Kluyveromyces lactis but Not Pichia fermentans Used as Adjunct Culture Modifies the Olfactory Profiles of Cantalet Cheese

Article Outline

• Abstract
• Introduction
• Materials and Methods
  • Cheese Manufacture and Sampling
  • Microbiological Analyses
    • Microbial Counts
  • Compositional Analyses
    • Sampling
    • Free AA
    • Individual FFA
    • Quantification of Volatile Compounds
  • Determination of Odorous Compounds
    • Extraction of the Volatile Compounds
    • 8W-GC-O
    • Identification of the Odorous Compounds
  • Flavor Assessment
    • Descriptive Evaluation by Trained Panel
  • Statistical Analyses
• Results
  • Lactic Starters and Yeast Adjuncts
  • Compositional Attributes
  • Volatile Composition
  • Odorous Compounds Analysis
  • Sensory Analyses
• Discussion
• Acknowledgements
• Supplementary data
• References
• Copyright

Abstract 

Yeasts are commonly detected in cheese. Two yeast species, Kluyveromyces lactis and Pichia fermentans, were isolated at high populations from raw-milk Cantal cheese, a French Protected Denomination of Origin hard cheese. To investigate the interest of these 2 species as adjunct cultures to promote flavor development of Cantalet cheese, they were added at 10⁵ cfu/mL to microfiltered milk. The global microbiological, biochemical, and flavor changes induced by the presence of the yeasts in cheese were determined. Adjunct yeasts were present at 10⁶ cfu/g in curd, declined to 10⁴ to 10⁵ cfu/g in cheese, and did not influence gross composition, content of free amino acids, or content of free fatty acids. By using 8-way gas chromatography-olfactometry in parallel with gas chromatography-mass spectrometry, 30 odorous compounds of Cantalet cheese were identified. The olfactory profiles of K. lactis cheeses contained significantly greater levels of 8 odorous compounds (ethanol, ethyl hexanoate, 4 aldehydes, and 2 branched-chain acids) compared with the control and P. fermentans cheeses. Sensory analysis of cheeses flavor discriminated K. lactis cheeses on only 2 attributes (acetaldehyde and alcohol odors). This study shows that yeast contribution is species-specific and that K. lactis, at a population of 10⁶ viable cells/g, can influence Cantalet cheese flavor.

Key words: Kluyveromyces lactis, Cantalet cheese, adjunct culture, odorous compound

Back to Article Outline

Introduction 

Yeasts are frequently detected within the microflora of various cheese types (Fleet, 1990), which can be attributed to their ability to grow at low temperatures, to assimilate and ferment lactose, assimilate organic acids such as succinic, lactic, and citric acids, and to resist high salt concentrations and cleaner products and sanitizers (Viljoen, 2001). Yeasts are common inhabitants of the surface of smear-ripened cheeses, where they grow during the initial stages of ripening, deacidifying the cheese surface, which allows the subsequent development of surface bacteria (Lenoir, 1984). As a result, they are used as adjunct cultures mainly in surface-ripened cheeses (Chamba and Irlinger, 2004). Yeasts can also be detected at levels as high as 10⁷ to 10⁸ cfu/g in the interior of cheese, where many species have been identified, the most commonly found being Debaryomyces hansenii, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia membranaefaciens, Pichia fermentans, Saccharomyces cerevisiae, Yarrowia lipolytica, and Geotrichum candidum (Fleet, 1990; Chamba and Irlinger, 2004).

In raw-milk Cantal cheese, a French Protected Denomination of Origin hard cheese that is manufactured in a similar way to Cheddar cheese, the population of yeasts enumerated reached 9 log₁₀ cfu/g of curd (I. De Freitas and N. Pinon; unpublished data). Kluyveromyces lactis and P. fermentans were the 2 main species identified, accounting for 80 and 10% of the isolates identified, respectively (I. De Freitas and N. Pinon; unpublished data). Earlier studies showed that most of the lactose-fermenting yeasts isolated from Cantal cheese were identified as K. lactis (formerly Saccharomyces lactis and Torulopsis sphaerica), and the non lactose-fermenting yeast species being identified as Pichia spp. and Rhodotorula spp. (Millet et al., 1974).

