Lait (1994) 74, 389·398© Elsevier/INRA

389

Original article

Carbon dioxide measurement in Swiss-type cheesesby coupling extraction and gas chromatography

F Girard 1, P Boyaval 2

1 Institut Technique du Gruyère, 73, rue de Saint-Brieuc, BP 6224, 35062 Rennes Cedex;2 Laboratoire de Recherches de Technologie Laitière, INRA, 65, rue.de Saint-Brieuc,

35042 Rennes Cedex, France

(Received 24 January 1994; accepted 27 June 1994)

Summary - The microbial production of carbon dioxide (C02) in Swiss-type cheeses is one of the majorevents during ripening. Ta beller control this formation of eyes, it is necessary ta be able ta evaluatethe CO2 in the body of the cheese. In this paper, we propose a method ta liberate the CO2 from thecheese sample dispersed in a citric alkaline solution in a blender and ta quantify the liberated gas, byacidification, by the direct coupling between the extraction and agas chromatography analyser. Theproposed method has a linearity between 0 ta 10 mmoll-1 of CO2, a coefficient of variation of 1.20%and a good repeatability.

carbon dioxide 1 cheese 1gas chromatography 1analytical method

Résumé - Détermination de la teneur en dioxyde de carbone des fromages de type Suissepar couplage extraction-chromatographie en phase gazeuse. La formation du gaz carbonique(C02) au sein des fromages à pâte pressée cuite est l'un des événements majeurs observables au coursde l'affinage. Le strict contrôle de la formation des yeux nécessite une méthode fiable de mesure duCO2 dissous dans la pâte. Dans cet article, nous décrivons une méthode qui permet d'extraire le CO2d'échantillons de fromage broyés dans une solution citrique alcaline et d'en mesurer la quantité àl'aide d'un chromatographe en phase gazeuse après acidification de la solution. La méthode proposéeprésente une parfaite linéarité de réponse entre 0 et 10 mmol rt ae CO2, un coefficient de variation de1,20% et une bonne répétabilité. De plus, elle est simple à mettre en œuvre.

dioxyde de carbone / fromage / chromatographie en phase gazeuse / méthode d'analyse

390 F Girard, P Boyaval

INTRODUCTION

ln France, the production of Emmentalcheese has reached 212 000 tons in 1993(CNIEL, 1994). This hard cooked cheese isthe first French cheese and the second, ona world-wide scale, after Cheddar. In thiskind of Swiss-type cheese, production ofcarbon dioxide (C02), due to lactate break-down by propionibacteria (Steffen et al,1987), plays one of the major roles duringthe ripening process by allowing eyes for-mation. In the cheese loaf, the amount ofCO2 is scattered as follows: 48% dissolvedin the body, 20% in the eyes and 32% dif-fused through the rind (Steffen et al, 1987).ln order to optimise the maturation processand so the quality of cheese, it is necessaryto measure the CO2 dissolved in the body ailalong the cheese processing.

ln the Iiterature, most of the data concer-ning CO2 produced by propionibacteria ina culture media or related to the bacterialmetabolism in cheese, are calculated fromthe final products or intermediate compoundconcentrations and from one or severalequations reflecting roughly the carbonmetabolism (Hettinga and Reinbold, 1972).Even using the work done by Wood (1981)on propionibacteria, these sketchy data donot permit to construct an accu rate carbonbalance. Moreover, none was carried outusing lactate as a substrate. CO2 measu-rements are always failing to reach the100% carbon recovery.

