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Key odorants of Lazur - a polish type mold-ripened cheese Ma#gorzata Anna Majcher, Kamila Myszka, Anna Grygier, Anna Gracka, and Henryk H. Jelen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04911 • Publication Date (Web): 01 Feb 2017 Downloaded from http://pubs.acs.org on February 3, 2017

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Journal of Agricultural and Food Chemistry

Key Odorants of Lazur - a Polish Type Mold-Ripened Cheese

Title running header: aroma-active compounds of mold-ripened cheese

Małgorzata A. Majcher1*, Kamila Myszka1, Anna Gracka1, Anna Grygier1, Henryk H. Jeleń1

1

Poznań University of Life Sciences, Faculty of Food Science and Nutrition, Wojska

Polskiego 31, 60-624 Poznań, Poland

*Corresponding author: phone: +48618487276; fax: +48618487314; e-mail: [email protected]

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ABSTRACT

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Application of gas chromatography olfactometry (GC-O) carried out on the volatile

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fraction isolated by solvent assisted flavor evaporation (SAFE) and solid phase

4

microextraction (SPME) from Lazur – mold-ripened cheese revealed 17 odor-active

5

compounds. The highest flavor dilution factor (FD) has been obtained for methanethiol

6

(2048) with burnt odor note and for 2(3)-methyl butanoic acid (2048) with cheesy,

7

pungent odor. Further quantitation of 15 most aroma-active compounds allowed for

8

calculation of their odor activity values (OAV). The highest OAV was obtained for

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methanethiol (500), 3(2)-methyl butanoic acid (321), 3-(methylthio)-propanal (210),

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2,3-butanedione (65), dimethyl trisulfide (22), butanoic acid (20), 1-octen-3-ol (18),

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(Z)-4-heptenal (14), dimethyl disulfide (14), dimethyl sulfide (13), phenylacetaldehyde

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(6), 2-ethyl-3,5-dimethyl pyrazine (5) and acetic acid (4). Aroma recombination

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experiment showed slight difference in the perception of cheesy/sweaty and

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moldy/musty notes. To verify influence of methyl ketones on aroma profile of mold-

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ripened cheese, recombinant has been additionally supplemented with the addition of 2-

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pentanone, 2-heptanone and 2-nonanone in concentrations determined in Lazur cheese.

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The aroma profile remained unchanged, which would suggest that methyl ketones, in

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this particular cheese do not play a significant role in the formation of aroma.

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KEYWORDS: mold-ripened cheese, aroma-active compounds, sulfur compounds, GC-

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O, aroma recombinate

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INTRODUCTION

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Blue cheeses are those type of cheeses whose matrix is grown through with Penicillium

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roqueforti mold resulting in blue, blue-greenish or blue-grayish spots or veins. They are

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widely appreciated for their specific texture and aroma which is formed in course of

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extensive proteolysis and lipolysis bringing pungent aroma and strong taste. Although

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the most well-known blue cheeses, with the longest tradition come from France

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(Roquefort), Italy (Gorgonzola), Great Britain (Stilton) or Denmark (Danish Blue), in

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recent years Poland as well has developed the production of mold-ripened cheeses. The

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most recognized have been produced for nearly 60 years and they are called Lazur Blue,

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Green or Silver, depending on the color of developed mold veins. Though the aroma of

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each of them is unique it is generally described as pungent and moldy.

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The investigations on the aroma of blue type cheeses have been performed for more

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than 100 years back, with the first research paper written on flavor of Roquefort cheese

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by James Currie in 1914.1 Since then, vast amount of studies has been performed on

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volatile composition as well as sensory qualities of mold-ripened cheeses, resulting in

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numerous published original papers as well as reviews which would be impossible to

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cite all. Although the number of volatiles as well as their chemical characteristic vary

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greatly in all of them, all authors agree that characteristic flavor of blue cheese is due to

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the high concentration of methyl ketones mainly 2-heptanone and 2-nonanone, which

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are formed during enzymatic lipolysis of free fatty acids.

