the axis origin. mixed thermophilic–mesophilic or mesophilic). Statistical analysis Val Ile Glu Trp 2 Pro A three-way split–splot analysis of variance (ANOVA) was ap. STAT- AAB and b-Ala and therefore had higher contents of these amino GRAPHICS Plus for Windows version 5..C. which repre- ville. In Fig. was made. Gly and Lys. were clustered on the positive side of PC1. were co-injected with the standard mixture solution to identify peaks in the standard solution. goat’s or cow’s) with the three types of culture (thermo- philic. Results and discussion and Ser (positive value). Principal component analysis (PCA) was used to reduce the dimensionality of the data and stepwise discriminant analysis was also applied to establish those amino acids capable of discrim- inating and classifying cheeses by the type of starter. Moreover. IL. Principal component analysis. Cheeses made with thermophilic culture were clustered near (SPSS for Windows version 14. The unidentified peaks were present. the type of 10. Consequently the second prin- USA). K. these peaks did not interfere with the identi- together they accounted for 51. Ser B_Ala Component 3 (10. 1986). 1. Plot of the free amino acids as a function of the two first principal components.42% of the total variability and was defined by Pro.1. Ala (negative values) 3. Pappa. because all cheeses. indicating a high were applied to fit the evolution of free amino acids with ripening positive correlation. (P < 0. Rock- acids. 4 values greater than 1. considering the type of culture (thermophilic. 1 are ripened 180 d old cheeses. these amino acids were ly- time. Manugistics Inc. TFAA AspThrHis Tau AlaCys Lys Ser plied to the data.0. since a great correlation be- Biplot tween the free amino acid contents was noticed. Fig. General had the highest contents of Pro and Ala (Fig. and was Met Asn Gly Trp Glu 0 TFAA mainly defined by TFAA.05). On the other hand.

990 * 0.914 *** P < 0. to 424 mg 100 g 1 DM in 60 day old cheese. 2004.997 *** 0.839 His *** 0. 1728 mg 100 g 1 DM. regardless of the type of milk and culture used. Hayaloglou. Val and after 180 days of ripening and re-increased rapidly until day 360.833 Asn *** 0.994 *** 0.988 *** 0.886 *** 0. 3. All two-way and three- way interactions between the factors were significant (P < 0. Effect of ripening time 800 Total amino acids mg/100g DM The results of the three-way split–splot ANOVA (Table 1) showed that the main effects.910 NS 0. The increase of TFFA at all stages of ripening.988 *** 0.937 *** 0.868 *** 0. increase of hydrophilic peptides and decrease of hydrophobic Edam cheese 1056 mg 100 g 1 DM and in Romano cheese peptides.953 Gly *** 0.939 *** 0. Ibanez.983 *** 0. Franco.05) 200 for the TFAA and for all individual free amino acids.991 Cys *** 0.938 Ala *** 0.509 *** 0.958 *** 0. and Ordonez (1995) reported an increase from et al.05).934 ** 0. to in Teleme cheese by Mallatou et al.901 *** 0.900 Thr *** 0.905 *** 0.988 *** 0.. 2001. & Xavier Mal- study. At the age of 180 nificantly from 295 mg 100 g 1 DM in 1 day old cheese to days. The proteolysis of as1-casein.932 *** 0.962 Tau *** 0.05.951 *** 0.970 Val *** 0.863 *** 0.994 *** 0.742 * 0.980 *** 0.897 *** 0. Effect size Sig.913 Glu *** 0.912 *** 0. Table 2 shows the concentrations of indi.989 *** 0.978 *** 0.938 *** 0. Effect size Sig. in tent.824 *** 0.5% of the TFFA.8 in most cases). Ibanez. decrease of the intact casein con.996 *** 0.912 *** 0.993 *** 0.980 *** 0. 0 60 120 180 vidual FAA of Teleme cheese made with different starter cultures Ripening day and milks during ripening. 