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Determination of free amino acid content in the Slovenian dry-cured ham »Kraški pršut« and product characterization

Determination of free amino acid content in the Slovenian dry-cured ham »Kraški pršut« and... doi:10.2478/v10014-012-0010-7 COBISS: 1.01 Agris category code: Q04 DetermInAtIOn Of free AmInO ACID COntent In the SLOvenIAn Dry-CureD hAm »KrAsKI prsut« AnD prODuCt ChArACterIzAtIOn Lucija JAnes 1, spela veLIKOnJA BOLtA 2, martin sKrLep 2, marjeta CAnDeK-pOtOKAr 3, maja prevOLnIK 3 received January 13, 2012; accepted may 23, 2012. Delo je prispelo 13. januarja 2012, sprejeto 23. maja 2012. Determination of free amino acid content in the Slovenian drycured ham »Kraski prsut« and product characterization The aim of this study was to characterize the chemical profile of dry-cured ham selected and processed in accordance with the Slovenian consortium rules for »Kraski prsut« and to investigate the differences between the Biceps femoris (Bf) and the Semimembranosus (Sm) muscle. The free amino acid content, moisture, salt, protein, total nitrogen, non-protein nitrogen, proteolysis index, intramuscular fat and dry matter were analysed in 135 samples of dry-cured hams including both muscles. A modified method for sample preparation was developed for determination of free amino acids in dry-cured ham. The method was validated for linearity, limit of detection, limit of quantification, precision and uncertainty. According to the validation parameters the method is appropriate for the determination of free amino acid content in dry-cured ham. higher content for several free amino acids and total free amino acids were determined in the Bf muscle compared to the Sm muscle. Key words: meat products / dry-cured ham / Kraski prsut / chemical composition / free amino acids / methods / postcolumn derivatization / muscles / Biceps femoris / Semimembranosus Vsebnost prostih amino kislin v kraskem prsutu in opis izdelka namen te studije je bil okarakterizirati kemijsko sestavo prsuta, ki je bil izbran in obdelan v skladu s slovenskimi pravili konzorcija za »Kraski prsut« in dolociti razlike med Biceps femoris (Bf) in Semimembranosus (Sm) misicama. Dolocili smo vsebnost prostih aminokislin, vlage, soli, beljakovin, skupnega dusika, ne-proteinskega dusika, proteolizni indeks, intramuskularne mascobe in suhe snovi v 135 vzorcih prsuta v obeh misicah. modificirali smo pripravo vzorcev za dolocanje prostih aminokislin v prsut. metodo smo validirali in dolocili obmocje linearnosti, mejo detekcije, mejo kvantifikacije, ponovljivost in obnovljivost. Glede na dobljene rezultate je metoda primerna za dolocanje prostih aminokislin v prsutu. visje vsebnosti nekaterih prostih aminokislin in skupnih prostih aminokislin so bile v misici Bf v primerjavi z misico Sm. Kljucne besede: mesni izdelki / prsut / Kraski prsut / kemijska sestava / proste aminokisline / metode / post-kolonska derivatizacija / misice / Biceps femoris / Semimembranosus INTRODUCTION Amino acids are essential to life and have many functions in metabolism. One important function is building blocks of proteins which are linear chains of amino acids (Gaull, 1989). During the processing of drycured meat products, e.g. dry-cured ham, meat proteins undergo intensive proteolysis resulting in relatively high amounts of free amino acids (toldrá and flores, 1998). Amino acids are beside nucleotides, peptides and inorganic salts important taste compounds in dry-cured meat products (macLeod, 1986). The proteolysis begins with the action of calpains which cause only partial proteolysis of cytoskeletal and myofibrillar proteins (Goll et al., 2003) followed by intensive action of cathepsin enzymes able to hydrolyse the majority of muscle proteins. The process of proteolysis is continued by breakdown of peptides as demonstrated by 1 Agricultural Institute of Slovenia, hacquetova ulica 17, SI-1000 Ljubljana, Slovenia, e-mail: lucija.janes@kis.si 2 Same address as 1 3 Same address as 1 and faculty of Agriculture and Life Sciences, university of maribor, pivola 10, SI-2311 hoce, Slovenia Acta argiculturae Slovenica, 100/1, 29­35, Ljubljana 2012 progressive decrease of middle size peptides (molecular weight ­ mW from 2700 Da to 4500 Da) and increase of smaller peptides with mW below 1200 Da (rodrígueznuñez et al., 1995). proteolysis continues with the generation of free amino acids. During the lengthy processing of dry-cured ham amino-peptidases degrade peptides and are responsible for amino acid generation which contributes to texture and flavour development (mcDonald and Barrett, 1986). free amino acids are highly correlated with the development of taste and flavour in dry-cured products (mcCain et al., 1968). Sweet taste is related to tryptophane, phenylalanine, histidine, tyrosine, leucine, glycine and alanine; bitter taste with valine, methionine is related to sea flavour, and the presence of glutamic acid is associated with characteristic meaty flavor (Kato et al., 1989). representative Slovenian mediterranean type of dry-cured ham is »Kraski prsut« which is similar to the well known Italian "prosciutto". This type of ham is characterized by dry salting and long maturation period during which characteristic sensory properties are developed due to the enzymatic processes involved (toldrá, 2002). Chromatographic procedures related to the analy sis of amino acidcontaining and aminecontaining ma trices were classified in three groups, i.e. simultaneous analysis of amino acids and amines from a single run, separate determination of amino acids and amines as different derivatives and quantisation of amines only, following the separation of amino acids from amines. The advantages and drawbacks of all three possibilities