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However, the newest studies which imply the molecular sensory approach on food

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aroma, are showing, that limited number of volatiles present in foods, so-called key

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odorants is able to interact with the human olfactory receptors generating aroma

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perception in the brain.2 To identify key odorants, gas chromatography olfactometry

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(GC-O) should be implied, followed by quantitation studies, which through comparison 3 ACS Paragon Plus Environment


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with odor threshold, lead to the calculation of odor activity values (OAV). Finally as a

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proof of good identification and quantitation, reconstitution experiments are carried out

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by mixing pure aroma compounds in the concentration determined in the food product

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in an appropriate matrix. Aroma reconstitution is widely accepted to finally proof the

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typical food aroma in regard to interaction between key aroma-active constituents.3

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There are only several research papers describing application of GC-O for analysis of

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blue cheese flavor. In 2000 Moio et al.4 has presented aroma-impact compounds of

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Gorgonzola cheese, then Qian et al.5 applied dynamic headspace GC-O for

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characterization of blue cheese aroma with specific emphasis on fat-derived compounds

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and finally Frank et al.6 with application of solid phase microextraction (SPME)

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combined with GC-O compared key aroma compounds of three different blue cheeses.

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Although there have been from 18 to 49 compounds identified by all three authors, only

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5 compounds are repeating in all them: methional, 2-heptanone, 2-nonanone, ethyl

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hexanoate and ethyl butanoate. Unfortunately only in one of those papers, quantitative

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results of key odorants were presented.4 More quantitative data could be obtained from

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Gallois7 report in which authors estimated, based on the addition of internal standard,

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the concentration of all volatiles in five different French blue type cheeses. In a more

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recent studies, number of papers report quantitative data on volatiles present in mold-

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ripened cheeses8-10 however to the best of our knowledge, there is no publication

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available successfully identifying the key odorants of mold-ripened cheese by a

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systematic approach using the Sensomics concept11 and in particular by using exact

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quantitative data to perform a final simulation of the overall aroma. Therefore, the aim

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of this study was identification of aroma-active compounds in a mold-ripened cheese -

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blue cheese type using Sensomic approach consisting of the identification of the most

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important odorants using gas chromatography-olfactometry (GC-O) and gas 4 ACS Paragon Plus Environment

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chromatography-mass spectrometry (GC/MS), followed by quantitation experiments

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with stable isotope dilution assay and, finally a simulation of the aroma by

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recombination experiments.

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MATERIALS AND METHODS

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Cheese samples. Green Lazur cheese samples were collected directly from the producer

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located in Nowe Skalmierzyce, Poland. It is made from pasteurized cow’s milk, first

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inoculated with mesophilic LAB (Lactococcus lactis, Leuconostoc spp.) and then

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coagulated with addition of rennet. At this point noble mold Penicillium roqueforti is

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introduced as well. For key odorants analysis cheese samples were collected after eight

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weeks of ripening and analyzed on the day of collection. Sampling was repeated three

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times, from three different cheese units. The gross composition of Lazur cheese has

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been determined as: moisture 44.3, fat 31.2, protein 20.6 (g/100g). The cheese is

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produced in a cylindrical shape with the weight of 3-6 kg.

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Chemical standards. Solvents, such as diethyl ether, methylene chloride and sodium

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sulfate were obtained from Sigma Aldrich (Poznań, Poland). The following reference

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aroma compounds were purchased from Sigma-Aldrich (Poznań, Poland): methanethiol,

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dimethyl sulfide, 2,3-butanedione, dimethyl disulfide, 2-methyl butanoic acid, 3-methyl

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butanoic acid, (Z)-4-heptenal, 3-(methylthio)-propanal, acetic acid, dimethyl trisulfide,

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1-octen-3-one, phenylacetaldehyde, butanoic acid, 2-ethyl-3,5-dimethylpyrazine, 2-

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pentanone, 2-heptanone, 2-nonanone, [2H7]-butanoic acid, [2H4]-acetic acid, [2H6]-

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dimethyl disulfide, [2H6]-dimethyl sulfide. The following compounds were purchased

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from Aroma LAB (Freising, Germany): [13C4]-2,3-butanedione, [2H3]-3-(methylthio)-

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propanal,

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dimethylpyrazine, [2H2]-2(3)-methyl butanoic acid. The purity of solvents and reference

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standards was no less than 99% and 97% respectively.