100 g 1 DM in180 day old cheese (P < 0. In Table 2 it can be seen that the con- Fig.960 *** 0.995 *** 0.990 *** 0.965 *** 0.984 *** 0. Javier Carballo. . Buriana and Farag (1983) found that the TFFA decreased From Table 2 and Fig.983 *** 0. type of culture (C) and ripening time (T) Amino acids M C T MC MT CT MCT Sig.954 Asp *** 0.979 *** 0.926 *** 0.948 *** 0.590 *** 0.961 *** 0. The levels of TFFA Fox. The same FAA were found in abundance cold room.982 *** 0. these compounds are released by proteolytic agents.823 *** 0.984 *** 0.634 *** 0.940 *** 0.977 *** 0.928 *** 0.958 *** 0.908 *** 0.967 Lys *** 0.908 *** 0.966 *** 0. in Cheddar cheese 1592 mg 100 g 1 DM.913 *** 0.938 *** 0.970 *** 0. Table 1 also shows the effect size (>0. Effect size Sig.493 *** 0. centrations of almost all FAA showed a clear tendency to increase with ripening time.869 *** 0. Pappa.824 Cit *** 0.990 *** 0.985 *** 0.993 *** 0.973 TFFA *** 0.959 *** 0. Effect size Sig. which has a high content Table 1 Significance levels and size effect of the three-way split–splot ANOVA for the effects.946 *** 0.994 *** 0.949 *** 0.925 *** 0.960 *** 0.990 *** 0.984 Phe *** 0. & McS- found in this study.974 *** 0. which describes the pro- 0 portion of total variability attributable to a factor.732 *** 0.683 *** 0.966 *** 0.903 *** 0. they accounted for 41.979 *** 0.942 *** 0. Zerfiridis.C.883 *** 0.001).903 *** 0.985 *** 0.994 *** 0.974 ** 0.05) and Tau. (2004). as was expected since.980 *** 0.740 Arg *** 0. in this 650 mg 100 g 1 DM (Tavaria.971 *** 0. Kand- arakis.900 Pro *** 0.915 *** 0. TFFA mean content of Teleme cheese increased sig.902 *** 0.894 *** 0.952 Bala *** 0.990 *** 0.434 *** 0. as well as in other cheeses (Hayaloglou. 600 ening time. 2003).926 *** 0.736 *** 0.986 *** 0.975 *** 0.939 *** 0.934 Leu *** 0. which was not significantly different with respect to starter addition (P > 0. Hannon. The FAA in Manchego cheese typically contribute the increase of nitrogen faction. K. type of culture and rip.993 *** 0. 1995).964 *** 0. Fig.907 *** 0. 3 presents the evolution of mean total free amino acids (TFAA) of Teleme cheese during ripening. Guven.989 *** 0.866 *** 0.916 Trp *** 0.993 *** 0.078 *** 0. were significant (P < 0. Sotirakoglou / Food Chemistry 111 (2008) 606–615 609 3.985 *** 0.668 ** 0. Vicente.962 *** 0.993 *** 0. Evolution of total free amino acids during the ripening time. as estimated by bal cheese.984 *** 0.861 * 0. Guven.965 *** 0.001) for the total free amino acids (TFAA) of Teleme cheese and for all of the individual free ami- no acids.970 *** 0.981 0. kind of milk. is in accordance with the results found by Mallatou et al.939 *** 0. (2004) and Polychroniadou and 492 mg 100 g 1 DM in 120 day old cheese and finally to 675 mg Vlachos (1979).996 *** 0.980 Ile *** 0.882 *** 0. Effect size AAB *** 0.986 *** 0. Barcina.982 *** 0.965 NS 0.943 *** 0. Lys were the major FAA of Teleme cheese at all stages of ripening. Effect size Sig.964 *** 0.989 *** 0.984 Tyr *** 0.872 *** 0.875 *** 0.953 *** 0. weeney.423 *** 0. cata.930 Orn *** 0.991 *** 0.985 *** 0.964 *** 0.934 *** 0. E. 4 it can be seen that Leu.577 *** 0. Barcina.849 *** 0. Phe.01.811 *** 0.936 *** 0.944 *** 0. Generally.911 *** 0. except Ala.921 Gaba *** 0.966 *** 0.971 *** 0.971 *** 0. Fox. Pappa.940 *** 0.952 *** 0.620 *** 0. during proteolysis. & Barron. regardless of the type 316 mg 100 g 1 DM on the day cheeses were transferred to the of milk and culture used. which did not change significantly during rip.957 *** 0.925 *** 0. are within the range reported from other stud. Effect size Sig. NS: not significant.979 *** 0. Glu. **P < 0. Barcina ies. Anifantakis.991 *** 0. 2005.966 *** 0.860 *** 0. 400 ening time (P > 0.2.598 *** 0.852 *** 0.973 *** 0. and Sotirakoglou (2006b) reported an 200 mg 100 g 1 DM in 1 day old to 1300 mg 100 g 1 DM in Idiaza- increase of proteolysis during cheese maturation.949 Met *** 0.940 *** 0.986 *** 0.796 *** 0.880 *** 0. kind of milk (M).001.990 *** 0.550 *** 0. & McSweeney.896 *** 0.909 *** 0.806 *** 0. *P < 0.867 *** 0.931 Gln *** 0.829 *** 0.996 *** 0.923 *** 0.930 Ser *** 0.988 *** 0.

