Parmesan Cheese

of 5
13 views
PDF
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Document Description
1136 J. Agric. Food Chem. 2003, 51, 1136−1140 Optimization of Solid Phase Microextraction Analysis for the Headspace Volatile Compounds of Parmesan Cheese JAE-HWAN LEE, RAYMOND DIONO, GUR-YOO KIM,† AND DAVID B. MIN* Department of Food Science and Technology, The Ohio State University, 2015 Fyffe Court, Columbus, Ohio 43210 Optimum conditions of solid phase microextraction (SPME) analysis of the headspace volatile compounds of Parmesan cheese in airtightly sealed 100-mL bottles were develope
Document Share
Document Tags
Document Transcript
  Optimization of Solid Phase Microextraction Analysis for theHeadspace Volatile Compounds of Parmesan Cheese J AE -H WAN L EE , R AYMOND D IONO , G UR -Y OO K IM , † AND D AVID B. M IN * Department of Food Science and Technology, The Ohio State University, 2015 Fyffe Court,Columbus, Ohio 43210 Optimum conditions of solid phase microextraction (SPME) analysis of the headspace volatilecompounds of Parmesan cheese in airtightly sealed 100-mL bottles were developed. The coefficientof variation of SPME analysis on the headspace volatile compounds of Parmesan cheese was 2%.The reproducibility of SPME was improved by a combination of sampling at - 10 ° C, controlling thesample temperature, and uniform magnetic stirring of samples during equilibrium and isolation steps.The sensitivity of SPME increased by 125% in total peak areas by a combination of 40 min ofsonication and 25% (w/v) sodium phosphate solution, compared with that of samples containingdeionized water only ( P  < 0.05). The addition of salt solution or sonication treatment in samplesincreased the headspace volatile compounds of cheese quantitatively without producing any newvolatile compounds. KEYWORDS: Cheese; solid phase microextraction; volatile compound analysis; salt effects; sonication INTRODUCTION Flavor is one of the most important characteristics thatdetermines the quality of cheese ( 1 ). Steam distillation, simul-taneous distillation extraction, static headspace, and dynamicheadspace have been used to study the volatile compounds incheese ( 2 - 5 ). Steam distillation can cause the loss of heatsensitive volatile compounds, and solvents can provide undesir-able artifacts during analysis ( 6  ). Although static headspaceanalysis is the simplest method for the determination of volatilecompounds, it lacks sensitivity ( 7  , 8 ). Dynamic headspaceanalysis can overcome the sensitivity problem of static head-space analysis. Dynamic headspace analysis is an expensive,time-consuming, labor-intensive method and is less reproduciblethan static headspace analysis ( 9 ).Solid phase microextraction (SPME) is an analytical techniquefor the isolation and concentration of volatile or nonvolatilecompounds in foods; it integrates sampling, extraction, con-centration, and sample introduction to gas chromatography (GC)or high-performance liquid chromatography ( 10 ). The principleof headspace SPME is an equilibrium partitioning of analytesamong the coating of solid phase or fiber, sample matrix, and/ or headspace ( 11 ). SPME has been used to analyze the volatilefraction of ewe’s milk cheese ( 12 ) and the headspace volatilecompounds in Cheddar cheese ( 13 ) and Swiss cheeses ( 14 ).Cryotrapping/SPME was designed to maximize the isolation andconcentration of volatile compounds from cheese ( 15 ). Eventhough cryotrapping/SPME is a sensitive method, the reproduc-ibility was not good enough due to the interference of trappedwater in the SPME solid phase ( 15 ). Studies of SPMEoptimization on cheese volatile compounds have been focusedon the selection of proper solid phases, exposure time, influenceof