[2H3]-1-octen-3-one,

[13C2]-phenylacetaldehyde,

[2H5]-2-ethyl-3,5-



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Isolation method. For isolation of aroma-active compounds two methods were used: by

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solvent assisted flavor evaporation (SAFE) described by Engel et al.12 and solid phase

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microextraction (SPME) described by Pawliszyn.13 Prior to SAFE extraction cheese

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samples (20 g) were frozen in liquid nitrogen, ground and extracted with methylene

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chloride (300 mL) for 2 hours each by shaking it in the horizontal shaker. After the

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volatiles were isolated by SAFE distillation extract was concentrated with a Kuderna

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Danish concentrator (Sigma-Aldrich) to about 400 µL. For SPME analysis 10 g of

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cheese sample, frozen in liquid nitrogen and ground, was placed in 20 ml headspace

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vials and capped with PTFE/silicon septa caps. Extraction of volatiles was performed

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with 2cm 50/30 µm CAR/PDMS/DVB fiber (Supelco) at 30 °C during 30 min using

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CTC combipal autosampler (Agilent Technologies).

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Gas Chromatography-Olfactometry (GC-O). Odor active compounds were identified

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both from SAFE extracts and SPME, by GC-O on an HP 5890 chromatograph using the

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following capillary columns: SPB-5 (30 m x 0.53 mm x 1.5 µm,) and Supelcowax 10

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(30 m x 0.53 mm x 1 µm); Supelco, Bellefonte, PA. The GC was equipped with an Y

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splitter dividing effluent 1:1 between olfactometry port with humidified air as a make-

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up flow, and a flame ionization detector. The operating conditions were as follows:

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helium flow 0.8 ml/min; for the SPB-5 column: initial oven temperature 40 °C (1 min),

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raised at 9 °C/min to 180 °C and at 20 °C/min to 280 °C. Operating conditions for the

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Supelcowax 10 column were as follows: initial oven temperature, 40 °C (2 min), raised

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to 240 °C at 9 °C/min rate, held for 2 min isothermally. For all peaks and flavor notes,

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retention indices were calculated to compare results obtained by GC/MS with literature

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data. Retention indices were calculated for each compound using homologous series of

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C7 – C24 n-alkanes.


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Gas Chromatography/ Mass Spectrometry. The chemical compounds were identified

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using Agilent Technologies 7890A GC coupled to a 5975C MSD with a Supelcowax-

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10 column (30 m x 0.25 mm x 0.25 µm) or SLB-5MS (25 m x 0.2 mm x 0.33 µm)

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column. Operating conditions for GC/MS were as follows: helium flow, 32.2 cm/sec;

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oven conditions were the same as for GC-O. Mass spectra were recorded in an electron

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impact mode (70 eV) in a scan range of m/z 33-350. Additionally, to confirm the

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identities of the compounds, samples were run on comprehensive gas chromatography

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mass spectrometry system - GCxGC-ToF-MS (Pegasus 4, LECO, St. Joseph, MI). The

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GC was equipped with a DB-5 column (25 m x 0.2 mm x 0.33 µm) and Supelcowax 10

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(1.2 m x 0.1 mm x 0.1 µm) as a second column. For two dimensional analysis

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modulation (liquid N modulator by ZOEX) time was optimized and set at 3 sec, mass

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spectra were collected at a rate 150 scans/sec. The transfer line was heated up to 280 °C,

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and the ion source was heated to 250 °C, respectively. For SPME fiber desorption,

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260 °C temperature has been used with splitless injection. For all the volatiles, except

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1,5-octadien-3-one, identification was performed by comparison of mass spectra,

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retention indices (RI), and odor notes on two columns of different polarities. For 1,5-

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octadien-3-one, the MS signal of the analyte was too weak to facilitate mass spectra

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comparison. In this case RI and odor notes of the compound were compared with

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literature data and used in tentative identification.

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Aroma Extract Dilution Analysis (AEDA). The flavor dilution factor (FD) of each of

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the odorants was determined by AEDA.14 The aroma extract (2 µL) was injected into

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the GC (Supelcowax 10) column in a splitless mode (1min) at 230°C. Odor-active

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regions were detected by GC-effluent sniffing (GC-O), and three panelists determined

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the description of the volatiles. The extract was than stepwise diluted by addition of

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methylene chloride, and each sample of the dilution series was analyzed until no aroma 7 ACS Paragon Plus Environment


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was perceivable at the sniffing port. The dilutions used in GC-O experiments were: 2, 4,

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8, 16, 32, 64, 128, 256, 512, 1024 and 2048-fold. Retention data of the compounds were

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expressed as retention indices (RI) on both columns.