42a 0.42a 8.71a 10.91a 16.61b 7.72a 0.92a 44.41b 2.31b 5.21a 37.21a 13.51b 24.41a 1.31a 1.42a 4.41a 4.12b 1–3 : For each age and each kind of milk.412a 10.02a 11.91a 6.81b 17.61b 2.82a 17.72a 4.71a 6.21a 3.21a 3.41a 16.02a 36.52a 46.01a 0.91a 0.12a 23.82a 7.91b 11.21a 10.13c 67.91a 0.42a 3.71a 1.01a 17.02a 0.42a 31.42a 21.02a 0.52a 32.71b 0.72c 4.02b 2. a–c : For each age and each kind of culture.72b 13.71c 8.91b 16.81b 7.53a 34.72a 34.81b 12.41a 1.93a 9.33b 0.52b Trp 2. .81a 0.812ab 12.81a 0.05) between kinds of milk.11 2.81a 6.13a 19.71a 0.72a 0.51a 15.71a 5.01b 6.32b Ala 3.52a 64.02a 11.05) between kinds of cultures.51a 13.22a 63.63a 17.91b 4.81a 3.92a b-Ala 16.21b Pro 7.82c 2.01b 28.53c Phe 22.81b 1.71a E.32a Arg 27. a mesophilic (M) or a mixture of thermophilic and mesophilic (MX) starter during ripening Age.21b 3.71a 14.01a 6.92a 2.52a 3.11b 24.11b 0.01a 6.61a 1.41a 1.21a 0.21a 14.81b 1.01a 3.712a 8.92a 16.81c 2.42b 7.41b 23.21b 3.31b 23.81b 6.21a 1.81b 4.11a 1.12ab 5.91b 1.42b 4.312b 15.212a 32.02ab 16.61a 13.42a 10.81ab 2. different letters indicate significant differences (P < 0. (days) 1 RB Milk S G C S G C Culture T MX M T MX M T MX M T MX M T MX M T MX M Asp 1.62a 0.41b 13.21b 1.51a 9.61ab 9.71a 10.21a 6.61b 3.03a 20.11a 3.92a 13.31a 1.62a 2.71b Met 18.91a 2.82a 9.21a 1.12a 1.81a Orn 14.91b 7.12a 1.33a 2.92a 8.51b 16.82a 5.31ab 1.01a His 4.63b 15.12b 4.42ab 7.93b 8.41a 25.02ab 13.02a 15.11a 11.83a 3.11b 2.62b 0.32b 7.62ab 2.01b 5.21a 3.52b 3.21a 13. A DM: Dry Matter.81a 43.92b 33.81c 8. goat’s (G) or cow’s (C) milk using a thermophilic (T).91b Asn 12. Pappa.22a 2.22a 3.92b 18.01b 16.82a 0.02a 8.51a 2.21b 5.01a 4.52a 35.72b 2.82b AAB 26.12a 6.22a 1.21b 7.11b 2.72c 36.02b 1.32a 16.01b Ile 40.42a 5.12a 10.02a 1.11a 29.51b 19. %.71a 33.01a 2.22a 5.02a 1.63c 18.212b 14.61a 1.33b 56.12a 32.93b 0.72b 3.22a 15.22a 8.03b 84.73a 43.52b 21.01a 2.52a 7.712a 0.81b 7.01c 3.52a 0.13a 29.412b 25.22a 0. Sotirakoglou / Food Chemistry 111 (2008) 606–615 Glu 30.92c 1.93a 28.52a 1.61a 1.12a 3.93a 21.93a 19.61a 14.62a 6.81a 6.71b Leu 23.43c Ser 3.82a 5.31a 49.81a 9.51a 8.71b 5.31b 10.02a 6.93b 1.41b 12.91a 2.31b 3.72a 0.21b 4.52a 9.33c 0.21c 6.12ab 0.02b 58.22b Gln 7.73b 17.91b 1. different numbers indicate significant differences (P < 0.02b 2.41c 22.92a 26.33a 23.81a 0.52a 8.22a 0.32a 34.81a 9.11b 16.22a 84.52a 11.01a 8.82a 12.02b 0.52a 3.02b 0.82a 6.92a 3.52b 33.71a 0.71b Lys 66.01c 4.81b 26.71a 3.51b 3.31a 0.02b 2.02a 20.32b Cit 0.61a 14.71ab 13.01a 8.91a 5.62a 29.01a 1.912a 23.11b 22.32b 22.312c 11.11b 1.81b 3.21a 29.11b 19.12a 12.81a 9.22a 3.42b 11.01b 0.72a 35.81a 25.51b 8.61b 5.21c 3.31c 37.21b 7.22a 0.22a 11.81c 5.91a 3.71a 1.93b Val 16.31a 6.91b 3.02b 7.52b 7.22a 9. 