were characterized on the basis of recovery, reproduc ibility values, time and cost phenomena of methods. Separation techniques were high performance liquid chromatography, capillary electrophoresis and ion or ionexchange chromatography followed by fluores cence, laserinduced fluorescence, coulometric, UV, indirect photometric and integrated pulse amperometric detection (MolnárPerl, 2003). The main objective of this work was to determine the free amino acid content and chemical parameters in the Slovenian dry-cured hams "Kraski prsut", to characterize the chemical profile of dry-cured hams and investigate the differences between Bf and Sm muscles. for the determination of free amino acids in dry-cured hams we used the method for the determination of free amino acids in animal feeding stuffs (ISO 13903, 2005). Due to homogeneity we had to modify the sample preparation, i.e. the weight of sample was enlarged from 5 to 10 g. to improve the extraction and prolong the precipitation of nitrogenous macromolecules the sample homogenates were maintained at 4 °C for 17 hours prior to the analyses. 30 MATERIALS AND METHODS 2.1 prepArAtIOn Of SAmpLeS The material used in the present study comprised 135 Slovenian dry-cured hams »Kraski prsut«. The selection of raw material and green ham evaluation is described by (skrlep et al., 2010). hams were selected and processed in accordance with the Slovenian consortium rules for dry ham »Kraski prsut«, i.e. green ham weight above 9.5 kg, subcutaneous fat thickness above 10 mm and non-pSe (i.e. pale soft and exudative) or non-DfD (i.e. dark, firm and dry) appearance. The processing consisted of two salting phases, resting phase, drying phase and maturation phase, i.e. 60 weeks in total. for the analyses 135 samples of central part of dry-cured Bf and 135 samples of Sm muscles were taken, vacuum packed and stored at -20 °C. prior to the analyses samples of drycured muscles were trimmed of connective and superficial fat tissue, cut in small pieces, frozen in liquid nitrogen, homogenised using a laboratory mill IKA m120 (IKA Werke, Staufen, Germany) and stored in plastic tubes at -20 °C until further use. 2.2 ChemICAL pArAmeterS for the determination of moisture content 5 g of the sample was mixed with equal amount of quartz sand and dried at 103 °C for 4 hours to constant mass. The loss of mass was recorded and expressed as a percentage of moisture in the sample (ISO 6496, 1999). Sodium chloride content was determined as described by (monin et al., 1997). 1 g of sample was mixed with 80 mL deionized water and boiled at 100 ºC for one hour. After cooling, 2 mL of 15% potassium ferrocyanide (99.0% purity, merck, Darmstadt, Germany) and 2 mL of 30% zinc acetate (99.5% purity, merck, Darmstadt, Germany) was added and diluted with deionized water to 100 mL. After filtration, the sodium chloride content was determined by potentiometric titration using 645 multiDosimat (metrohm, herisau, Switzerland). total nitrogen content was determined with Kjeldahl method (ISO 5983-2, 2005) using the Kjeltec 2300 nitrogen analyser (foss Analytical, hileroed, Denmark). preparation of sample for the determination of nonprotein nitrogen was performed as described by (monin et al., 1997). 2.5 g of sample was homogenised in 25 mL of deionized water and centrifuged. Afterwards, 10 mL of 20% trichloroacetic acid (99.5% purity, merck, Darmstadt, Germany) was added, stirred well and let to stabilise for 60 min at room temperature. After centrifugation the supernatant was filtered and 15 mL of filtrate used for determination of nitrogen as described for total nitrogen (ISO 5983-2, 2005). Intramuscular fat content was determined by standard method (ISO 1443, 1973) using Büchi extraction System B-811 (Büchi Labortechnik AG, flawil, Switzerland). 2.3 DetermInAtIOn Of free AmInO ACID COntent 2.3.1 ChemICALS AnD reAGentS Deionized water produced by a milli Q system (millipore, Bedford, mA, uSA), sodium citrate dihydrate, 2,2'-thiodiethanol, sodium hydroxide, 5-sulfosalicylic acid dihydrate (all 99% purity), phenol (99.5% purity) and hydrochloric acid (37% purity) were used (Sigma Aldrich, St. Louis, mO, uSA). Standard compounds of amino acids: leucine, tyrosine and phenylalanine (all 98% purity), aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cystine, valine, methionine, isoleucine, lysine monohydrochloride, histidine monohydrochloride monohydrate, arginine monohydrochloride and internal standard norleucine (all 99% purity) were used (Sigma Aldrich, St. Louis, mO, uSA). 2.3.2 prepArAtIOn Of StAnDArD tIOnS AnD SAmpLeS SOLu- ten g of the prepared sample was weighed into a conical flask and 100 mL of extraction mixture (c = 0.1 mol/L hCl, containing 2% 2,2'-thiodiethanol) was added. The mixture was shaken for 60 min using a mechanical shaker. When the sediment settled down, 10.0 mL of the supernatant was transferred into a 100 mL beaker, 5.0 mL of 6% 5-sulfosalicylic acid solution was added by stirring and stirred for 5 min with the magnetic stirrer. The homogenates were maintained at 4 °C for 17 hours and filtered through Black ribbon filter (Whatman, Clifton, nJ, uSA). 10.0 mL of filtrate was transferred into 100 mL beaker and ph adjusted to 2.20 using 1 m sodium hydroxide solution. The solution was quantitatively transferred into a 100 mL volumetric flask, 0.5 mL of internal standard norleucine (50 µmol/mL) was added and made up to the mark with sodium citrate buffer (c = 0.2 mol/L, ph 2.20). The extract was filtered in vials through 0.2 µm membrane filters ptfe (Whatman, Clifton, nJ, uSA). 