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Quantitation by Stable Isotope Dilution Assays (SIDA) and standard addition (SA)

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method. For majority of the compounds (12) stock internal standards of the respective

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labeled isotopes were prepared in diethyl ether and added on the ground cheese sample

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in the concentration similar to that of the relevant analyte present and extracted using

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SPME method as described before. Volatiles were analyzed by GCxGC-ToF-MS

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monitoring the intensities of the respective ions given in Table 1. For all compounds

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response factors were calculated in the standard mixture of labeled and unlabeled

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compound in known concentration. The concentrations in the sample were calculated

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from the peak area of the analyte and its corresponding internal labeled standard

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obtained for selected ions.15 For the remained aroma-active compound, (Z)-4-heptenal,

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as well as for the methyl ketones: 2-pentanone, 2-heptanone, 2-nonanone, standard

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addition method has been used.16 Linearity for the standard curve was calculated as the

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regression coefficient (r2) and for (Z)-4-heptenal presented in Table 1. For the methyl

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ketones: r2 equaled to 0.997, 0.996, 0.997 for 2-pentanone, 2-heptanone, 2-nonanone

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respectively. The calculation using both quantification methods was done with Chroma

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TOF software.

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Aroma recombination. For cheese aroma recombination stock solution of 13 aroma

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compounds: methanethiol, 3 and 2 – methyl butanoic acid, 3-(methylthio)-propanal,

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2,3-butanedione, dimethyl trisulfide, butanoic acid, 1-octen-3-ol, (Z)-4-heptenal,

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dimethyl disulfide, dimethyl sulfide, phenylacetaldehyde, 2-ethyl-3,5-dimethyl pyrazine

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and acetic acid, was prepared in deodorized sunflower oil and mixed in the

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concentration levels equal to those determined in mold-ripened cheese. 8 ACS Paragon Plus Environment

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Sensory evaluation. Sensory analyses of mold-ripened cheese samples as well as

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recombinants were evaluated by 10 members panel experienced in descriptive analysis.

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For aroma profile analyses, the intensities of eight odor qualities (cheesy/sweaty, milky,

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buttery, rancid, moldy/musty, mushroom, pungent, and fruity) were rated on a 10 cm

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linear scale from 0 (none) to 10 (very strong). The odor descriptors were determined in

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preliminary tests which involved panelists discussion over Lazur cheese aroma in order

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to define the most common descriptors among word groups. The 8 g of cheese sample

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or recombinant were placed in 100 mL glass containers and presented to the panelists.

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They were presented at room temperature in three separate sessions. The results from

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linear scale were converted into numerical values for data analysis. The similarity

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between the recombinant and the original Lazur cheese was evaluated using a numerical

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category scale from 0 (no difference) to 6 (very large difference).

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RESULTS AND DISCUSSION

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Sensory characterization of Lazur cheese. In the first experiment the mold-ripened

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cheese has been subjected to aroma profile analysis. For that reason eight odor

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descriptors: cheesy, milky, buttery, rancid, moldy, mushroom, pungent and fruity,

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chosen in a preliminary section were evaluated by the experienced sensory panel.

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Results presented in Figure 1 illustrate that aroma of Green Lazur cheese is mostly

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described by cheesy and moldy descriptors followed by pungent, mushroom, rancid and

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buttery notes.

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Identification of key aroma compounds in a mold-ripened cheese by the means of

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GC-O AEDA analysis.