610 Table 2a 1 Changes of amino acid contents (mg 100 g DMA) of Teleme cheese made from sheep’s (S).61a 3.02b 0.82c 0.91a 0.12a 1.11a 6. K.12c 5.41b 7.31a 6.31a 3.42a 3.92a 16.71a 3.112a 3.12a 11.21a 2.02a 9.62a 7.81a 4.72a 38.61b 6.21a 8.02a 9.02a 4.21b 5.62a 35.21b 13.11b 6.11b 38.52b 3.52a 2.91b 2.C.81a 8.32a 11.41a 52.512a 26.42a 0.01b 5. B R: Day cheeses were transferred to cold room.81a 1.02a 2.81a 4.82b 8.21b 11.71b 23.22a 1.33a 6.33a 19.53b 55.02a 0.31b 5.32a 16.12a 26.51c Tyr 11.02a 10.01b 14.92b 5.32b 23.11a 18.92a 7.31b 16.91b 14.32b 17.23a 7.22a 11.11a 2.71a 11.31a 8.11b 1.91a 73.52a 15.41a 2.112c 19.22a 10.43b 2.11a 35.312a 7.51a 2.81ab 19.81b 68.03b 0.712a 2.11b 17.82b 3.41b 3.81b 19.11a 2.92a 15.12a 5.62a 40.61a 11.43c Gaba 3.51a 0.91b 14.32b 8.72a 6.91a 13.81a 8.81b 5.81a 17.92a 1.02a 4.72b 1.12c Tau 2.42a 1.21b 9.01a 1.11a 11.62a 2.212a 3.41a 21.52a 3.01c 37.81b 15.81b 12.52a 0.12a 7.51a 12.71a 5.31c 2.12c 8.01a 0.11b 10.412b 4.12b Gly 6.31a 3.61a 6.02c 4.43c Cys 6.12a 4.81a 0.71b 28.01a 0.23a 51.012a 1.92a 7.12b 11.61a 4.51ab 6.01b 38.11b 4.42a 20.11b 31.12b 12.71a 6.71a 13.82b Thr 5.83a 22.61a 1.81b 24.

91a 7.41b 2.21a 20.73ab 4.62a 13.51a 11.73a 43.42b 13.91c 15.32a 7.51b 1.12b 12.91a 0.91a 18.61b 8.02b 17.21b 2.92b 40.52a 30. different letters indicate significant differences (P < 0.12a 12.41a AAB 46.52b 12.61a 30.81b 19.11a 2. a–c : For each age and each kind of culture. Pappa.52a 28.11a 3.01b 25.92b 16.62c 2.52a 0.41a 0.01ab 3.52a 6.05) between kinds of cultures.71a 21.212a 26.72b 1.91b 20.02a 23.91a 4.61b 0.11a 21.52a 10.31b 27.12a 2.61c 2.83a 22.91a 3.52a 36.71a 8.01a 3.71a 0.01b 101.92a 20.21a 18.22a 6.12b 8.91b 1.52a 8.42b 16.51b 0.01b 2.31a 13.22b 3.03b 9.32b 67. K.41a 5.62c 9.41a 28.21a 5.81b 17.61a 7.01a 2.33b 5.92a 6.512a 7.22a 3.53b Ile 35.11a 30.12c 7.82a 1.41a 31.71a 4.62a 3.71b Ala 4.61a 0.92a 3.92b 3.51b 10.91a 3.91b 14.71a 19.91b 17.52b 4.81b 36.31a 4.72b 30.02c 114.21a 15.42a 19.52b 4.31a 3.61b 3.01a 28.81a 11.51a 9.31b 13.42a 12.91b 30.12b 4.03c Lys 59.91b 10.32a 2.52a 38.02a 5.81a 7.93a 37.02b Ser 5.01b 5.41b 3.31a 9.71a 38.91b 5.12a 134.41a 23.82b 26.53a 35.62c Met 20.01a 27.81a 2.22a 3.12b 0.01a 78.92ab 0.51b 77.53b Cys 6.22b 5.81a 2.11a 4.81b Gly 5.01a 4.21c 24.41a 0.91a 4.12b 8.41a 20.01a 23.11b 135.81a 3.31a 49.11a 1.11a 5.62b 0.21a 29.11a 1.31a 6.21ab 13.22a 6.11b 3.21b 14.91a 0.81a 0.51a 9.62b 23.22a 4.12b 3.62a 0.81b Trp 7.41b 6.11a 