absorption temperature, and the ratio of sample and headspacevolume ( 12 , 13 ). However, few studies have been reported onthe development of both reproducible and sensitive SPMEconditions on the headspace volatile compounds due to therelatively low concentration and uneven distribution of volatilecompounds in cheese and the continuous generation andtransformation of volatile compounds during the ripening stage.The objective of this study was to optimize the SPMEconditions to increase reproducibility and sensitivity for theanalysis of the headspace volatile compounds in Parmesancheese. MATERIALS AND METHODS Materials. Parmesan cheese was donated from Antigo Cheese Co.(Antigo, WI) and stored in a - 10 ° C cold room. A manual SPMEfiber holder unit, 75 µ m (diameter) Carboxen/poly(dimethylsiloxane)(CAR/PDMS), serum bottles, Teflon-coated rubber septa, and aluminumcaps were purchased from Supelco (Bellefonte, PA). Sodium chloride(NaCl), sodium phosphate (NaH 2 PO 4 ), potassium chloride (KCl),sodium sulfate (Na 2 SO 4 ), ethanol, acetic acid, butanoic acid, 2-hep-tanone, benzaldehyde, hexanoic acid, limonene, sorbic acid, 2-nonanone,undecane, octanoic acid, 2-undecanone, decanoic acid, and dodecanoicacid were purchased from Sigma (St. Louis, MO). Effects of Sample Size on Reproducibility. All samples wereprepared at - 10 ° C in a cold room in the Department of Food Scienceat The Ohio State University. Cheese was cut with a cheese cutter intosmall pieces ∼ 1 mm in diameter at - 10 ° C to increase the surfacearea. To study the effects of sample size, 10, 15, 20, 25, and 30 g of prepared Parmesan cheese with 10, 15, 20, 25, and 30 mL of deionized * Author to whom correspondence should be addressed [telephone (614)292-7801; fax (614) 292-0218; e-mail Min.2@osu.edu]. † Present address: Department of Animal Food Science and Technology,Kangwon National University, Hyoja2-Dong, Chunchon, Kangwon-Do,Korea. 1136 J. Agric. Food Chem. 2003, 51, 1136 − 1140 10.1021/jf025910+ CCC: $25.00 © 2003 American Chemical SocietyPublished on Web 01/23/2003  water, respectively, were put in a 100-mL bottle with a magnetic stirringbar (8 × 25 mm). To study the ratio of cheese to water contents, samplesof 25 g of prepared cheese with 20, 25, or 30 mL of deionized water,respectively, were put in a 100-mL bottle with a magnetic stirring bar(8 × 25 mm). Sample bottles were prepared in triplicate and sealedairtight with aluminum caps and Teflon-coated rubber septa. Samplebottles wrapped in aluminum foil were kept in a - 10 ° C cold roomuntil use. Headspace Volatile Compound Analysis by SPME. Sample bottleswere put in a new flavor isolation apparatus that can regulate thetemperature and stirring of a sample ( Figure 1 ). The sample bottleswere kept for 10 min at 50 ° C in the new flavor isolation apparatus tomelt the cheese. Sample was magnetically stirred in the new flavorisolation apparatus for 20 min at 50 ° C to homogenize the sample andto accelerate equilibrium of headspace volatile compounds betweenthe cheese matrix and the headspace. The 75 µ m CAR/PDMS fibertrapped the headspace volatile compounds for 30 min at 40 ° C duringstirring. The volatile compounds isolated by CAR/PDMS were desorbedin the injector port of a GC (Hewlett-Packard 5890 GC). Effects of Salt Concentration. NaCl was added into deionized waterto obtain 5, 20, and 25% (w/v) and saturated salt solution. To studythe effects of salt concentration on the sensitivity of SPME, headspacevolatile compounds from Parmesan cheese samples with deionizedwater, 5, 20, and 25% (w/v), or saturated NaCl solution were analyzedby SPME-GC. Effects of Salt Types. NaCl, NaH 2 PO 4 , KCl, or Na 2 SO 4 was addedinto deionized water to obtain 25% (w/v) solutions. To