The first attempt to measure CO2 gene-rated by micro-organisms was performedby Leichman (1896). Then, different well-known authors, generally working on lac-tic acid bacteria (Freudenreich and Orla-Jensen, 1906; Orla-Jensen, 1919; Allen,1939), developed special apparatuses, butnone of them appeared reliable.

ln 1945, Gibson and Abdel-Malek mea-sured CO2 production by incubating cul-tures in a volumetric f1ask with a calibrated

neck. This method was used to differentiateheterofermentative lactic acid bacteria, orga-nisms producing a detectable quantity ofgas, from other lactic acid bacteria or spore-forming bacilli. They demonstrated that theamount of CO2 formed during fermentationof sugars depends on several factors suchas sugar (nature and concentration) andbuffering capacity of the medium. There-fore, this method was not quantitative. Gra-vimetric, manometric and titrimetric methodswere used until 1978 (Sandine et al, 1957;Robertson, 1957, 1962; Hoglund et al, 1972;Flückiger et al, 1978). More recently, Bossetet al (1986) measured the partial pressure ofCO2 in various Iiquid fermented milk pro-ducts using a gas-sensitive CO2 electrode.ln a review article, Dixon and Kell (1989)described more than fifteen methods used inestimation of determination of CO2 du ringfermentations, in the liquid or gas phase. In1992, a simple method based on the che-mical reaction of CO2 with a specific indi-cator layer packed into gas diffusion tubeswas described by Kneifel and Gretner. Mohret al (1993) adapted the method of King andMabbitt (1982) who used gas chromato-graphy to measure CO2 that was added tomilk in order to preserve it, by lowering thepH. Gas chromatographic methods werepreviously developed (Kreula and Moisio,1970; Ross, 1987) but required a samplepreparation of 17 to 24 h, neglected thesolubility of CO2 in aqueous solutions anddid not account for the difference of pres-sure among the bottles.

Data on the level of CO2 dissolved in thebody of hard-cooked cheese, importantduring ripening, are not available in the fac-tories, probably because of the difficultiesencountered to measure il. Therefore,attempts were made to develop methods inthe beginning of the century (Clark, 1917).Hoestettler (1944) estimated the CO2 inEmmental cheese by drawing the gas offunder vacuum. Hiscox et al (1941) estima-tOOthe dissolved CO2 of Cheddar cheese in

lCO2 measurement in Swiss-type cheese

connexion with the estimation of volatileacids by subtraction of values obtained withaerated samples from the values obtainedwith non-aerated samples. In 1953, Swartlingand Willart, working on Herrqârt cheese,developed a titrimetric method. The samplewas emulsified in alkali, acidified and CO2was carried away in CO2-free air from whichit was absorbed in a standard solution ofbarium hydroxide. The excess of bariumhydroxide was measured using titrated acid.Robertson (1957, 1962) adapted the pre-vious method to measure the CO2 content ofNew-Zealand Cheddar cheese. Later on, inthe eighties, a Swiss team (Bosset et al,1980) carried on with a new method todetermine CO2 in food products with parti-cular application to cheese. Samples wereemulsified, under reduced pressure, in analkaline citrate medium using a mixer. Car-bon dioxide was quantitatively displacedinto the headspace by addition of an excessof sulphuric acid. Measurements were car-ried out with a specifie non-dispersive infra-red detector. Afterwards, Bosset et al (1989)described an analyser based on gas chro-matographie separation with thermalconductivity detection which can be usedfor the analysis of gas atmosphere overdairy products such as canned milk pow-der, canned condensed milk, cheese packedin cans or with plastic film and yogurt. Anenzymic procedure was used for determi-ning CO2 in blood serum (Forrester et al,1976). The procedure was adapted by Crowand Martley (1991) for the measurement ofCO2 dissolved in Cheddar cheese. But noneof these methods can be routinely used.Titrimetric methods are not accu rate (10%).Enzymic techniques are much more accu-rate (0.4%) however, such a kind of cheeseas Emmental shows marked variations ofCO2 concentration within the loaf of cheeseso the sample (5 g) becomes absolutely notrepresentative. Methods using infrareddetector require, like titrimetric methods, therelease and measurement of the totalamount of CO2 (accuracy: 2%). The vapor

391

must be maintained constant because of itsinterference with the measurements.

The aim of the present investigation wasto determine CO2 dissolved in Swiss-typecheese. The method was chosen, takinginto account the different methods alreadystudied. CO2 was estimated using agas/solid chromatography analyser equip-ped with a thermal conductivity detector,after treatment of the sam pie according tothe method of Bosset et al (1979, 1980).