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To evaluate which compounds are responsible for the aroma of a mold-ripened cheese

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AEDA technique was applied to the aroma concentrates obtained using SAFE method



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(Table 2). Gas chromatography olfactometry analysis revealed 17 compounds with FD

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factors ranging from 16 to 2048. Among them the burnt/sulfuric and cheesy/pungent

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odor showed the highest FD factor followed by the cabbage/garlic (1024), boiled potato

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(512) and cheesy (512) odor notes. In addition, a sulfuric/onion and rancid smelling

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odorants had FD factors at the level of 256. To identify compounds responsible for the

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odor notes retention indices on two columns with different polarity have been estimated

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and compared with literature data and reference compounds. For each compound mass

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spectrometry identification has been performed, obtaining clear spectrum for 16

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compounds, Table 2. Following this procedure, compounds with the most intense aroma

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were identified as methanethiol and 2(3)-methyl butanoic acid. Further compounds with

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high FD factors were dimethyl sulfide, 3-methylthio-propanal and butanoic acid. The

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sulfuric/onion smell was caused by dimethyl disulfide and the rancid odor was

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attributed to (Z)-4-heptenal. These results are partially with the agreement of Qian et

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al.5 work, where authors also obtained high FD for 3-methylthio propanal, butanoic acid

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and for 3-methyl butanoic acid. Apart from this authors identified diacetyl, unknown

210

with baked aroma and 2-heptanone as major aroma compounds of blue cheese.5 On the

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other hand no detection of methanethiol nor dimethyl sulfide was reported in Qian et

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al.5 studies, methanethiol however was identified in blue cheese studied by Frank et al.6

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Differentiations in recognition of sulfur highly volatile compounds may be due to the

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diverse extraction method used in each research, as they are known to be very

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challenging in isolation and identification.

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For the odor-active compounds which had the highest FD factor quantitative

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measurement was applied. The results presented in Table 3 show large differences in a

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concentration range of each odorant. The highest concentration has been noted for

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acids: butanoic acid (2750 µg/kg), acetic acid (3254 µg/kg) and 2(3)-methyl butanoic 10 ACS Paragon Plus Environment

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acid (1605 µg/kg) and the lowest for the 2-ethyl-3,5-dimethyl pyrazine and dimethyl

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sulfide. Majority of the already published studies presented free fatty acids as the most

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abundant compounds in blue type cheeses, however the concentration of each acid vary

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in different cheeses, sometimes with the substantial difference even in the same variety

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of cheese. Alejwin et al.8 estimated concentration of acetic acid and butanoic acids as

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556 and 718 mg/kg in a Danish Blue cheese, whereas Trihhas et al.9 also in a Danish

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Blue calculated concentration of acetic acid as 13 mg/kg, much higher than in Lazur

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cheese. According to data gathered in a review on soft cheeses, concentration of

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butanoic acid can vary from 53 to 1448 mg/kg of blue cheese.17 The differences in the

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concentration of acid in mold-ripened cheeses are probably due to the various ripening

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time and different fat composition.18 High levels of acids in blue type cheeses are

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caused by the lipolytic activity of the molds, which is much more advanced in this

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cheese type rather than for example surface ripened cheeses or semi hard cheeses.18

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Although many research studies report trace levels of 2,3-butanedione in blue type

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cheeses, in our studies concentration of 2,3-butanedione in Lazur cheese has been

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determined as 652 µg/kg which gave considerably high OAV value of 65. In addition in

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the research studies of Qian et al.5 and Frank et al.6, buttery smelling 2,3-butanedione

237

have been similarly detected at olfactometry port with great intensity. Furthermore,

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quantitative data on sulfur compounds shows their relatively high concentration in

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Lazur cheese, ranging from 15 to 165 µg/kg. Regarding low odor thresholds (OT) of

240

sulfur compounds, their contribution to the aroma of Lazur cheese seems to be

241

understandable. As mentioned before, sulfur compounds have not been often reported in

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the studies of blue cheese aroma. Nevertheless, Gallois et al.7 examined volatiles of

243

different French blue cheeses and showed that monitored levels of methanethiol,

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dimethyl sulfide and dimethyl disulfide distinguish their aroma profile. 3-methylthio11 ACS Paragon Plus Environment


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propanal has been also reported as major compound in blue type cheese. It has been

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identified in the work of Moio et al.4 in Gorgonzola cheese, also in several French blue

247

cheeses.6,7 It may be formed in a course of enzymatic and non-enzymatic breakdown of

248

methionine, which is major sulfur amino acid in mold-ripened cheeses.19

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Honey-like smelling phenylacetaldehyde has been present in Lazur cheese in the

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concentration of 128 µg/kg, similar to the concentration in a 4 weeks ripened Danish

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blue cheese9 but twice higher than in French blue cheese.7

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To evaluate the aroma influence of the individual compound each concentration was

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divided by the respective odor threshold according to odor activity value concept

254

(OAV).3 For all the compounds values of the odor threshold in sunflower oil were used.