2.3.3 pOSt-COLumn DerIvAtIzAtIOn AnD ChrOmAtOGrAphIC COnDItIOnS Stock standard solutions of amino acids were pre pared by dissolving the weighed solid standard in so dium citrate buffer (c = 0.2 mol/L, pH 2.20) in the con centration range from 2.5 to 50.0 µmol/mL and further diluted with the same buffer to obtain working solu tions of 0.25 µmol/mL for injection into chromatograph equipped with the post column derivatization instrument. Table 1: Gradient program for eluants Preglednica 1: Parametri gradientne elucije Step 0 1 2 3 4 5 6 7 time (min) 0 10 32 56 56.1 58 58.1 70 Interval (min) 0 10 22 24 0.1 1.9 0.1 11.9 Ionexchange chromatography followed by post column derivatization with ninhydrin was used (Spack man et al., 1958; Grunau and Swiader, 1992). Ninhydrin reacts with primary amino acids and hydrindantin to form Ruhemann`s Purple which is detectable at 570 nm. Ninhydrin reacts with secondary amino acids to form a yellow complex detectable at 440 nm (Ruhemann, 1910). Agilent 1200 Series chromatograph with diode array detector (Agilent Technologies, Palo Alto, CA, USA) and postcolumn derivatization instrument Pinna cle PCX (Pickering Laboratories, Mountain View, CA, USA) were used for the separation of amino acids, their detection and quantification. The separation was per formed at 48 °C using sodium cation exchange column (8 µm, 3.0 mm × 250 mm) with sodium guard column Sodium eluant ph 3.28 (%) 100 100 0 0 0 0 100 100 Sodium eluant ph 7.50 (%) 0 0 100 100 0 0 0 0 Sodium column regenerant (%) 0 0 0 0 100 100 0 0 (2.0 mm × 20 mm, Pickering Laboratories, Mountain View, CA, USA). The ninhydrin reaction was carried out at 130 °C with a reactor volume of 500 µL and flow rate 0.3 mL/min. The mobile phase consisted of sodium elu ant pH 3.28, sodium eluant pH 7.50 and sodium column regenerant (Pickering Laboratories, Mountain View, CA, USA). The gradient used is shown in Table 1. The col umn was equilibrated 10 min prior to the next analysis. The flow rate was 0.3 mL/min and the injection volume was 10 µL. The wavelength for detection was at 570 nm for primary amino acids and at 440 nm for secondary amino acids. culated with f test and multiple linear regression. Calibration curves were linear in the ranges shown in table 2 (r2 from 0.9942 to 0.9998). for each amino acid limit of detection (LOD) and limit of quantification (LOQ) were calculated by using "least squares" method. LOD were from 2 to 5 mg/100 mg of dry-cured ham and LOQ from 5 to 18 mg/100 mg of dry-cured ham (table 2). 3.1.2 preCISIOn, unCertAInty AnD reCOvery RESULTS AND DISCUSSION 3.1 methOD vALIDAtIOn 3.1.1 LIneArIty, LImIt Of DeteCtIOn, LImIt Of QuAntIfICAtIOn Linearity was verified by measuring the standard solutions of amino acids in five concentration levels for the calibration curve and five repetitions for each concentration level. Linearity and range were determined and cal- repeatability and reproducibility were determined within the period of 10 days; two samples of dry-cured ham were prepared each day (ISO 5725-2, 1994). Standard deviation of repeatability and standard deviation of reproducibility were calculated. Dispersion of results was checked with Cochran test and outliers with Grubbs test. uncertainty of repeatability and uncertainty of reproducibility were calculated by multiplying the standard deviation of repeatability and the standard deviation of reproducibility by Student's t factor for 9 degrees of freedom and 95% confidence level (t95; 9 = 2.262). The results are given in table 3. The recovery was checked on spiked samples in six Table 2: Linearity, linear range, LOD and LOQ of the method for the determination of free amino acid content in dry-cured ham "Kraski prsut" Preglednica 2: Linearnost, obmocje linearnosti, meje detekcije in meje kvantifikacije za metodo za dolocanje vsebnosti prostih amino kislin v kraskem prsutu Amino acid Aspartic acid Threonine Serine Glutamic acid proline Glycine Alanine Cystine valine methionine Isoleucine Leucine tyrosine phenylalanine Lysine histidine Arginine r2 0.9996 0.9997 0.9998 0.9997 0.9994 0.9997 0.9995 0.9997 0.9996 0.9996 0.9997 0.9997 0.9996 0.9997 0.9942 0.9988 0.9998 Lin. range (mg/100 g) 8­502 7­448 5­397 8­551 18­608 5­284 5­335 16­901 7­445 9­558 8­493 9­492 12­684 11­620 6­549 7­235 9­659 LOD (mg/100 g) 2 2 2 2 5 2 2 5 2 3 2 3 4 3 2 2 3 LOQ (mg/100 g) 8 7 5 8 18 5 5 16 7 9 8 9 12 11 6 7 9 Table 3: Standard deviations of repeatability (sr), repeatability limit (r), standard deviations of reproducibility (sR), reproducibility limit (R), uncertainty of repeatability (Ur) and uncertainty of reproducibility (UR) of the method for the determination of free amino acid content in dry-cured ham "Kraski prsut" (in mg/100 g dry matter) Preglednica 3: Standardni odkloni ponovljivosti (sr), meje ponovljivosti (r), standardni odkloni obnovljivosti (sR), meje obnovljivosti (R), negotovost ponovljivosti (Ur) in negotovost obnovljivosti (UR) metode za dolocanje vsebnosti prostih amino kislin v kraskem prsutu (v mg/100 g suhe snovi) Amino acid Aspartic acid Threonine Serine Glutamic acid proline Glycine Alanine Cystine valine methionine Isoleucine Leucine tyrosine phenylalanine Lysine histidine Arginine means of the levels 593 418 523 1153 491 399 678 16 593 271 464 693 281 430 1074 275 525 sr 23 13 13 26 13 9 15 1 16 8 12 16 7 10 22 6 12 r 74 42 42 83 42 29 48 3 51 26 38 51 22 32 70 19 38 sR 25 21 27 47 30 18 29 2 28 12 25 33 15 20 46 15 22 r 80 67 86 150 96 58 93 6 90 38 80 106 48 64 147 48 70 ur 52 29 30 58 28 20 33 2 37 18 27 37 17 22 50 13 27 uR 58 46 61 106 68 40 66 5 63 28 56 75 35 44 103 34 49 repetitions using the internal standard norleucine. The calculated recovery for norleucine was from 95.0% to 97.2%. 