MATERIALS AND METHODS

Analytical system

The analytic system is iIIustrated in figure 1. Thesample was introduced in a modified stainlesssteel gas proof 1-1 blender (Waring S 1021,Standa Industrie, Caen, France) equipped witha two-speed motor. The cover was modified bywelding five connexions (5 cm long and a 5 mmdiameter). The first one was equipped with aBourdon depression-meter (0 to -760 mbar, Har-tereau, Rennes, France). The second one wasconnacted to the controlled vacuum system. Thethird one was connected to the reference gas(C02 1% ± 0.02, v/v, in nitrogen, Aga, France).The fourth one was connected to the gas chro-matograph and the last one was equipped with ateflon tunnel. CO2 was analyzed in a Varian gaschromatograph (3400, equipped with a 250 IIIautomatic injection loop, Les Ulis, France). Sepa-ration took place in a packed coIumn (3.66 m x 3.2mm; Haye Sep, N,80/100 Mesh) at 105°C withhighly pure hydrogen as gas vector (N55, Alpha-gaz, France, flow rate 50 ml-1, pressure 2.8 x105 Pa. A thermal conductivity detector was usedat 140°C. The results were analyzed and storedin a microcomputer equipped with the softwareGold (Beckman, USA). Each analysis took 4.5min. Between each sample, 10 min were requiredin order to clean the column (at 140°C).

Sampling and sample preparation

Each piace of cheese was a 3 kg parallelepipedicblock of 9 cm width, with both rinds, taken in the

392

CO2

REFEREN( EGAS

F Girard, P Boyaval

BLENDER

COMPUTER

Fig 1. Equipment used for the determination of CO2 in cheese.Équipement mis en œuvre pour la mesure du CO2 dans les fromages.

central part of the loaf, kept in a sealed bag, underlow pressure, at + 4°C. Six to eight samples from30 to 50 9 were collected in each block with achee se borer (1.8 x 18 cm, in stainless steel).

The sample was then immersed and finelydispersed during storage in 60 9 of a citric alkalinesolution (weighted exactly) (sodium hydroxide inpellets, 10 9 1-1 (Merck, pro analysi, Darmstadt,Germany) and tri-sodium citrate, 50 9 1-1 (Merck,Germany), dissolved in a freshly distilled, sterili-zed water, cooled under low pressure to avoidair redissolution) and stored in gas-proof vials.Just before analysis, water was added to thesam pie in order to supplement the cheese weightto 100 9 (to avoid variations of volume in the blen-der; Bosset et al, 1980).

Ana/ysis

The sample was then placed in the blender, whichwas subsequently connected to vacuum (-450 ±50 mbar). The blender was then isolated; the grin-ding was performed under partial vacuum for 2 min.After 1.5 min, 50 ml of sulfuric acid (2.5 mol 1-1,Merck, Darmstad, Germany) was added from theteflon funnel. The blender was gently tumed backto atmospheric pressure then isolated before seve-rai automatic injections were finally carried out,

The four first ones were used to drain the 1.0ml volume of the tubing between the blender and

the injection loop and discarded. The four nextones were then collected and the value was thearithmetic mean of these four injections.

The result was then calculated from the fol-lowing equation:

% CO2 x (vol (blender)-vol(chee se solution)) x 1000

y mol C0t1<g of cheese100 x 25220

x mass of cheese (g)

with vol = volume

R = 25.22 ml COimmol CO2 at 20°C and under720 mbar.

Calibration of the ana/ytie system

This procedure was done weekly. The stabilityof this calibration along this period has been veri-fied. In order to discard completely the air of theblender, vacuum was firstly applied (-700 mbar)and then highly pure CO2 (1%, v/v) was added init, until atmospheric pressure was restored. Thisstep was repeated three times. Afterwards, acontinuous CO2 flow rate was allowed to sweepthe blender to be sure to maintain CO2 at theatmospheric pressure (otherwise, an overpres-sion can alter the quality of the calibration) andthat the multiple injections did not create any

CO2 measurement in Swiss-type cheese

depression aise detrimental for the analysis. Nineinjections were then carried out in which the fivelast were taken into account to calibrate the appa-ratus. Six calibrations were performed (standarddeviations: 0.025 for CO2 at 1%).