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They were collected from the literature.20 The highest OAV among identified volatiles

256

was determined for methanethiol (500), despite its rather low concentration of 30 µg/kg.

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High OAVs were also calculated for the cheesy/pungent smelling 2(3)-methyl butanoic

258

acid (321) and 3-methylthio-propanal (210) with boiled potato odor quality.

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Furthermore, 2,3-butanedione with buttery note, dimethyl trisulfide with cabbage like

260

aroma, butanoic acid and 1-octen-3-ol with cheesy and mushroom odor notes had OAV

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of 65, 22, 20 and 18 respectively. These results suggest that compounds with sulfuric,

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cheesy, pungent, buttery, cabbage and mushroom smell contribute mostly to the aroma

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of the mold-ripened cheese. OAVs higher than one were also determined for (Z)-4-

264

heptenal, dimethyl disulfide, dimethyl sulfide, phenylacetaldehyde, 2-ehyl-3,5-dimethyl

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pyrazine and acetic acid.

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The opinion on the importance of methyl ketones in formation of the unique

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flavor of Blue cheese dates back to mid 60-ties and 70-ties.21 Anderson and Day22

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observed two major methyl ketones in Blue cheese – 2-nonanone and 2-heptanone. Jolly

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and Kosikowski23 stated that methyl ketones and especially 2-heptanone and 212 ACS Paragon Plus Environment

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nonanone are responsible for it. In 1979 King and Clegg24 observed the formation of

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blue cheese flavor paralleled by methyl ketones, fatty acids and secondary alcohols

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formation in a slurry system. Fruity, floral and musty notes are associated with such

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ketones as 2-octanone, 2-nonanone, 2-decanone and 2-undecanone, whereas Blue

274

cheese notes are attributed to 2-heptanone.25 These authors reviewed 11 publications in

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which a variety of 2-methylketones were identified as important odorants in such

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cheeses as Cheddar, Grana Padano, Ementaler, Gorgonzola, Camembert, Flor de Guia,

277

water buffalo Mozzarella, Gruyere, Ragusano.

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Lipases released by P. roqueforti are contributors to the development of blue cheese

279

flavor.26 The addition of 2-heptanone to food product containing small amount of Blue

280

cheese greatly enhances Blue cheese flavor.27 Formation of free fatty acids is considered

281

as a limiting step in the formation of 2-heptanone.28 P. roqueforti converts free fatty

282

acids, released in the process of triglycerides lipolysis, into corresponding

283

methylketones as a way to detoxify metabolites that are harmful to them. If fatty acids

284

were absent in their environment, their metabolism shifts to formation of

285

sesquiterpenes.29

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In contrast to earlier information on the profile of blue veined cheese aroma

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compounds in our study no 2-methylketones were identified among potent odorants in

288

analyzed cheese, though they were identified in volatile compounds fraction. 2-

289

pentanone, 2-heptanone and 2-nonanone were identified in Lazur chees in

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concentrations of 1.32, 2.48 and 0.65 mg/kg as quantified using standard addition

291

method described in Materials and methods section. OT for these compounds that can

292

be found in the literature differ substantially depending not only on the medium used for

293

its determination, but also for the same medium the concentrations can be very different.

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For 2-pentanone OT (mg/kg) in oil is 61, whereas for cheese is 288. For 2-heptanone it 13 ACS Paragon Plus Environment


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is 1.5 in oil and 27 in cheese. For 2-nonanone its OT is 0.1 in oil, 1.7 in air and

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interestingly 7.7 in ripe cheese and 116 in cheese.20 In water OTs for 2-heptanone are

297

even more diverse and range from 0.001 to 3.73 mg/kg.20

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Figure 1 shows the comparison of sensory profile of mold-ripened cheese and

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the recombinant of flavor compounds prepared in oil according to results in Table 3.

300

The oil was previously used for preparation of matrix for cheese aroma recombinant

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preparation,30, 31 as it is a complex task to mimic cheese matrix with aroma compounds

302

removed. The overall similarity between Lazur cheese and recombinate was ranked with

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2.1 on a scale from 0 (no difference) to 6 (very large difference). The only differences

304

noted between cheese and recombinant were observed for cheesy/sweaty and

305

moldy/musty notes. This would suggest that some compounds might be omitted in the

306

recombinant, though the differences between samples (Figure 1) were not very high.