Yeasts can play a role in the ripening process, either directly or via their interactions with the other cheese microorganisms. The most studied species are D. hansenii, K. lactis, Y. lipolytica, S. cerevisiae, and G. candidum. Yeasts can either positively contribute to the formation of cheese flavor, or be responsible for yeasty flavors and other off-flavors (Jakobsen and Narvhus, 1996). Other defects caused by the presence of spoilage yeasts are gas production, discoloration, and changes of texture. Kluyveromyces lactis is known for its ability to assimilate lactose and its enzymatic potential such as aminopeptidase activity (Lenoir, 1984). The interactions between bacteria and yeasts may result in the stimulation or the inhibition of the growth of one or both populations (Viljoen, 2006). Yeasts, however, are considered to stimulate the growth of lactic acid bacteria, via the synthesis of growth factors such as vitamins (Addis et al., 2001; Viljoen, 2006).

Yeasts are able to produce a variety of volatile odorous compounds in dairy products such as esters (mainly ethyl acetate), volatile organic sulfur compounds derived from the conversion of Met, various compounds of the catabolism of aromatic and branched-chain amino acids, and methylketones (Spinnler et al., 2001; Arfi et al., 2004). As a result, yeasts have the potential to contribute to the formation of cheese flavor (Lenoir, 1984). The potential of Pichia spp. to produce volatiles in cheese has not been studied.

The present work aimed to determine the interest of 2 yeasts, K. lactis and P. fermentans, isolated from the dominant yeast population of a typical raw milk Cantal cheese, as adjunct cultures to promote the formation of flavor of Cantalet cheese. We characterized the global microbiological, biochemical, and sensory changes induced by the presence of yeasts in microfiltered milk Cantalet cheese, with a particular focus on the formation of odorous compounds, determined by using a recently developed multi gas chromatography-olfactometry (GC-O) device (Berdagué et al., 2007).

Back to Article Outline

Materials and Methods 

Cheese Manufacture and Sampling 

Cantalet cheese is a small Cantal cheese, with a weight of 8 to 10kg and a cylindrical shape (20 to 22cm in diameter×approximately 20cm high). The manufacturing process is close to that of Cantal cheese (De Freitas et al., 2007). Six Cantalet cheeses, 2 experimental and 1 control repeated twice on 2 consecutive days, were manufactured from microfiltered skim milk and pasteurized cream. Skimmed milk (∼350L) and pasteurized (80°C for 20s) cream (∼40L) were refrigerated and carried at 4°C to the manufacture plant. Skimmed milk was then heated at 40°C and microfiltered by microfiltration. The pilot installation Bactocatch type (MFS1, Alfa Laval, Les-Clayessous-Bois, France) was equipped Sterilox ceramic membrane with a porosity gradient of mean diameter 1.4μm (1P1940, length 1.02m, filtration area of 0.24m², Société des Céramiques Techniques, Tarbes, France). Pasteurized cream was added to the microfiltration permeate to adjust the fat content to 3.3%, constituting cheese milk. The milk was then divided into 3 vats of 100L each and heated to 31°C. Lactic starters and yeast cultures were then added. Calf rennet (33 mL/100L of milk) was added 10min later. Coagulation took around 25min, and the curd was cut for 10min. The mixture of curd and whey was then directed toward a pressing vat in which whey drainage and pressing took place at room temperature. The curd was turned and pressed 6 times in pressure cycles with an increase from 1.8×10⁵ to 6.0×10⁵ Pa for 60min. Then, the pressed curd was matured overnight at 17°C to a pH of about 5.1 to 5.2. The matured curd was then ground and mixed with 2.0% of dry salt. One hour later, the salted curd was molded and pressed for 48h (pressure was increased from 0.9×10⁵ to 5.0×10⁵ Pa) at 16°C. After removal from the molds, the cheeses were ripened under controlled conditions (10°C and 95% relative humidity).

The control cheeses were made by adding only commercial lactic starters (F-DVS DCC-260 and CHN19, Chr. Hansen, St-Germain Les Arpajon, France), to obtain a global population of 10⁵ cfu/mL in cheese milk. Starters contained lactococci, leuconostocs, Streptococcus thermophilus, Lactobacillus helveticus, and Lactobacillus casei. The 2 experimental cheeses were made by adding the same lactic starters and the yeasts K. lactis F41 no. 1 (Kl) or P. fermentans F31 no.2 (Pf) (2 strains isolated from raw milk Cantal cheese, from LFO, Villefranche de Lauragais, France) inoculated at 10⁵ cfu/mL in cheese milk. For each cheese trial, 5 samples were analyzed: microfiltered milk and cream mixture (T₀), curd after maturation (T_14h), cheese after demolding (T_3d), and cheeses after 45 and 95 d of ripening. Samples were stored at −20°C before analyses, except those for volatile compound and FFA analyses, for which samples were kept at −80°C. Preliminary results obtained by enumerating yeasts in the same Cantalet cheese sample either freshly sampled or after being stored for 15 d at −20°C showed that counts did not differ by more than 0.5 log₁₀ cfu/g.