Linearity of the method

ln order to evaluate the Iinearity of the method,solutions of sodium carbonate 114 mmol 1-1 and228 mmoll-1 (Merck, Darmstadt, Germany) wereused to liberate CO2 in the blender by addition ofsulphuric acid (same procedure as for sampi es):

Na2C03 + H2S04 -> H20 + CO2 '" + Na2S04

RESULTS

Limit of detection

The Iimit of detection was calculated at 6.310-3 mmol1-1 of CO2 (0.016%) on the baseof a signal-to-noise ratio of 3 (from a chro-matogram of CO2 at 1% in nitrogen). TheCO2 content of the air of the laboratory wasmeasured. A set of values of 0.040% ±0.010 was easily detected. This value is inclose agreement with the value of 0.031 % ±0.005 of the literature (Kuiperg, 1976). Itwas not possible to test lower concentra-tions as calibrated gas was not availablebelow this limit. Lower gas concentrationcould be prepared by dilution of the refe-rence gas. But, as already demonstratedby Bosset et al (1989), the biggest error willbe in the preparation of the diluted gas ratherthan in the measurement itself.

Linearity of the method

Figure 2 iIIustrates the response of the com-plete device to the Iiberation of CO2 fromsodium carbonate. The regression line canbe described by the equation:

393

QJV!Coc-V!QJ~~Eu2QJ

"'C

3 time (min)3.4Fig 2. Example of a chromatogram obtained bythis method.Exemple de chromatogramme obtenu à l'aidede cette méthode.

peak area = 0.64 x mmol CO2 + 0.12

(with r= 0.998)

which is usable for the conversion of peakarea in content of carbon dioxide in thesam pie (before data reduction to 1 kgsample weight). The method has a goodlinearity between 0 to 10 mmol t"of CO2•

Repeatability of the method

ln order to analyse this characteristic, 330 ±20 9 of a cheese block were prepared inthe citric alkaline solution kept in a gas-tightglass bottle. Six series of analysis were per-formed with this preparation. The resultsare presented in table 1. The mean value is24.1 mmol of CO2 for 1 kg of cheese. Thecoefficient of variation (CV) is 1.2%.

The application of such a method to thedetermination of the carbon dioxide contentof cheese samples needs a blank whichmust be prepared and measured beforeeach cheese sample (especially if the valueof this blank and of the sample are lime-dependent as is the case in this type of mea-surement). Moreover, the stability of the gascontent of the sam pie between samplingand measurement must be ensured in orderto present an interest for the professionals.

394 F Girard, P Boyaval

Table 1. Repeatability of CO2 measurement incheese. Example for six analyses of the samechee se preparation.Évaluation de la répétabilité de la mesure de CO2dans les fromages. Exemple de la répétition (6fois) de la mesure sur une même préparation defromage.

Analysis mmol CO;/kg of cheese

123456

24.523.824.223.624.224.1

mean (X) = 24.15D = 2.9CV= 1.20%

The blank contains ail the constituentsexcept the cheese. The value of this blankhas been systematically subtracted fromthe value determined with the sam pie.

Importance of the gas tightnessof the bottle« used for the storage

When the plastic sampling bottles were usedto keep the citric alkaline solution alone, theresults were systematically higher th an forthe samples kept in glass bottles, leading toerrors from 2 to 15% (table Il). Cheese solu-tions kept in plastic bottles were not stable interm of CO2 content. These increasingvalues are mainly due to the carbonatation of

Table Il. Influence of storage time and storage conditions (vials) between the immersion in the citric alka-line solution and analysis.Influence du temps de conservation et des conditions de conservation de l'échantillon dans la solutioncitrique alcaline avant analyse.