study the effectsof salt types on the sensitivity of SPME, headspace volatiles fromParmesan cheese samples with deionized water, 25% NaCl, NaH 2 PO 4 ,KCl, or Na 2 SO 4 solution were analyzed by SPME-GC. Effects of Sonication. To determine the effects of sonication timeon the sensitivity of SPME, sample bottles, which were kept for 10min at 50 ° C in the new flavor isolation apparatus without stirring andthen magnetically stirred for 20 min at 50 ° C in the new flavor isolationapparatus, were sonicated for 0, 10, 20, 30, 40, 50, and 60 min in aFischer Scientific ultrasound water bath (Shelton, CT). A constant waterlevel of 2000 mL was maintained for each sonication treatment. Thewater temperature in the ultrasound water bath was maintained at 40 ( 0.5 ° C during sonication. Sample bottles in a 40 ° C water bathwithout sonication were used as controls. After sonication, an equi-librium step was introduced for 10 min at 40 ° C in the new flavorisolation apparatus with stirring. Combination Effects of Sonication and Salt. To study thecombination effects of sonication and salt on the sensitivity of SPME,Parmesan cheese samples with 25% (w/v) NaH 2 PO 4 solution weresonicated for 40 min at 40 ° C. The headspace volatile compounds incheese samples with deionized water only, 25% NaH 2 PO 4 solution only,or 25% NaH 2 PO 4 solution with 40 min of sonication were isolated bySPME-GC. Conditions of Gas Chromatography. A Hewlett-Packard 5890 gaschromatograph was equipped with a 0.75 mm i.d. glass injection liner,a flame ionization detector, and a 30 m × 0.25 mm i.d., 1.0 µ m film,DB-5 column, from J&W Scientific (Folsom, CA). The oven temper-ature was held at 40 ° C for 2 min and increased from 40 to 160 ° C atthe rate of 6 ° C/min and from 160 to 210 ° C at 10 ° C/min. Thetemperatures of the injector and detector were 250 and 300 ° C,respectively. The flow rate of nitrogen carrier gas was 1.0 mL/min.The isolated volatile compounds in the solid phase of SPME weredesorbed at 250 ° C for 2 min. Identification of Volatile Compounds in Parmesan Cheese. Theheadspace volatile compounds in samples of cheese only, cheese withdeionized water, cheese with 25% NaH 2 PO 4 solution, and cheese withdeionized water and sonication treatment were isolated by CAR/PDMS,separated by GC, and identified by MS. A Hewlett-Packard 5971Amass selective detector equipped with a Hewlett-Packard 59822Bionization gauge controller was used. All mass spectra were obtainedat 70 eV and an ion source temperature of 220 ° C. Identification of compounds was made by the combination of NIST mass spectra andgas chromatographic retention times of standard compounds. Heliumcarrier gas at 0.9 mL/min and an HP-5 column (30 m × 0.25 mm i.d.,0.25 µ m thick) from Agilent Technologies (Palo Alto, CA) were used.The GC conditions for GC-MS were the same as the gas chromato-graphic analysis conditions described previously. Statistical Analysis. One-way analysis of variance and Tukey’smultiple comparisons were used to analyze the data. A P value of  e 0.05was considered to be significant. All statistical analyses were conductedwith Minitab 12.1 (Minitab Inc., State College, PA). RESULTS AND DISCUSSION Effects of Sample Preparation Temperature and SampleSize on Reproducibility. The coefficient of variation of SPMEanalysis on the headspace volatile compounds in Parmesancheese was 2% for total GC peak areas. The reproducibility of SPME could be achieved by a combination of sampling at - 10 ° C, controlling the sample temperature, and uniform magneticstirring of samples during equilibrium and isolation steps.A new flavor isolation apparatus, which can control thetemperature and the magnetic stirring speed of a sample, wasdesigned for this study to