3.2 AnALySIS Of Dry-CureD hAm SAmpLeS free amino acid content and chemical parameters were determined in 135 Slovenian dry-cured hams »Kraski prsut« for 2 different muscles. The results of chemical parameters are given in table 4. In the Sm muscle the moisture content and proteolysis index are lower in comparison with the Bf muscle, but protein and total nitrogen content were observed to be higher. higher quantity of total nitrogen can be explained by the difference in dry matter and moisture content. however, the Table 4: Chemical parameters for the BF and SM muscle in dry-cured hams "Kraski prsut" (mean ± standard deviation) Preglednica 4: Kemijski parametri za misici BF in SM v kraskem prsutu (povprecje ± standardni odklon) parameter moisture (%) naCl (%) protein (%) total nitrogen (g/kg) non-protein nitrogen (g/kg) proteolysis index (%) Intramuscular fat (%) Dry matter (g/kg) Bf muscle 58.5 ± 1.5 7.6 ± 0.7 29.5 ± 1.2 47.2 ± 1.9 12.6 ± 1.0 26.8 ± 1.8 3.0 ± 0.9 415 ± 14 min­max 54.5­65.2 5.9­9.4 26.0­33.6 41.6­54.0 7.0­14.7 16.1­31.1 1.6­6.2 377­455 Sm muscle 49.0 ± 2.3 6.7 ± 0.7 39.0 ± 2.3 62.4 ± 3.6 11.8 ± 1.1 18.9 ± 1.8 4.5 ± 1.2 510 ± 23 min­max 43.5­56.2 4.5­8.3 32.0­43.9 51.2­70.2 7.8­14.4 13.3­24.1 2.1­8.8 438­565 concentration of salt is higher in the Bf muscle. According to the literature, this can be explained by the fact that salt consequently moves to the areas with higher moisture content where more salt can be dissolved (Arnau et al., 1995; monin et al., 1997). higher proteolysis index and non-protein nitrogen contents were observed in the Bf muscle. As this is an internal muscle, it is reached by the salt penetration much later and the desiccation is smaller than in the Sm muscle (Arnau et al., 1995). Both factors, the salt content and the water availability, are crucial for the activity of muscle proteolytic enzymes and consequently the generation of non-protein nitrogen (toldrá, 2002). table 5 shows the contents of free amino acids in dry-cured hams "Kraski prsut" for the Bf and Sm muscles. The amino acid content in "Kraski prsut" is in the middle of the range of values reported for other sorts of dry-cured hams (toldrá and flores, 1998). Considering the results calculated on dry matter basis, the content of free amino acids is higher in the Bf muscle, which is in accordance with the result for the proteolysis index, also found to be higher in the Bf muscle in the present ex- periment as well as in other reports (monin et al., 1997; Candek-potokar et al., 2002). The exceptions were only the content of cystine (lower in the Bf muscle) and the content of methionine and phenylalanine (lower in the Sm muscle, though not statistically significant). regarding the differences in the dry-cured ham procedures (length, temperature conditions, amount of salt added, raw materials) between the manufacturers and countries of origin, it is difficult to compare our results obtained for free amino acid content with the available literature data. CONCLUSION Chemical profile of dry-cured ham selected and processed in accordance with the Slovenian consortium rules for »Kraski prsut« was characterized and differences between the Bf and Sm muscle were investigated. for the determination of free amino acids in drycured hams we used the method for the determination of free amino acids in animal feeding stuffs and modified the sample preparation. validation of the method Table 5: The free amino acid content (in mg/100 g dry matter) in dry-cured hams "Kraski prsut" for the BF and SM muscle (mean ± standard deviation) Preglednica 5: Vsebnost prostih amino kislin (v mg/100 g suhe snovi) v kraskem prsutu za misici BF in SM (povprecje ± standardni odklon) Amino acid Aspartic acid Threonine Serine Glutamic acid proline Glycine Alanine Cystine valine methionine Isoleucine Leucine tyrosine phenylalanine Lysine histidine Arginine total Bf muscle 450 ± 19 350 ± 17 447 ± 23 1007 ± 40 446 ± 27 345 ± 16 622 ± 27 33 ± 5 523 ± 24 215 ± 9 385 ± 21 566 ± 26 293 ± 17 342 ± 16 907 ± 39 234 ± 14 445 ± 17 7610 min­max 318­591 273­441 332­593 834­1213 348­578 268­431 472­789 4­80 406­655 145­296 280­524 431­758 218­381 253­475 723­1139 184­288 249­593 SB muscle 391 ± 17 297 ± 15 379 ± 20 849 ± 34 370 ± 22 290 ± 13 519 ± 23 46 ± 7 470 ±22 211 ± 9 361 ± 20 529 ± 24 279 ± 16 341 ± 16 789 ± 34 213 ± 12 338 ± 13 6670 min­max 301­529 214­412 327­557 666­1090 300­566 218­388 393­673 6­130 363­660 143­367 252­598 383­849 209­398 235­578 632­1076 173­300 200­530 Sign. *** *** *** *** *** *** *** *** *** n.s. *** *** *** n.s. *** *** *** *** fisher test value Sign., significance level: ***, p < 0.001; **, p <0.01; *, p<0.05; n.s., non significant has shown good repeatability, reproducibility and linearity. According to the validation parameters the method is appropriate for the determination of free amino acid content in dry-cured ham. from the results obtained in the present work it can be concluded that the proteolysis index, the content of moisture, salt, non-protein nitrogen, and free amino acids are higher in the Bf muscle compared to the Sm muscle. The results concerning the free amino acid content reflect the chemical composition of dry-cured ham and the differences between both investigated muscles. ACKNOWLEDGEMENTS The research was funded by the grants of the research project truefOOD ("traditional united europe food"), an Integrated project financed by the european Commission under the 6th framework programme for rtD, contract no. fOOD-Ct-2006-016264. In Slovenia this study was also partly made with the grants of Slovenian research Agency for the applied project L4-9468. The authors would also like to express their thanks to industrial partners taking part in this study farme Ihan d.d., turolense Ganadera S.A. (pig producers), KrAS d.d. and mIp d.d., Grupo monte nevado (dry ham processors), Agroalimentaria teruel (abattoir) and pyragena france (dry ham processor). The authors also thank zvonko Bregar for his contribution to the present article. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Agriculturae Slovenica de Gruyter