Day of mmol of CO;/kg of cheesestorage

Glass bottle Plastic bottle

Sample 1 Sample2 Blank ~ Sample3 Blank ~

0 18.0 20.4 1.6 16.4; 18.8 21.5 1.6 19.917.1 15.5 21.6 t + 6 h = 2.0 20.0

ND 20.5 1.6 18.9 ND ND2 18.0 ND 1.9 16.1 22.3 2.3 20.0

1.78 15.9 24.4 22.1

3 ND 20.5 1.8 18.7 ND ND20.9 19.1

4 17.9 ND 1.7 16.2 23.1 2.0 21.117.7 16.0 23.1

7 17.9 21.0 1.6 16.3 2.28 2.8 20.019.4 2.47 21.9

ND: not determined. Blank = citric alkaline solution without cheese. t. = sample value - blank value. Some trialshave been performed twice (two values).ND : non déterminé. Blank = solution citrique alcaline sans échantillon de fromage. t. = valeur de l'échantillon aprèssoustraction du témoin. Certains échantillons ont fait l'objet de 2 séries de dosages conduisant à 2 valeurs.

CO2 measurement in Swiss-type cheese

the citric alkaline solution through the contai-ner: 0.16 to 0.28 mmoll-1 CO2 in 7 days ofstorage (table Il). On the opposite, when thesamples were kept in glass bottles, weobserved no difference in CO2 measure-ments du ring 7 days of storage (table Il).

Sampling

The losses of CO2 between the cheesesamples extracted from the loaf are fast andconsiderable due to the acidic medium ofcheese body (H2C03 -> H20 + CO2) andto the difference of partial pressure of CO2between cheese body and ambient air. Theylead to a substantial decrease of the CO2value. In order to show this phenomenon,several cheese bores were not immediatelyimmersed in the citric alkaline solution but letin the laboratory atmosphere 12 h beforedispersion/dissolution treatment. The nor-mally treated samples (of the same cheeseblock) presented 31.0 mmol CO2/kg ofcheese (CV: 3.0%) while the other one pre-sented only 8.7 mmol CO2/kg of cheese.

This CV of 3.7% is high in comparisonwith the CV of 1.2% of the method. It is pro-bably due to the difference of the surfaceexchange cheese-air between the differentsamples (eyes number, size and heteroge-neity of the samples).

Heterogeneity of the CO2 contentof the cheese

The heterogeneity of the CO2 content withina cheese block is evidenced in table III. TheCV varied from 0.9 to 8.1 %, value higherth an the 1.2% of the repeated analysis ofthe same cheese preparation (table 1).These variations in the measurement of theCO2 content are due much more to sam pieheterogeneity than to the repeatability ofthe method.

395

Table III. Heterogeneity of the CO2 content of thecheese body.Hétérogénéité du CO2 dissous dans la pâte.

5amples X mmol/kg 50 CV (%) nofcheese

A 23.3 1.2 5.1 6B 22.6 1.8 7.9 5C 24.9 0.9 3.6 60 23.3 1.9 8.1 5E 25.0 0.9 3.6 6F 31.0 0.3 0.9 3

Each value is the average of 3-6 measurements for thesame piace of cheese. The samples were taken fromdiflerent cheeses (A-F).Chaque échantillon est caractérisé par sa moyenne X.son écart type (5D) et son coefficient de variation (CV)calculé sur la base de 3, 5 ou 6 mesures (n) effectuéessur la même meule de fromage. Les échantillons A à Fproviennent de meules différentes de la même sorte defromage (5wiss-type).

PEAK

AREA

5

4

3

2

5 10 15 nvnol CO2

Fig 3. A standard curve of gas chromatographiepeak areas of CO2 derived from solutions ofsodium bicarbonate of different molarities.Un exemple des courbes d'étalonnage obtenuesavec des solutions de bicarbonate de différentesmolarités.