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Based on literature data 2-methylketones were the compounds believed to be crucial

308

odorants of blue veined cheese. A recombinant with the addition of 2-pentanone, 2-

309

heptanone and 2-nonanone in concentrations determined in Lazur cheese was prepared

310

and assessed by sensory panel. Surprisingly the aroma profile remained practically

311

unchanged (Figure 1). This would suggest that methyl ketones in this particular cheese

312

do not play any significant role in the formation of Lazur aroma. Moreover, this would

313

also suggest that the OTs of these ketones are rather high, therefore calculated potential

314

OAVs remain low (Table 4). Possibly eight weeks ripening period which was used for

315

Lazur cheese, quite short compared with some other blue cheeses (Roquefort – over

316

three months) could explain the low lipolysis and lack of methyl-ketones production.

317

Another explanation in differences of moldy/musty odor between recombinant and

318

Lazur cheese might be possibilities of interaction among odorants or between cheese

319

matrix and odorants. 14 ACS Paragon Plus Environment

Page 15 of 25


320

ACKNOWLEDGEMENTS

321

This research was financed by the Polish National Science Center project: N

322

N312 157 134.

323 324

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Page 19 of 25

408


FIGURE CAPTIONS Figure 1. Sensory aroma profiles of a Lazur cheese (  mold ripened cheese), its recombinant (- - - recombinant) and its recombinant with addition of methyl ketones – MK (• • • recombinant+MK).



Page 20 of 25

TABLES Table 1. Method of quantification, quantification ions, response factor and regression coefficient of calibration curves used for concentration calculations of 17 key odorants prese method of quantitationa SIDA

quant. ionsb 48

dimethyl sulfide

SIDA

62

2

2,3-butanedione

SIDA

86

13

SIDA

94

2

SA

68

compound methanethiol

dimethyl disulfide (z)-4-heptenal

labeled standards 2 H6

ion ISc

Rf/r2d

68

1.3

68

0.98

C4

90

0.94

H6

100

1.3

H6

-

-

0.996

H3

73

1.1

1-octen-3-ol

SIDA

99

2

dimethyl trisulfide

SIDA

126

2

H6

132

1.1

acetic acid

SIDA

60

2

H4

64

1.2

3-(methylthio)-propanal

SIDA

104

2

H3

107

1.1

2-ethyl-3,5-dimethylpyrazine

SIDA

135

2

H6

141

1.3

butanoic acid

SIDA

73

2

H7

77

1.2

phenylacetaldehyde

SIDA

120

13

C2

122

0.9

87

2

H2

89

1.1

2(3)-methyl butanoic acid

SIDA

nt in the moldripene d chees e.

a – SIDA – stable isotope dilution assay, SA – standard addition, b – ions of analytes used for quantification, c- ions of internal standards (labeled isotopes) used for quantification, d - Rf-response factor between analyzed compound and its internal standard (labeled isotope), r2regression coefficient


Page 21 of 25


Table 2. Key aroma compounds identified in a mold-ripened cheese.

1. 2. 2. 3. 4. 5 6 7 8 9 10 11 12 13 14 15 16 17

Odorc

Compoundd

Supelcowax-10

RIa SPB5

burnt cabbage, garlic buttery sulfuric, onion rancid mushroom popcorn geranium cabbage vinegar boiled potatoes roasted, earthy fatty, green earthy cheesy honey cheesy, pungent phenolic, smoky

methanethiol dimethyl sulfide 2,3-butanedione dimethyl disulfide (Z)-4-heptenal 1-octen-3-ol 2-acetyl-1-pyrroline 1,5-octadien-3-onee dimethyl trisulfide acetic acid 3-(methylthio)-propanal 2-ethyl-3,5-dimethylpyrazine 2-nonenal unknown butanoic acid phenylacetaldehyde 2(3)-methyl butanoic acid 2-methoxy phenol

695 720 990 1070 1242 1290 1342 1375 1367 1450 1455 1495 1527 1580 1620 1650 1660 1870

Key Odorants of Lazur, a Polish Mold-Ripened Cheese - Journal of

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