Microbiological Analyses 

Cheese samples were prepared as previously described (De Freitas et al., 2005). Mesophilic aerobic microflora were enumerated on PCA (Biokar Diagnostics, Beauvais, France), lactococci on M17 agar (Difico, BD Diagnostics, Pont de Claix, France) incubated for 7 d at 15°C, thermophilic streptococci on M17 agar (Difco) incubated for 1 d at 43°C, thermophilic lactobacilli on de Man, Rogosa, and Sharpe, pH 5.4, incubated for 2 d at 43°C under anaerobiosis, and facultative heterofermentative lactobacilli on FH agar, incubated for 3 d at 37°C under anaerobiosis, as previously described (De Freitas et al., 2005). The use of M17 at 15°C for lactococci enumeration was adapted from the IDF Standard to improve the selective recovery of lactococci from mixed lactic acid bacteria populations, because it is known that M17 is poorly selective. Yeasts were enumerated on oxy-tetracycline glucose agar (Biokar Diagnostics) after incubation for 5 d at 24°C. Mean values resulting from duplicate plates are given on the log₁₀ basis.

Compositional Analyses 

For all analyses, an inner cheese sample was grated, thoroughly mixed, and used directly for pH, TS content, and fat determinations. pH was measured by using a pH meter (HI 9025, Hanna Instruments, Vila do Conde, Portugal); TS was determined in triplicate by drying cheese samples (2g) mixed with sand at 102°C for 7h (FIL-IDF, 1987b); fat was determined in duplicate by the gravimetric method (FIL-IDF, 1987a). Thirty grams of grated cheese was homogenized with 60g of distilled water by mixing in an ice bath for 2×1min at 20,500rpm using an Ultraturrax blender (Janke & Kunkel, Staufen, Germany). The homogenates were frozen at −20°C before analysis of acidity, ethanol, and lactose. Lactose contents were determined by an enzymatic method through the use of Boehringer kits (Ref. 10176303, R-Biopharm, Darmstadt, Germany).

Individual free AA (FAA) were determined in duplicate from a homogenate of 5g of grated cheese with 10g of lithium citrate buffer, 0.5 M, pH 7.0. An aliquot of the homogenate was deproteinized by addition of a 10% sulfosalicylic acid solution (1:4, wt/wt), incubated for 1h at 4°C, and centrifuged at 1,000×g at 4°C for 15min. The supernatant was filtered on a 0.2-μm pore diameter membrane, and diluted (1:4, vol/vol) with lithium citrate buffer, 0.2 M, pH 2.2. Amino acids were analyzed using an amino acid analyzer (AlphaPlus series 2, Pharmacia, Uppsala, Sweden).

Free fatty acid contents were determined at 95 d of ripening after extraction according to the method of De Jong and Badings (1990). Two independent extractions of cheese lipids were performed using aminopropyl solid-phase extraction columns (500 mg/3mL; Phenomenex, Torrance, CA) and a vacuum manifold (Macherey Nagel, 52313 Duren, Germany). Individual FFA (from C2:0 to C18:3) were quantified by GC using a Varian 3800 gas chromatograph with flame-ionization detection, a Varian 1079 universal capillary injector, a Varian 8410 liquid auto sampler, and Varian Star operating software (Varian Analytical Instruments, Harbor City, CA). A fused-silica semicapillary column BP21 (SGE Europe, Courtaboeuf, France; 25 m×0.53 mm×0.5μm film thickness) was used. Direct cold on-column injection took place at 65°C, the oven temperature increased from 65°C to 240°C at a rate of 10°C/min, and was then held at 240°C for 20min. The flame-ionization detection temperature was 250°C. The flow rate of the carrier gas (hydrogen) was 9.7 mL/min at 65°C.