provide constant volatile compoundisolation conditions ( Figure 1 ). Water temperature, water level,and the magnetic stirring speed of a sample were kept constantin the new flavor isolation apparatus for each sample treatment.The water level in the apparatus can be maintained byminimizing the evaporation of water using a water bath cover.This new apparatus can be used to measure the headspacevolatile compounds from other aqueous food samples and toanalyze light-sensitive samples when the constant-temperaturewater bath and a sample bottle are wrapped with aluminum foilto make dark conditions.Magnetic stirring plays an important role in increasing thereproducibility of SPME by accelerating the mass transferbetween the sample and solid phase and shortening theequilibrium time ( 11 , 16  ). Preliminary study showed that thecoefficient of variation from samples containing only cheesewithout magnetic stirring was > 15% due to the unevendistribution of volatile compounds in cheese samples (data notshown).Sample preparation at - 10 ° C also is important in achievingreproducibility of SPME. Preliminary study showed that thecoefficients of variation of volatile compounds from samplesprepared at 5 and 25 ° C were 11 and 19%, respectively (datanot shown).Effects of sample size and the ratio of sample to headspacevolume in a 100-mL sample bottle on the headspace volatilecompounds in Parmesan cheese by SPME are shown in Table1 . As the amount of cheese increased from 10 to 15, 20, 25,and 30 g in 1:1 ratio of sample to headspace volume, total peak areas increased from 2.30 to 3.56, 5.21, 6.99, and 8.07 (1 × Figure 1. Flavor isolation apparatus, which can control temperature andmagnetic stirring speed of a sample for SPME analysis. Optimization of SPME Conditions for Cheese Volatiles J. Agric. Food Chem., Vol. 51, No. 5, 2003 1137  10 5 ), respectivelt, and the coefficient of variation in total peak areas ranged from 0.9 to 6.4%.The total peak areas of samples containing 25 g of cheesewith 20, 25, and 30 mL were 6.85, 6.99, and 5.67 (1 × 10 5 ),respectively. The 20% decrease in total peak areas in the samplesof 25 g of cheese with 30 mL of water may be due to theincrease of water vapor pressure at the current analysis condi-tions. Water vapor competes with volatile compounds towardthe active sites of SPME solid phases and decreases the totalpeak areas in aqueous foods such as orange juice ( 17  ). Themixture of 25 g of cheese and 25 g of water was chosen as theoptimum sample size for this study, considering the bestreproducibility. The optimum ratio of sample to headspacevolume for SPME varies depending on the sample’s character-istics. The ratio of orange juice to headspace was 1:5 ( 17  ), andthat of soybean oil to headspace was 2:1 ( 18 ). Effects of Salt Concentration. Relative total peak areas fromParmesan cheese samples with 0, 5, 20, 25%, and saturated NaClsolution are shown in Figure 2 . As salt concentration increasedfrom 0 to 25%, relative total peak areas increased significantlyby 70% ( P < 0.05). The amount of headspace volatiles producedby samples with saturated NaCl was 15% less than that producedby samples with 25% NaCl.Electrolytes, such as salt, in an aqueous system can influencethe phase boundary properties and decrease the solubility of hydrophobic compounds by competing water molecules, whichis called “salting out” ( 19 , 20 ). Although the efficiency of volatile compound extraction depends on the concentration of salt solution, a 20 - 30% (w/v) salt concentration was reportedto be sufficient to give the best sensitivity for most volatilecompounds ( 20 ). A salt concentration of 25% was chosen asthe optimum level for this study. Effects of Salt Types. Effects of 25% NaCl, KCl, NaH 