11.62b 9.51a 44.23c 70.93a 140.22b Phe 67.51b 34.31a 8.11c 12.51a 5.23c 46.31a 38.51a 13.02a 3.91b 4.42a 36.72a 1.63b 22.13b 3.02a 0.41c 41.72a 14.41a Asn 5.71c 7.41a 40.912a 1.31a 17.61b 19.81a 1. goat’s (G) or cow’s (C) milk using a thermophilic (T).72b 21.52a 27.92a 9.02a 71.01b 11.12a 1.61a 37.62a 0.92a 32.01b 5.31a 2.82a 7.312a 16.21a 24.21a 40.81b 5.51a 1.31a 11.11a Thr 15.02b 22.02a 5.31b 14.72ab 23.01c 84.61a 15.31a 8.02c 10.12b 19.52a 11.41a 5.31a 13.81a 1.22b 22.82a 18.61b 15.21a 3.83b 8.22b 23.71a 51.51a 11.51b 15.11a 2.21b 33.51b 15.412a 2.43b 18.81b 6.91a 12.82b 20.12a 0.92a 12.42a 1.22a 1.52a 8.32a 0.91b 11.91a 4.51a 1.82b 6.11a 1.012a 22.53b 3.71a 5.71a 4.51c 28.21a 3.03a 41.31a 3.63a 103.82a 37.91a 14.71a 25.72a 12.41b 11.01a 2.12a 13.22a 5.73a 31.31a 4.92a 3.32b 8.02a 5.82b 2.71b 18.02b 9.42a 8. 60 120 (days) Milk S G C S G C Culture T MX M T MX M T MX M T MX M T MX M T MX M E.61b 3.02b 1.91a 48.Table 2b 1 Changes of amino acids content (mg 100 g DMA) of Teleme cheese made from sheep’s (S).41a 13.73b 48.21b 9.12a 12.81c 40.63a 28.01a 11.61a 6.43b Glu 122.11b 11.01b 9.41a 8.11a 9.82c 6.91a 24.01a 38.62a 6.42a 38.01b 22.93a 1–3 : For each age and each kind of milk.42a 18.51b 1.31a 0.22b 12.21a 4.41a 3.5a Arg 29.41a 16.61c b-Ala 23.21a 8.62c 59.52a 26.01ab 3.812b 3.51a 5.11b 7.41a 16.42a 6.05) between kinds of milk.82a 7.11a 81.81a 4.41b 10.01b 7.23b 4.012a 64.11b 0.42b 58.11a 11.91a 91.72c 3.51a His 13.11a 20.22a 4.22a 15.41b 34.11b 6.91b 6.01a 52.82a Tau 3.01b 77.11a 51.02ab 10.02b 52.32ab 21.93b 17.12a 12.63ab 9.71a 6.72a 2.42a 42. a mesophilic (M) or a mixture of thermophilic and mesophilic (MX) starter during ripening Age.42b 3.01a 3.21ab 7.22a 20.01a 30.41a 4.42a 9.51a 2.92a 21.71a 3.61a 6.82c Val 28.21b 34.31a 4.41b 13.32b 16.72b 6.21a 10.62a 13.03a 12.41b 9.03a 0.71a 3.11c Leu 16.11b 1.32a 3.03a 9.71a 3.11a 4.83c 66.41a 19.91b 157.22c 61.21a 3.31a 20.01a 1.11b 4.92b 5.71b 46.412b 25.81a 3.412a 132.42b Cit 3.62a 11.01a 1.12b 6.61a 16.71c 38.03a 43.81ab 8.01a 0. %.42a 13.12b 11.C. A DM: Dry Matter.12b 2.91a 11.912a Tyr 6.91b 5.51a Gln 8.02a 10.41a 26.112a 15.72a 7.02b 0.312a 14.41b 37.82a 6.61b 26.62b 1.71a 28.11b 33.12b 3.51b 0.73b 4.91a 2.42ab 0.11a 12.61a 2.31a 0.91b 3.91b 17. Sotirakoglou / Food Chemistry 111 (2008) 606–615 Asp 5.72b 48.02a 13.22a 4.23b Gaba 3. 611 .72a 72.72a 5.81a 5.01c 147.61a 8.31a 4.11a 6.62b 51.72c 3.12b 34.73b 3.72c 11.02b 1.32a 1. different numbers indicate significant differences (P < 0.01a 8.512a 10.93a 31.52a 2.81b Pro 8.01a 3.01a 6.42c 10.52b 32.61a 18.91b 16.41a 4.33b Orn 7.