Determination of free amino acid content in the Slovenian dry-cured ham »Kraški pršut« and product characterization

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doi:10.2478/v10014-012-0010-7 COBISS: 1.01 Agris category code: Q04 DetermInAtIOn Of free AmInO ACID COntent In the SLOvenIAn Dry-CureD hAm »KrAsKI prsut« AnD prODuCt ChArACterIzAtIOn Lucija JAnes 1, spela veLIKOnJA BOLtA 2, martin sKrLep 2, marjeta CAnDeK-pOtOKAr 3, maja prevOLnIK 3 received January 13, 2012; accepted may 23, 2012. Delo je prispelo 13. januarja 2012, sprejeto 23. maja 2012. Determination of free amino acid content in the Slovenian drycured ham »Kraski prsut« and product characterization The aim of this study was to characterize the chemical profile of dry-cured ham selected and processed in accordance with the Slovenian consortium rules for »Kraski prsut« and to investigate the differences between the Biceps femoris (Bf) and the Semimembranosus (Sm) muscle. The free amino acid content, moisture, salt, protein, total nitrogen, non-protein nitrogen, proteolysis index, intramuscular fat and dry matter were analysed in 135 samples of dry-cured hams including both muscles. A modified method for sample preparation was developed for determination of free amino acids in dry-cured ham. The method was validated for linearity, limit of detection, limit of quantification, precision and uncertainty. According to the validation parameters the method is appropriate for the determination of free amino acid content in dry-cured ham. higher content for several free amino acids and total free amino acids were determined in the Bf muscle compared to the Sm muscle. Key words: meat products / dry-cured ham / Kraski prsut / chemical composition / free amino acids / methods / postcolumn derivatization / muscles / Biceps femoris / Semimembranosus Vsebnost prostih amino kislin v kraskem prsutu in opis izdelka namen te studije je bil okarakterizirati kemijsko sestavo prsuta, ki je bil izbran in obdelan v skladu s slovenskimi pravili konzorcija za »Kraski prsut« in dolociti razlike med Biceps femoris (Bf) in Semimembranosus (Sm) misicama. Dolocili smo vsebnost prostih aminokislin, vlage, soli, beljakovin, skupnega dusika, ne-proteinskega dusika, proteolizni indeks, intramuskularne mascobe in suhe snovi v 135 vzorcih prsuta v obeh misicah. modificirali smo pripravo vzorcev za dolocanje prostih aminokislin v prsut. metodo smo validirali in dolocili obmocje linearnosti, mejo detekcije, mejo kvantifikacije, ponovljivost in obnovljivost. Glede na dobljene rezultate je metoda primerna za dolocanje prostih aminokislin v prsutu. visje vsebnosti nekaterih prostih aminokislin in skupnih prostih aminokislin so bile v misici Bf v primerjavi z misico Sm. Kljucne besede: mesni izdelki / prsut / Kraski prsut / kemijska sestava / proste aminokisline / metode / post-kolonska derivatizacija / misice / Biceps femoris / Semimembranosus INTRODUCTION Amino acids are essential to life and have many functions in metabolism. One important function is building blocks of proteins which are linear chains of amino acids (Gaull, 1989). During the processing of drycured meat products, e.g. dry-cured ham, meat proteins undergo intensive proteolysis resulting in relatively high amounts of free amino acids (toldrá and flores, 1998). Amino acids are beside nucleotides, peptides and inorganic salts important taste compounds in dry-cured meat products (macLeod, 1986). The proteolysis begins with the action of calpains which cause only partial proteolysis of cytoskeletal and myofibrillar proteins (Goll et al., 2003) followed by intensive action of cathepsin enzymes able to hydrolyse the majority of muscle proteins. The process of proteolysis is continued by breakdown of peptides as demonstrated by 1 Agricultural Institute of Slovenia, hacquetova ulica 17, SI-1000 Ljubljana, Slovenia, e-mail: lucija.janes@kis.si 2 Same address as 1 3 Same address as 1 and faculty of Agriculture and Life Sciences, university of maribor, pivola 10, SI-2311 hoce, Slovenia Acta argiculturae Slovenica, 100/1, 29­35, Ljubljana 2012 progressive decrease of middle size peptides (molecular weight ­ mW from 2700 Da to 4500 Da) and increase of smaller peptides with mW below 1200 Da (rodrígueznuñez et al., 1995). proteolysis continues with the generation of free amino acids. During the lengthy processing of dry-cured ham amino-peptidases degrade peptides and are responsible for amino acid generation which contributes to texture and flavour development (mcDonald and Barrett, 1986). free amino acids are highly correlated with the development of taste and flavour in dry-cured products (mcCain et al., 1968). Sweet taste is related to tryptophane, phenylalanine, histidine, tyrosine, leucine, glycine and alanine; bitter taste with valine, methionine is related to sea flavour, and the presence of glutamic acid is associated with characteristic meaty flavor (Kato et al., 1989). representative Slovenian mediterranean type of dry-cured ham is »Kraski prsut« which is similar to the well known Italian "prosciutto". This type of ham is characterized by dry salting and long maturation period during which characteristic sensory properties are developed due to the enzymatic processes involved (toldrá, 2002). Chromatographic procedures related to the analy sis of amino acidcontaining and aminecontaining ma trices were classified in three groups, i.e. simultaneous analysis of amino acids and amines from a single run, separate determination of amino acids and amines as different derivatives and quantisation of amines only, following the separation of amino acids from amines. The advantages and drawbacks of all three possibilities were characterized on the basis of recovery, reproduc