Twenty-one neutral volatile compounds were preselected on the basis of 1) previous analyses of volatile compounds in Cantal cheese (De Freitas et al., 2007) and 2) volatile compounds that were reported to be produced in cheese by yeasts, such as products from the catabolism of methionine and of branched-chain AA, ethanol, and ethyl esters (Spinnler et al., 2001; Arfi et al., 2004; Leclercq-Perlat et al., 2004). Selected compounds included 6 alcohols (ethanol, propanol, 2-butanol, 2-methyl-1-propanol, 3-methyl-butanol, and 2-methyl-butanol), 7 ketones (2,3-butanedione, 2-butanone, 2-pentanone, 2,3-pentanedione, 3-methyl-2-pentanone, 2-heptanone, and 2-nonanone), 5 esters (ethyl acetate, ethyl butanoate, ethyl hexanoate, ethyl octanoate, and 3-methylbutyl acetate), 2 aldehydes (ethanal and 3-methyl-butanal), and 1 sulfur compound (dimethyldisulfide). These selected neutral volatile compounds were extracted, identified, and quantified using a first dynamic headspace (DH)-GC-MS method. The DH-GC-MS equipment comprised a Tekmar 3000 headspace device (Tekmar Inc., Cincinnati, OH) coupled to a GC HP5890A (Agilent Technologies, Massy, France) and MS HP 7972A (Agilent Technologies). A 20-g sample was homogenized with 80g of refrigerated solution of boiled distilled water by mixing for 2min at 20,500rpm using an Ultraturrax blender. A 7-g sample of this cheese homogenate (± 0.05g) was used for each headspace GC-MS analysis, performed in duplicate. Samples were purged with ultrapure helium gas (35 mL/min) at 65°C for 15min to isolate headspace volatiles, which were adsorbed on a Vocarb 3000 trap (Supelco, Bellefonte, PA). The trapped compounds were thermally desorbed at 250°C over 4min and cryofocused at −100°C before being injected by heating at 270°C. They were separated on an Agilent Technologies HP5 capillary column (60 m×0.32 mm×1.0μm film thickness) as previously described (Thierry et al., 2005b). Peaks were quantified by the areas of the total ion current (TIC). To avoid the approximation related to the commonly used internal standard calibration, volatiles were quantified (ng/g) using an external calibration method based on the addition of standards to the cheese homogenate (Thierry et al., 2005b). Volatiles were identified by comparison of mass spectra and retention times with those of authentic standards purchased from Sigma-Aldrich (St. Quentin Fallavier, France). Ethanol was detected by DH-GC-MS, but it was quantified by an enzymatic method through the use of Boehringer kits (Ref. 10176290).

Determination of Odorous Compounds 

For 8-way GC-olfactometry (8W-GC-O) analyses, volatile compounds were extracted using a second DH-GC method. This DH-GC method was used in parallel to identify the candidate chemical entities to account for the odorous zones previously detected by 8W-GC-O, by using an independent single GC-O/MS set-up. A 3-g aliquot of grated cheese was placed on glass wool at the bottom of a Pyrex extractor (M3, Maillères, Aubière, France) of a Tekmar headspace device (Tekmar Inc.). The extractor was maintained at ambient temperature and purged for 60min with a helium stream at a flow rate of 60 mL/min (Air Liquide, He/U purity: 99.995%). The trap (Tenax TA 60/80, Supelco) was operated at 30°C. The dry purge step was set at 3min. The volatile components were then desorbed from the trap at 180°C for 10min using helium (Air Liquide, He/N55 purity: 99.9995%) and cryofocused at −150°C before splitless injection at 220°C.

The volatile components were separated on an RTX-5 capillary column (60 m×0.53 mm×1.5μm film thickness, Restek, Evry, France). The chromatograph oven temperature was programmed as follows: 5min isothermal at 40°C, 4°C/min increase to 205°C, and 5min isothermal at 205°C. The duration of the GC-O analyses was 2,100s; the helium flow rate was 8 mL/min. The 8W-GC-O device previously described in detail was designed to distribute the volatile components separated by chromatography to 8 sniffing ports synchronously and evenly (Berdagué et al., 2007). Thus, 8 sniffers can detect the substances eluted in a single chromatographic separation simultaneously and under identical conditions, thereby improving the quality of the data obtained. The compounds separated by GC (Hewlett Packard 4890D, Agilent Technologies) were transferred via a transfer line heated at 210°C from the end of the capillary column to the effluent divider of the 8W-GC-O device. The total olfactory signal (TOS), corresponding to the overall response of the sniffer panel, was expressed by the sum of the intensities of each individual aromagram from sniffer 1 to sniffer 8 against time. To study the information in the vocabulary items, the VIDEO-Sniff (Berdagué and Tournayre, 2004) method was used, which breaks up the TOS into olfactory classes. The deconvolution of the olfactory signal to classes facilitates a consensual description of the odor of the olfactory peaks.