2 -PO 4 , or Na 2 SO 4 solution on the volatile compounds in Parmesancheese are shown in Figure 3 . Addition of Na 2 SO 4 , KCl, NaCl,and NaH 2 PO 4 solution increased the total peak areas by 23, 51,70, and 126%, respectively, compared with samples withdeionized water. NaH 2 PO 4 showed the highest increases in theheadspace volatile compounds. The phosphate ion from NaH 2 -PO 4 can chelate the calcium ion in cheese and change the ratioof calcium to phosphate concentration, which can solubilize thecoagulated milk proteins and loosen the cheese matrix ( 21 ). Theloosened structure would accelerate the release of volatilecompounds trapped inside the matrix of the cheese, whereasNa 2 SO 4 , KCl, and NaCl may just compete for water with thevolatile compounds without changing the structure of the cheesematrix. Effects of Sonication. Effects of sonication times of 0, 10,20, 30, 40, 50, and 60 min on the headspace volatile compoundsin Parmesan cheese are shown in Figure 4 . Total peak areas of Parmesan cheese with sonication were significantly greater thanthose of samples in a water bath without sonication from 10 to40 min ( P < 0.05). Total peak areas in Parmesan cheeseincreased by 70% up to 40 min of sonication and decreasedsignificantly after 40 min ( P < 0.05), which may be due to theincrease of water vapor pressure in the headspace of samplebottles. Sonication can loosen the structures of sample matrixand release the volatile compounds physically trapped in thematrix. Sonication treatment has been reported to increase theamount of polyaromatic hydrocarbons in headspace from watersamples ( 11 ). Forty minutes of sonication was chosen for furtherexperiments in this study. Combination Effects of Sonication and Salt. Effects of thecombination of sonication and NaH 2 PO 4 on the total peak areas Table 1. Effects of Sample Size and the Ratio of Sample toHeadspace Volume in a 100-mL Sample Bottle on the HeadspaceVolatile Compounds in Parmesan Cheese by SPME cheese (g)/water (mL)sample vol/headspace voltotal GC peakareas in electroniccount a  (1 × 10 5 )coefficient ofvariation (%)10:10 20:80 2.30 ± 0.10a 4.315:15 30:70 3.56 ± 0.12b 3.420:20 40:60 5.21 ± 0.19c 3.625:20 45:55 6.85 ± 0.10e 1.425:25 50:50 6.99 ± 0.06e 0.925:30 55:45 5.67 ± 0.12d 2.130:30 60:40 8.07 ± 0.52f 6.4 a  Mean ± SD ( n  ) 3). Different letters indicate a significant difference ( P  <0.05). Figure 2. Effects of 0, 5, 20, 25% (w/v), and saturated NaCl solution onthe headspace volatile compounds in Parmesan cheese by SPME. Barswith different superscripts are significantly different ( P  < 0.05). Figure 3. Effects of sodium chloride, potassium chloride, sodiumphosphate, and sodium sulfate solution on the headspace volatilecompounds in Parmesan cheese by SPME. Bars with different superscriptsare significantly different ( P  < 0.05). 1138 J. Agric. Food Chem., Vol. 51, No. 5, 2003 Lee et al.  in cheese are shown in Figure 5 . Total peak areas in cheesetreated with sonication only, 25% NaH 2 PO 4 solution only, andthe combination of sonication and NaH 2 PO 4 solution increasedby 70, 126, and 125%, respectively, compared with sampleswith deionized water only. Effects of a combination of soni-cation and salt were not significantly different from those of samples with salt solution only ( P > 0.05). Thus, for timeefficiency reasons, salting with 25% NaH 2 PO 4 solution only isrecommended. Identified Headspace Volatile Compounds in Parmesan. Identified headspace volatile compounds from Parmesan cheesewithout water, with deionized water, with 25% NaH 2 PO 4 solution, or with deionized water and sonication are shown in Table 2 . Addition of deionized water, 25% NaH 2 PO 4 solution,and sonication treatment changed the headspace volatile