5). Asn of the amino acids Leu.11b 61.51a 17.41a 3.01a 8.11a 30.52a 62.21c 0.22a 15.62 34.22a 59.62a 5.01b 97.91a 3.92a 60.71a 2.12a 1.41a 39.12b 23.31a 4.52b 81.42b Thr 31.11a 38.53a 18.71a 23.31a 25.41ab 11. Four dis- criminant functions were statistically significant (P < 0. goat’s (G) or cow’s (C) milk using a thermophilic (T).02a 7.51a 33.92a 6.71b 24.72a 4.21a 21.22a 5.72b 7.32b 12.03a 56. (days) 180 Milk S G C Culture T MX M T MX M T MX M Asp 15. its content represented less AAB ALA ARG ASN ASP BALA CIT CYS GABA GLN GLU GLY HIS ILE LEU LYS MET ORN PHE PRO SER TAU THR TRP TYR VAL than 2% of the TFAA content.92b 72. 1993).23c Asn 49.52a 7.08%).12a 5.52b 5. ripening. which predominates over the proteolysis of b-casein.91b 2.62a 9.C.72b b-Ala 25.11c 14. The percentage of in the formation of a-ketoacids while the a-ketoacidacceptor.71a 3.312 16.82b 37.32a 14.91b 19.32a 1.62c 6.42a 38.62c Gaba 1.32a Leu 36.11c Cys 5.92c 11.52a 63.91a 5. Leu and Met contributed mainly to discriminant function D2 Teleme cheese samples had high contents of Glu (Fig.22a 34.32a 17.12b 81.42b 30.52b 15. different numbers indicate significant differences (P < 0.05) between kinds of milk.11b 23.02a 34. A similar decrease may not be completely separated.41a 3.03c 1–3 : For each age and each kind of milk.41b 6. which caused the mis- other free amino acids. 4).51b 3.23a 29.62b 1.02a 108. As ripening is a continuous process.61c 57.41a 50.02a 16.51a 4.23a 7.63b 25.61a 16.31b 46.21a 5.61b 3. centroids corresponding to R.62a 3.21a Ile 34.02a 7. a–c : For each age and each kind of culture.21a 22.2 mg 100 g 1 DM in the 1 day old cheese and reached 26.91b 7.05)..42c Cit 33. Some overlapping between R.83b 30. at 180 days of ripening.41a 5.71a 43.61b 1.0 mg 100 g 1 DM in 180 day old mg/100g DM 40 cheese.41a 18.82a 21.02 23.91c 36.0%.92a 9.03a 15.32b 8.71a 1.31b 5.612 E.03a 112.61a 5.51b 0.81a 0.01b 31.82c Met 10.52a 27.31a 23.81b 7. Glu decreased with cheese ripening and classification.62c 23. 60 and 120 day old samples were Pro mean concentration was 13.12b 1.61b 36.41a 16.41a 36.31b 24.112a 46.12b 4.41a 22.81c 34.6 mg 0 100 g 1 DM in 180 day old cheese).51a 6. Phe and Val. K.8 mg 100 g 1 DM in 1day old cheese to 11.33a 63.212b 45. the groups presented a maximum around day 60 or 120.21b 67.11b 1.82b 19. The distances between the group with cheese ripening was found for AAB. is transformed to the corresponding amino acid of ripening was 80. Despite the fact that the mean concentration 10 of this amino acid increased