To identify the candidate chemical entities to account for odorous zones, an independent single DH-GC-O/MS setup composed of a chromatograph (GC 6890, Agilent Technologies), a mass detector (MSD 5973 Inert, Agilent Technologies), and a homemade sniffing port was used. The split between sniffing port and MS was 1:1. The purge and trap and the chromatographic conditions were the same as those used for 8W-GC-O, except for the capillary column used (RTX-5, 60 m×0.32 mm×1.0μm film thickness, Restek) and the helium flow rate (2 mL/min). The volatile components were ionized by electronic impact at 70eV. Ions were scanned over the range 18<m/z<220amu and identified using databases of spectra (NIST/EPA/NIH 2005 V2.0d http://www.nist.gov/srd/nist1a.htm; Masslib 1999, MSP Köfel, Koeniz, Switzerland), relative retention indices, and odors (Flavor-Base 2004, Leffingwell and Associates, Canton, GA; Flavornet 2006, www.flavornet.org; LRI & Odor Database 2006, www.odour.org.uk).

The computer coupling between the 8W-GC-O set-up and the independent GC-O/MS system made it possible to obtain an alignment that was sufficiently precise for candidate structures to be matched with olfactive peaks. This coupling was obtained by importing the aromagram of the TOS (in the netCDF exchange format) into the mass spectrometry software MSDChem Agilent D.01.02 (Agilent Technologies), to place the TOS in parallel with the TIC or with specific ions. Before export, the 8W-GC-O aromagram retention time was resynchronized with the independent GC-O/MS aromagram/chromatogram retention time using olfactory standards (occurring naturally in the mixture or added specifically). To localize the standards accurately by mass spectrometry, a search based on specific ions was necessary, because of the frequent occurrence of coelutions. The best alignment was calculated using a third-order polynomial regression model between the start of the odor-active peaks and the start of the mass spectrometry peaks.

Flavor Assessment 

Sensory evaluations of ripened cheeses were performed by Les Maisons du Goût (Bourgen-Bresse, France) as previously described (Thierry et al., 2005a). A panel of 18 highly experienced trained assessors, selected for their ability to memorize various odor and flavor attributes, participated in the sensory analysis. The cheeses were evaluated in duplicate by each assessor, at an interval of 2 d. On the day of assessment, the core cheese samples were cut into 30-g sticks, placed in disposable flasks, and allowed to warm to 15°C for 2h before evaluation. Cheese odor (nasal perception) and flavor (defined as the global perception of aroma and taste when samples are put in the mouth) intensities were rated from 0 (none) to 10 (intense) by each panelist. Four attributes of taste (salty, sweet, acidic, and bitter) and 53 attributes of odor (nasal perception) and aroma (retronasal perception) were used to describe Cantalet cheeses. For each of these attributes, the assessors indicated whether the corresponding odor, aroma, and taste were perceived or not, allowing a frequency of perception (number of “presence” responses out of the total number of responses; i.e., 18 assessors×2 replicates=36) to be calculated and expressed as a percentage. The numbers of attributes cited to describe odor and flavor were also determined. This method of sensory evaluation was devised to show a maximum of odor and aroma attributes, whereas classical sensory evaluation by scaling the intensity of odor/aroma attributes considers only a limited number of attributes. This method proved useful to compare the impact of different starters or to show the sensory particularities of Protected Denomination of Origin cheeses (J. F. Clément, Les Maisons du Goût, Bourgen-Bresse, France; personal communication). Data were collected and analyzed using FIZZ software (Biosystèmes, Couternon, France).

Statistical Analyses 

Averaged concentration data of duplicate analyses of each compound were used for statistical analysis. One-way ANOVA were performed by using the GLM procedure of Statgraphics Plus (Statistical Graphic Corp., Englewood Cliffs, NJ) to determine the effect of the addition of yeasts on the concentration data of each compound. The Fisher's least significant difference (LSD) test was used to determine which means were significantly different at a 95% confidence level.

In the same way, the Fisher test was used to determine which of the olfactory zones of the 3 TOS corresponding to the 3 cheese treatments were significantly influenced by the addition of yeasts. An ANOVA was performed in each of the 2,100 scans (S_i) of the aromagrams within this model: S_i = μ + yeast effect_p + judge effect_j + ɛ_{i, p, j, n} (with μ = average effect, p=3, j=8, and n=2). The 2,100 ANOVA thus calculated made it possible to build an “ANOVAgram” to identify the zones of aromagrams that were most influenced by the treatment (here the addition of adjunct yeasts).