ibility values, time and cost phenomena of methods. Separation techniques were high performance liquid chromatography, capillary electrophoresis and ion or ionexchange chromatography followed by fluores cence, laserinduced fluorescence, coulometric, UV, indirect photometric and integrated pulse amperometric detection (MolnárPerl, 2003). The main objective of this work was to determine the free amino acid content and chemical parameters in the Slovenian dry-cured hams "Kraski prsut", to characterize the chemical profile of dry-cured hams and investigate the differences between Bf and Sm muscles. for the determination of free amino acids in dry-cured hams we used the method for the determination of free amino acids in animal feeding stuffs (ISO 13903, 2005). Due to homogeneity we had to modify the sample preparation, i.e. the weight of sample was enlarged from 5 to 10 g. to improve the extraction and prolong the precipitation of nitrogenous macromolecules the sample homogenates were maintained at 4 °C for 17 hours prior to the analyses. 30 MATERIALS AND METHODS 2.1 prepArAtIOn Of SAmpLeS The material used in the present study comprised 135 Slovenian dry-cured hams »Kraski prsut«. The selection of raw material and green ham evaluation is described by (skrlep et al., 2010). hams were selected and processed in accordance with the Slovenian consortium rules for dry ham »Kraski prsut«, i.e. green ham weight above 9.5 kg, subcutaneous fat thickness above 10 mm and non-pSe (i.e. pale soft and exudative) or non-DfD (i.e. dark, firm and dry) appearance. The processing consisted of two salting phases, resting phase, drying phase and maturation phase, i.e. 60 weeks in total. for the analyses 135 samples of central part of dry-cured Bf and 135 samples of Sm muscles were taken, vacuum packed and stored at -20 °C. prior to the analyses samples of drycured muscles were trimmed of connective and superficial fat tissue, cut in small pieces, frozen in liquid nitrogen, homogenised using a laboratory mill IKA m120 (IKA Werke, Staufen, Germany) and stored in plastic tubes at -20 °C until further use. 2.2 ChemICAL pArAmeterS for the determination of moisture content 5 g of the sample was mixed with equal amount of quartz sand and dried at 103 °C for 4 hours to constant mass. The loss of mass was recorded and expressed as a percentage of moisture in the sample (ISO 6496, 1999). Sodium chloride content was determined as described by (monin et al., 1997). 1 g of sample was mixed with 80 mL deionized water and boiled at 100 ºC for one hour. After cooling, 2 mL of 15% potassium ferrocyanide (99.0% purity, merck, Darmstadt, Germany) and 2 mL of 30% zinc acetate (99.5% purity, merck, Darmstadt, Germany) was added and diluted with deionized water to 100 mL. After filtration, the sodium chloride content was determined by potentiometric titration using 645 multiDosimat (metrohm, herisau, Switzerland). total nitrogen content was determined with Kjeldahl method (ISO 5983-2, 2005) using the Kjeltec 2300 nitrogen analyser (foss Analytical, hileroed, Denmark). preparation of sample for the determination of nonprotein nitrogen was performed as described by (monin et al., 1997). 2.5 g of sample was homogenised in 25 mL of deionized water and centrifuged. Afterwards, 10 mL of 20% trichloroacetic acid (99.5% purity, merck, Darmstadt, Germany) was added, stirred well and let to stabilise for 60 min at room temperature. After centrifugation the supernatant was filtered and 15 mL of filtrate used for determination of nitrogen as described for total nitrogen (ISO 5983-2, 2005). Intramuscular fat content was determined by standard method (ISO 1443, 1973) using Büchi extraction System B-811 (Büchi Labortechnik AG, flawil, Switzerland). 2.3 DetermInAtIOn Of free AmInO ACID COntent 2.3.1 ChemICALS AnD reAGentS Deionized water produced by a milli Q system (millipore, Bedford, mA, uSA), sodium citrate dihydrate, 2,2'-thiodiethanol, sodium hydroxide, 5-sulfosalicylic acid dihydrate (all 99% purity), phenol (99.5% purity) and hydrochloric acid (37% purity) were used (Sigma Aldrich, St. Louis, mO, uSA). Standard compounds of amino acids: leucine, tyrosine and phenylalanine (all 98% purity), aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cystine, valine, methionine, isoleucine, lysine monohydrochloride, histidine monohydrochloride monohydrate, arginine monohydrochloride and internal standard norleucine (all 99% purity) were used (Sigma Aldrich, St. Louis, mO, uSA). 2.3.2 prepArAtIOn Of StAnDArD tIOnS AnD SAmpLeS SOLu- ten g of the prepared sample was weighed into a conical flask and 100 mL of extraction mixture (c = 0.1 mol/L hCl, containing 2% 2,2'-thiodiethanol) was added. The mixture was shaken for 60 min using a mechanical shaker. When the sediment settled down, 10.0 mL of the supernatant was transferred into a 100 mL beaker, 5.0 mL of 6% 5-sulfosalicylic acid solution was added by stirring and stirred for 5 min with the magnetic stirrer. The homogenates were maintained at 4 °C for 17 hours and filtered through Black ribbon filter (Whatman, Clifton, nJ, uSA). 10.0 mL of filtrate was transferred into 100 mL beaker and ph adjusted to 2.20 using 1 m sodium hydroxide solution. The solution was quantitatively transferred into a 100 mL volumetric flask, 0.5 mL of internal standard norleucine (50 µmol/mL) was added and made up to the mark with sodium citrate buffer (c = 0.2 mol/L, ph 2.20). The extract was filtered in vials through 0.2 µm membrane filters ptfe (Whatman, Clifton, nJ, uSA). 