com-pounds in Parmesan cheese quantitatively but did not produceany new volatile compounds, which were not present in cheesesamples. Low molecular weight and water-soluble compoundsincluding ethanol, acetic acid, and butanoic acid decreased inthe samples of deionized water, compared with cheese-onlysamples due to the solubility of these compounds in water.Addition of salt or sonication treatment to cheese samplesincreased some of the headspace volatiles quantitatively but notqualitatively.Most of the identified volatile compounds were reportedpreviously ( 2 , 22 ). High molecular weight and relativelynonpolar compounds including decanoic and dodecanoic acids,which were reported by simultaneous distillation and extractionmethod ( 2 ) but not by purge-and-trap technique ( 2 , 22 ), can bedetected by CAR/PDMS SPME-GC analysis.In conclusion, a reproducible and sensitive SPME conditionfor the analysis of headspace volatiles in cheese was developed.Reproducibility of SPME was achieved by a combination of sampling at - 10 ° C, using 25 g of cheese with 25 mL of waterin a 100-mL sample bottle, controlling the sample temperature,and uniform magnetic stirring of samples during equilibriumand isolation steps using a flavor isolation apparatus. Additionof NaH 2 PO 4 solution increased the sensitivity of analysis of volatile compounds in cheese significantly. The reproducibleand sensitive conditions of SPME analysis in this study can beused for the analysis of the cheese volatile compounds duringcheese manufacturing and aging to enhance the flavor qualityof cheese. LITERATURE CITED (1) Adda, J. Flavour of dairy products. In De V elopment in Food Fla V or  ; Birch, G. G., Lindley, M. G., Eds.; Elsevier AppliedScience: London, U.K., 1986; pp 151 - 172. Figure 4. Effects of sonication on the headspace volatile compounds inParmesan cheese by SPME. Figure 5. Combination effects of salt solution and sonication on theheadspace volatile compounds in Parmesan cheese by SPME. Bars withdifferent superscripts are significantly different ( P  < 0.05). Table 2. Identified Headspace Volatile Compounds in ParmesanCheese without Water, with Deionized Water, with 25% NaH 2 PO 4 Solution, and with Sonication volatile compound a  cheeseonlywithdeionizedwaterwith 25%NaH 2 PO 4 solutionwithsonicationethanol GC,MS 27.2 b  19.6 29.5 28.61,3-pentadiene MS 12.8 10.2 13.2 12.12-methylfuran MS 3.2 2.1 3.9 3.7acetic acid GC, MS 32.6 21.5 36.4 30.52-pentanone MS 0.7 1.1 1.2 1.1butanoic acid ethyl ester MS 0.8 1.2 1.6 1.3butanoic acid GC,MS 7.5 3.9 9.5 8.71,2-dimethylbenzene MS 0.3 0.3 0.5 0.32-heptanone GC,MS 6.9 4.2 7.5 6.8benzaldehyde GC , MS 0.4 0.5 0.7 0.5hexanoic acid GC,MS 18.5 18.1 21.2 20.5limonene GC,MS 0.4 0.4 0.5 0.4benzenacetaldehyde MS 0.6 0.7 0.9 0.8sorbic acid GC,MS 4.7 4.5 6.2 4.22-nonanone GC,MS 7.8 8.2 10.3 9.4undecane GC,MS 0.3 0.4 0.6 0.5octanoic acid GC,MS 3.8 4.2 4.8 4.3octanoic acid ethyl ester MS 1.8 0.9 2.3 2.1octanoic acid isopropyl ester MS 0.2 0.3 0.4 0.32-undecanone GC,MS 0.3 0.3 0.4 0.3decanoic acid GC,MS 0.7 0.7 0.9 0.8dodecanoic acid GC,MS 0.2 0.3 0.4 0.3 a  Superscript GC,MS represents a volatile identified by both GC retention timeof a standard compound and GC-MS. Superscript MS represents a volatile identifiedby GC-MS only. b  Mean value of peak area of a compound in ion count (1 × 10 7 )by SPME-GC-MS ( n  ) 3). Optimization of SPME Conditions for Cheese Volatiles J. Agric. Food Chem., Vol. 51, No. 5, 2003 1139
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks
SAVE OUR EARTH

We need your sign to support Project to invent "SMART AND CONTROLLABLE REFLECTIVE BALLOONS" to cover the Sun and Save Our Earth.

More details...

Sign Now!

We are very appreciated for your Prompt Action!

x