--------- -------

396 F Girard, P Boyaval

DISCUSSION

The proposed method can be comparedwith the others already published in the lite-rature for CO2 measurements in cheese.This method used a detector which hasbeen proven to present an excellent Iinearresponse from 0 to 10 mmoll-1 of CO2 witha high sensitivity and a detection Iimit of 6.310-3 mmoll-1. Bosset et al (1980) presen-ted a CV of 1.66% (here 1.2%; n = 6) with astandard deviation of 0.0934 (n = 10) (here0.029 which is comparable to the value0.033 (n = 3) obtained by Hoglund et al,1972). The standard deviation of the enzy-matie method was 1 mmol/kg of cheese(mean value: 11 mmol/kg; n = 6) for esti-mation of CO2 in Cheddar cheese (Crowand Martley, 1991). The gas chromatogra-phie technique developed by Ross (1987)had a CV of 8.0% (n = 3) for within-dayvariation.

The sampling from 30 to 50 9 of Emmen-tal cheese with a cheese borer is a com-mon practice in industry and is already usedto evaluate the eyes formation in the loavesand to taste the cheese. So, cheese sam-pling by this method could easily be intro-duced in the factories. The studies ofSwartling and Willart (1953) for Herrqârdcheese, Robertson (1957) and Flückiger etal (1978) for Swiss-type cheeses as weil asBlanc et al (1980) for Swiss Gruyère cheesehave clearly established that the carbondioxide concentration decreased towardsthe outer surface of the cheese loaf due toits pk value (pka of CO2 = 4.6). So Robert-son (1957) had decided to take, as asample, only the deepest end of the plug,discarding the upper part considered as toonear from the surface and so having a lowerCO2 content than the inner part of thecheese. This concentration gradient of car-bon dioxide is not the only reason of theheterogeneity of the sam pie. The cheeseborer does not allow to take sam pie with'complete eye'. Most of the eyes are eut

through, so their particular gas content isimmediately lost into the laboratory atm os-phere. The cheese body surrounding theeyes has probably a slightly different CO2content than the rest of the body. So, thedensity of the eyes in the bore is probably ofimportance in the CO2 measurement.

Based on the results of Bosset et al(1980) and our own results, we proposedto immediately immerse and weigh thissample, in a citric alkalinesolution prepa-red in agas proof glass bottle. This bottlecan be stored and subsequently analysed ormailed to the centrallaboratory for measu-rement without any problem for severalweeks.

This effect of carbonatation of sodiumhydroxide is weil known but its importance isparticularly evidenced when low levels ofCO2 must be measured as is the case inour method. Moreover, the preparation ofthis alkaline solution must be done withfreshly distilled water and weil protectedcaustic tablets. In a proper gas proof glasscontainer, this solution can be kept 2 weeks.The only possibility to take into account thisinterference effect is to determine and sub-tract the corresponding blank. This methodallows the investigation of carbon dioxideformation in hard and semi-hard cheesesIike Emmental, but also for cheeses withlower eyes formation (Comté, Gouda, Mimo-lette). This information is of great interestto follow the propionic acid fermentation insuch cheeses.

This method presents some limitations:only four samples can be measured perhour and the analysis must be performedby well-trained technicians. In order toincrease the quality of the measurementsday-to-day temperature and atmosphericpressure variations should be taken intoaccount.

The elaboration of a robust method tofollow CO2 formation in cheese body openswide fields of research: i) investigations onthe propionic acid formation during cheese

CO2 measurement in Swiss-type cheese

manufacturing and especially du ring ripe-ning, leading to a better control of this crucialperiod; ii) comparisons of potential forma-tion of CO2 by different strains of propionicstarters in true cheese environ ment; and iii)examination of the CO2 content of thecheese body after cutting of the loaf andbefore wrapping, to better control the CO2content of the gas added in the packaging.

This point would allow to subsequentlydiminish the bringing back of cheese parts tothe facto ries, mainly due to problems ofappearance of wrapped cheese (blowingby CO2 release in the plastic bag or stic-king of the plastic film on the cheese por-tion, leading to a wet appearance). Thispoint was recently examined in Canada byFedio et al (1994).

ACKNOWLEDGMENTS

The authors gratefully acknowledge JV Meudec(ITG) and A Fauvel (Entremont) for the cheesesampling facilities, C Corre and C Dupuis for theïrhelp du ring the experiments and JL Maubois andJR Kerjean for the excellent facilities extendedta us to realize this work.

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