2.3.3 pOSt-COLumn DerIvAtIzAtIOn AnD ChrOmAtOGrAphIC COnDItIOnS Stock standard solutions of amino acids were pre pared by dissolving the weighed solid standard in so dium citrate buffer (c = 0.2 mol/L, pH 2.20) in the con centration range from 2.5 to 50.0 µmol/mL and further diluted with the same buffer to obtain working solu tions of 0.25 µmol/mL for injection into chromatograph equipped with the post column derivatization instrument. Table 1: Gradient program for eluants Preglednica 1: Parametri gradientne elucije Step 0 1 2 3 4 5 6 7 time (min) 0 10 32 56 56.1 58 58.1 70 Interval (min) 0 10 22 24 0.1 1.9 0.1 11.9 Ionexchange chromatography followed by post column derivatization with ninhydrin was used (Spack man et al., 1958; Grunau and Swiader, 1992). Ninhydrin reacts with primary amino acids and hydrindantin to form Ruhemann`s Purple which is detectable at 570 nm. Ninhydrin reacts with secondary amino acids to form a yellow complex detectable at 440 nm (Ruhemann, 1910). Agilent 1200 Series chromatograph with diode array detector (Agilent Technologies, Palo Alto, CA, USA) and postcolumn derivatization instrument Pinna cle PCX (Pickering Laboratories, Mountain View, CA, USA) were used for the separation of amino acids, their detection and quantification. The separation was per formed at 48 °C using sodium cation exchange column (8 µm, 3.0 mm × 250 mm) with sodium guard column Sodium eluant ph 3.28 (%) 100 100 0 0 0 0 100 100 Sodium eluant ph 7.50 (%) 0 0 100 100 0 0 0 0 Sodium column regenerant (%) 0 0 0 0 100 100 0 0 (2.0 mm × 20 mm, Pickering Laboratories, Mountain View, CA, USA). The ninhydrin reaction was carried out at 130 °C with a reactor volume of 500 µL and flow rate 0.3 mL/min. The mobile phase consisted of sodium elu ant pH 3.28, sodium eluant pH 7.50 and sodium column regenerant (Pickering Laboratories, Mountain View, CA, USA). The gradient used is shown in Table 1. The col umn was equilibrated 10 min prior to the next analysis. The flow rate was 0.3 mL/min and the injection volume was 10 µL. The wavelength for detection was at 570 nm for primary amino acids and at 440 nm for secondary amino acids. culated with f test and multiple linear regression. Calibration curves were linear in the ranges shown in table 2 (r2 from 0.9942 to 0.9998). for each amino acid limit of detection (LOD) and limit of quantification (LOQ) were calculated by using "least squares" method. LOD were from 2 to 5 mg/100 mg of dry-cured ham and LOQ from 5 to 18 mg/100 mg of dry-cured ham (table 2). 3.1.2 preCISIOn, unCertAInty AnD reCOvery RESULTS AND DISCUSSION 3.1 methOD vALIDAtIOn 3.1.1 LIneArIty, LImIt Of DeteCtIOn, LImIt Of QuAntIfICAtIOn Linearity was verified by measuring the standard solutions of amino acids in five concentration levels for the calibration curve and five repetitions for each concentration level. Linearity and range were determined and cal- repeatability and reproducibility were determined within the period of 10 days; two samples of dry-cured ham were prepared each day (ISO 5725-2, 1994). Standard deviation of repeatability and standard deviation of reproducibility were calculated. Dispersion of results was checked with Cochran test and outliers with Grubbs test. uncertainty of repeatability and uncertainty of reproducibility were calculated by multiplying the standard deviation of repeatability and the standard deviation of reproducibility by Student's t factor for 9 degrees of freedom and 95% confidence level (t95; 9 = 2.262). The results are given in table 3. The recovery was checked on spiked samples in six Table 2: Linearity, linear range, LOD and LOQ of the method for the determination of free amino acid content in dry-cured ham "Kraski prsut" Preglednica 2: Linearnost, obmocje linearnosti, meje detekcije in meje kvantifikacije za metodo za dolocanje vsebnosti prostih amino kislin v kraskem prsutu Amino acid Aspartic acid Threonine Serine Glutamic acid proline Glycine Alanine Cystine valine methionine Isoleucine Leucine tyrosine phenylalanine Lysine histidine Arginine r2 0.9996 0.9997 0.9998 0.9997 0.9994 0.9997 0.9995 0.9997 0.9996 0.9996 0.9997 0.9997 0.9996 0.9997 0.9942 0.9988 0.9998 Lin. range (mg/100 g) 8­502 7­448 5­397 8­551 18­608 5­284 5­335 16­901 7­445 9­558 8­493 9­492 12­684 11­620 6­549 7­235 9­659 LOD (mg/100 g) 2 2 2 2 5 2 2 5 2 3 2 3 4 3 2 2 3 LOQ (mg/100 g) 8 7 5 8 18 5 5 16 7 9 8 9 12 11 6 7 9 Table 3: Standard deviations of repeatability (sr), repeatability limit (r), standard deviations of reproducibility (sR), reproducibility limit (R), uncertainty of repeatability (Ur) and uncertainty of reproducibility (UR) of the method for the determination of free amino acid content in dry-cured ham "Kraski prsut" (in mg/100 g dry matter) Preglednica 3: Standardni odkloni ponovljivosti (sr), meje ponovljivosti (r), standardni odkloni obnovljivosti (sR), meje obnovljivosti (R), negotovost ponovljivosti (Ur) in negotovost obnovljivosti (UR) metode za dolocanje vsebnosti prostih amino kislin v kraskem prsutu (v mg/100 g suhe snovi) Amino acid Aspartic acid Threonine Serine Glutamic acid proline Glycine Alanine Cystine valine methionine Isoleucine Leucine tyrosine phenylalanine Lysine histidine Arginine means of the levels 593 418 523 1153 491 399 678 16 593 271 464 693 281 430 1074 275 525 sr 23 13 13 26 13 9 15 1 16 8 12 16 7 10 22 6 12 r 74 42 42 83 42 29 48 3 51 26 38 51 22 32 70 19 38 sR 25 21 27 47 30 18 29 2 28 12 25 33 15 20 46 15 22 r 80 67 86 150 96 58 93 6 90 38 80 106 48 64 147 48 70 ur 52 29 30 58 28 20 33 2 37 18 27 37 17 22 50 13 27 uR 58 46 61 106 68 40 66 5 63 28 56 75 35 44 103 34 49 repetitions using the internal standard norleucine. The calculated recovery for norleucine was from 95.0% to 97.2%. 