continuously throughout the ripening period (from 4.05) between kinds of cultures. %.42b 5.53a 27.53a 105.21c 16.11a 2.02b 56.51b 19.72a 3. different letters indicate significant differences (P < 0.63b 15.82c 26.51a 42.22a 42.51a 4.33c Val 94.01a 17. Cys and Ala.12c 10.42b 28.72c 9.712c Phe 38.01b 9.01b 34.01a 70. Gaba (which is produced from Glu by a decarboxylase and is 20 present in low-quality cheeses) was found at low concentrations in Teleme cheese.02a 14.92ab Ala 4.51a 7.01b 2.11c 8.41b 28.52a 34.51c 29.42a 46.41b 12.42b 52.23a 9.02b 29.81a 7. Sotirakoglou / Food Chemistry 111 (2008) 606–615 Table 2c Changes of amino acids content (mg 100 g 1 DMA) of Teleme cheese made from sheep’s (S).72c 7.41c 92.03a 7.22a 5.92b Tyr 31.02a 24. . 4.01a 5.43a 31. Amino (35%).31b 14.51b 30.02a 20.3 mg 100 g 1 DM in 180 day old samples.62a 14. for discriminating among the stages of ripening (Fig.212a 2.72b 4. a mesophilic (M) or a mixture of thermophilic and mesophilic (MX) starter during ripening Age. Arg is responsible for unpleasant or bitter taste (Polo et al. Glu and Leu to discriminant function D3 (7.82a 79. (Fig.61b 48.41b 44.13c Ser 16.83b Lys 150.61b Gly 14.62a 6.81a 0.912b Tau 6.92a 26.93a 63.21a 23.23b 5.01c 105.92b 60. 50 The mean concentration of Arg was 14.42b 66. 1985).2 mg 100 g 1 DM in the 1 day lower than the distances corresponding to 1 and 180 day old old cheese and increased to 24.72c 32.91a 14.01a 89. 2001).12c 7.21a 13.01c 112. occurs during the first days of and Tau contributed the most to discrininant function D1(54%).81b 5.02b Glu 86. often the samples that were classified into the correct group by the stage a-ketoglutarate.62c Trp 7.11b Arg 39.01c 3.42a 5.612b Gln 32. Free amino acid mean content of Teleme cheese.32a 27.51b 7.92a 6.92a 5.82a 7.12b 25.71c Orn 14.11b 0. Stepwise discriminant analysis selected only nine amino acids Fig. 5).01b 53. therefore it is important that this amino acid concen- 30 tration does not increase with ripening time. cheese.61a 20. Pappa.93a 9.81a 8.01b 74.61a 53.71c Pro 34.22ab 8.212b AAB 34. 60 Glu (Yvon & Rijnen.66%) and Orn acid transamination is catalyzed by aminotranferases and results and Leu to discrininant function D4 (2.61b 41.01b 9.02b 0.12a His 52. The low concentration of Pro in Teleme cheeses could be 60 because this amino acid is normally released as a dipeptide (Prit- chard & Coolbear. In contrast to TFAA and most of the and 120 day old samples was observed.512b 1. A DM: Dry Matter.72a 0.62a 9.22a 1.01b 6.412a 0.23b 132.