3.2 AnALySIS Of Dry-CureD hAm SAmpLeS free amino acid content and chemical parameters were determined in 135 Slovenian dry-cured hams »Kraski prsut« for 2 different muscles. The results of chemical parameters are given in table 4. In the Sm muscle the moisture content and proteolysis index are lower in comparison with the Bf muscle, but protein and total nitrogen content were observed to be higher. higher quantity of total nitrogen can be explained by the difference in dry matter and moisture content. however, the Table 4: Chemical parameters for the BF and SM muscle in dry-cured hams "Kraski prsut" (mean ± standard deviation) Preglednica 4: Kemijski parametri za misici BF in SM v kraskem prsutu (povprecje ± standardni odklon) parameter moisture (%) naCl (%) protein (%) total nitrogen (g/kg) non-protein nitrogen (g/kg) proteolysis index (%) Intramuscular fat (%) Dry matter (g/kg) Bf muscle 58.5 ± 1.5 7.6 ± 0.7 29.5 ± 1.2 47.2 ± 1.9 12.6 ± 1.0 26.8 ± 1.8 3.0 ± 0.9 415 ± 14 min­max 54.5­65.2 5.9­9.4 26.0­33.6 41.6­54.0 7.0­14.7 16.1­31.1 1.6­6.2 377­455 Sm muscle 49.0 ± 2.3 6.7 ± 0.7 39.0 ± 2.3 62.4 ± 3.6 11.8 ± 1.1 18.9 ± 1.8 4.5 ± 1.2 510 ± 23 min­max 43.5­56.2 4.5­8.3 32.0­43.9 51.2­70.2 7.8­14.4 13.3­24.1 2.1­8.8 438­565 concentration of salt is higher in the Bf muscle. According to the literature, this can be explained by the fact that salt consequently moves to the areas with higher moisture content where more salt can be dissolved (Arnau et al., 1995; monin et al., 1997). higher proteolysis index and non-protein nitrogen contents were observed in the Bf muscle. As this is an internal muscle, it is reached by the salt penetration much later and the desiccation is smaller than in the Sm muscle (Arnau et al., 1995). Both factors, the salt content and the water availability, are crucial for the activity of muscle proteolytic enzymes and consequently the generation of non-protein nitrogen (toldrá, 2002). table 5 shows the contents of free amino acids in dry-cured hams "Kraski prsut" for the Bf and Sm muscles. The amino acid content in "Kraski prsut" is in the middle of the range of values reported for other sorts of dry-cured hams (toldrá and flores, 1998). Considering the results calculated on dry matter basis, the content of free amino acids is higher in the Bf muscle, which is in accordance with the result for the proteolysis index, also found to be higher in the Bf muscle in the present ex- periment as well as in other reports (monin et al., 1997; Candek-potokar et al., 2002). The exceptions were only the content of cystine (lower in the Bf muscle) and the content of methionine and phenylalanine (lower in the Sm muscle, though not statistically significant). regarding the differences in the dry-cured ham procedures (length, temperature conditions, amount of salt added, raw materials) between the manufacturers and countries of origin, it is difficult to compare our results obtained for free amino acid content with the available literature data. CONCLUSION Chemical profile of dry-cured ham selected and processed in accordance with the Slovenian consortium rules for »Kraski prsut« was characterized and differences between the Bf and Sm muscle were investigated. for the determination of free amino acids in drycured hams we used the method for the determination of free amino acids in animal feeding stuffs and modified the sample preparation. validation of the method Table 5: The free amino acid content (in mg/100 g dry matter) in dry-cured hams "Kraski prsut" for the BF and SM muscle (mean ± standard deviation) Preglednica 5: Vsebnost prostih amino kislin (v mg/100 g suhe snovi) v kraskem prsutu za misici BF in SM (povprecje ± standardni odklon) Amino acid Aspartic acid Threonine Serine Glutamic acid proline Glycine Alanine Cystine valine methionine Isoleucine Leucine tyrosine phenylalanine Lysine histidine Arginine total Bf muscle 450 ± 19 350 ± 17 447 ± 23 1007 ± 40 446 ± 27 345 ± 16 622 ± 27 33 ± 5 523 ± 24 215 ± 9 385 ± 21 566 ± 26 293 ± 17 342 ± 16 907 ± 39 234 ± 14 445 ± 17 7610 min­max 318­591 273­441 332­593 834­1213 348­578 268­431 472­789 4­80 406­655 145­296 280­524 431­758 218­381 253­475 723­1139 184­288 249­593 SB muscle 391 ± 17 297 ± 15 379 ± 20 849 ± 34 370 ± 22 290 ± 13 519 ± 23 46 ± 7 470 ±22 211 ± 9 361 ± 20 529 ± 24 279 ± 16 341 ± 16 789 ± 34 213 ± 12 338 ± 13 6670 min­max 301­529 214­412 327­557 666­1090 300­566 218­388 393­673 6­130 363­660 143­367 252­598 383­849 209­398 235­578 632­1076 173­300 200­530 Sign. *** *** *** *** *** *** *** *** *** n.s. *** *** *** n.s. *** *** *** *** fisher test value Sign., significance level: ***, p < 0.001; **, p <0.01; *, p<0.05; n.s., non significant has shown good repeatability, reproducibility and linearity. According to the validation parameters the method is appropriate for the determination of free amino acid content in dry-cured ham. from the results obtained in the present work it can be concluded that the proteolysis index, the content of moisture, salt, non-protein nitrogen, and free amino acids are higher in the Bf muscle compared to the Sm muscle. The results concerning the free amino acid content reflect the chemical composition of dry-cured ham and the differences between both investigated muscles. ACKNOWLEDGEMENTS The research was funded by the grants of the research project truefOOD ("traditional united europe food"), an Integrated project financed by the european Commission under the 6th framework programme for rtD, contract no. fOOD-Ct-2006-016264. In Slovenia this study was also partly made with the grants of Slovenian research Agency for the applied project L4-9468. The authors would also like to express their thanks to industrial partners taking part in this study farme Ihan d.d., turolense Ganadera S.A. (pig producers), KrAS d.d. and mIp d.d., Grupo monte nevado (dry ham processors), Agroalimentaria teruel (abattoir) and pyragena france (dry ham processor). The authors also thank zvonko Bregar for his contribution to the present article.

Journal

Acta Agriculturae Slovenicade Gruyter

Published: Jun 1, 2012

References