.14%). Orn (as well as Cit. 7). Moreover.001). goat’s and cow’s milk.mesophilic) culture.44%) 2. AAB and GABA) did not originate from case- in. 1996). but accumulated as a metabolic product of microorganisms.7 cheese (P < 0.05).05). Linear discriminant functions (D1 and D2) for cheese samples made from mean concentration of Orn in cheeses made from ewe’s milk was sheep’s. Stepwise discriminant analysis selected 18 amino acids for dis- criminating among the three kinds of milk (Fig. 120.001) than that of cow’s (13. 60 According to Mehaia and Al-Kanhal (1992). Pappa.81%) while TFAA and Asn contributed mainly to discriminant function Fig.9 old cheese. TFAA and -7 -5 -3 -1 1 3 5 Phe contributed the most to discrininant function D1 (80.14%) sheep’s (380 mg 100 g 1 DM) milk cheese. Total free amino acid mean content in cheeses made with sheep’s. D2 (19. In this 180 study. there was an increase in peptidase PepS) with respect to Lc. Effect of type of culture Total amino acids mg/100g DM 1000 The mean concentration of TFAA in Teleme cheese made with 800 the thermophilic culture increased from 366 mg 100 g 1 DM in 1day old cheese to 877 mg 100 g 1 DM in 180 day old cheese.9 Ripening time cheese had a mean content of 16. in 180 day 0.01) than those of goat’s (460 mg 100 g 1 DM) or cow's D2 (19. Tau (which origi- D2 (35. 1995).4. Two discrimi- -3. lactis and shows higher specific the mean TFAA concentration from 263 mg 100 g 1 DM in 1 day old cheese to 590 mg 100 g 1 DM in 180 day old cheese. it is known that S.C. Tau was found in low concentrations in goat’s milk cheeses.9 R other two types of milk.05). In contrast.05). The highest TFAA contents of Teleme cheeses made with the Fig. Cheeses made with the mesophilic 0 60 120 180 culture had significantly lower (P < 0. could be due to an earlier lysis of thermophilic starter bacteria that could have enhanced the early release of intracellular enzymes in cheese 3. Both D1 and D2 were significant (P < 0. 3.2 mg 100 g 1 DM and was significant lower (P < 0.0 mg 100 g 1 DM and was signif- 1 icantly higher (P < 0. regardless of the type of milk used. Mallatou et al. The kind of milk overall statistical analysis showed that.1 et al. in caprine Plot of Discriminant Functions cheese from 326 mg 100 g 1 DM to 652 mg 100 g 1 DM and in bo- 4. the TFAA in cow’s milk sheep's cheese had a mean content 481 mg 100 g 1 DM and was significant goat's 2. As it can be observed in Fig. 8).001) than that of the cheeses made from the 4.3 not differ (P > 0. 7. Centroids as well as in ewe’s and cow’s milk cheeses (<1% of TFAA. Freitas et al.7 vine cheese from 297 mg 100 g 1 DM to 784 mg 100 g 1 DM. (1998) found that the TFFA content in Picante cheese made from sheep’s milk was lower than the cheese made from goat’s milk too. 5. 6. E.1 mg 100 g 1 DM) milk cheese. sheep's milk Generally. regardless of the kind of milk and culture used). Both D1 rectly classified according to the kind of milk (100% of the total and D2 were significant (P < 0. the overall statistical analysis showed that the mean 200 goat's milk content of TFAA in Teleme cheese made with the thermophilic cul- ture was 551 mg 100 g 1 DM and was significantly higher than cow's milk 0 that in mixed (421 mg 100 g 1 DM) or mesophilic (350 mg 100 g 1 DM) culture (P < 0. on the corresponding days (Fig. D1 (54. K. 60. -7 -4 -1 2 5 8 Orn has been found in Teleme cheeses from the first day (Table D1 (80. cases). (2004) found that the TFAA contents of Teleme cheeses made from ewe’s or goat’s milk did -3. in 600 Teleme cheese made with the mixed (thermophilic. The Fig.. Linear discriminant functions (D1 and D2) for cheese samples at 1. Sotirakoglou / Food Chemistry 111 (2008) 606–615 613 Plot of Discriminant Functions 8. thermophilic culture.001). from 273 mg 100 g 1 DM to 619 mg 100 g 1 DM and in 400 cheese made with the mesophilic culture from 246 mg 100 g 1 DM to 530 mg 100 g 1 DM.86%).3 (1983) found that the TFFA in ovine Teleme cheese was higher than that of bovine cheese. .001) mean concentration of Ripening day TFAA than had cheeses made with the other two types of culture.3. Buriana and Farag -1.1 nating functions were statistically significant (P < 0. 6 shows the evolution of TFAA for the three types of milk possesses two additional peptidases (an oligopeptidase and amino- during ripening time.86%) 2). the cheeses were cor- 180 days of ripening as well as the day cheeses entered the cold room (R).9 120 nates from Cys) is the most abundant FAA in goat’s milk. goat’s and cow’s milk. 1998) and Idiazabal cheese (Barcina et al. In ovine cheese. Effect of type of milk matrix and could have increased the rate of proteolysis (Chapot- Chartier.7 higher (P < 0. 7. Low Tau contents have also been reported for Picante cheese (Freitas -1. Orn in goat’s milk 6. thermophilus Fig. Cheese made with Centroids goat’s milk was also significant higher than that of sheep’s milk 0.

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