Sensory data were analyzed by using FIZZ software (Biosystèmes). The ability of odor and flavor intensity scores and richness to discriminate between cheeses was investigated using 1-way ANOVA. The Newman Keuls test was used to determine which means were significantly different at a 95% confidence level. Only odor/aroma attributes having a perception frequency over 10% for at least 1 cheese, were subjected to statistical analyses. Perception frequencies lower than 10% (less than 4 presence responses out of 36) can be considered as the noise of the method. The statistical significance of differences between the frequencies of this attribute in the 3 cheeses was investigated using a χ² test, by using STATITCF version 5 (ITCF, Paris, France).

Back to Article Outline

Results 

Lactic Starters and Yeast Adjuncts 

Figure 1 shows the changes occurring in the starter and adjunct populations. Total mesophilic bacteria counts were 2.9 and 2.7 log₁₀ cfu/mL in pasteurized creams, and 1.1 and 1.4 log₁₀ cfu/mL in microfiltered milks on the 2 d of manufacture, respectively. Yeasts were not detected in microfiltered milks or in pasteurized creams. As expected, after addition of the starter and adjuncts, the population of yeasts was 5.11±0.09 and 5.07±0.03 log₁₀ cfu/mL in Kl and Pf cheese milks, respectively, whereas yeasts were not detected in the control cheese milks. Yeasts counts were stable during manufacture (5.87±0.04 and 5.80±0.07 after maturation at T_14h, and 5.99±0.19 and 5.94±0.08 log₁₀ cfu/g at T_3d in Kl and Pf cheeses, respectively). During the ripening period, yeast populations decreased to 4.1 and 4.7 log₁₀ cfu/g in Kl and Pf cheeses at 45 d, respectively, and then increased to 5.4 and 5.0 log₁₀ cfu/g at 95 d. All the colonies of yeasts on agar plates prepared from Kl and Pf cheese samples had the same morphotype, showed the same morphology under microscopic observation, and corresponded to those of K. lactis and P. fermentans, respectively. The dominant populations of yeasts in Kl and Pf cheeses thus represented the added yeast species throughout the ripening period. In contrast, colonies of different sizes, colors, and aspects were observed on the oxytetracycline glucose agar plates made from the control cheeses. These contaminating yeasts probably originated from the plant environment, and increased in cheese during acidification to reach 2.7 log₁₀ cfu/g at T_3d. Thereafter, yeast counts of the control cheeses decreased to 1.8 log₁₀ cfu/g at 45 d. Some significant differences of the populations of lactococci and thermophilic lactobacilli (P=0.02 and P=0.01, respectively) in the 3 cheeses were observed at 45 d of ripening (Figure 1). Hence, the cultivable populations of lactococci decreased by 81% in the control cheeses during the 45-d ripening period, vs. 16 and 57% decreases in Kl and Pf cheeses, respectively.

• 
  • View Large Image
  • Download to PowerPoint

• Figure 1. 

  Changes in the microbial populations of lactic starters and yeast adjuncts throughout the ripening in 3 Cantalet cheeses manufactured without (control) and with addition of yeasts (Kluyveromyces lactis and Pichia fermentans) as adjunct cultures.

Compositional Attributes 

The pH of matured curd (T_14h) was 5.16±0.09, 5.18±0.01, and 5.12±0.04 in the control, Kl, and Pf cheeses, respectively, showing that the addition of yeasts did not modify the rate of acidification. In agreement, the addition of yeasts did not influence the concentrations of residual lactose at 3 d, which were similar to that found in 3-d-old Cantal cheese (De Freitas et al., 2007). Lactose concentrations were thus not further determined during the ripening. There were no significant differences of gross composition between the control and the 2 experimental Cantalet cheeses (Table 1), demonstrating that the addition of yeasts did not modify the characteristics of the cheese. All cheeses showed the same changes over the ripening: increase in TS and fat contents due to loss of moisture, and increase in pH, acidity, and FAA concentrations due to proteolysis (Table 1). Five individual FAA (Pro, Arg, Orn, Phe, and Leu) showed slight but significant differences of concentrations between the control and the experimental cheeses at 45 d of ripening only. The control and Kl cheeses had 11 to 16% greater concentrations of Pro, Phe, and Leu than did Pf cheeses (P<0.05). Arginine was at 3- to 5-fold greater concentrations in the control cheeses than in Kl and Pf cheeses (P<0.01), and Orn was at a 14% greater concentration in Kl cheeses than in the other cheeses (P=0.02; data not shown). The Cantalet cheese composition values were in the range of those of Cantal cheese, as expected (De Freitas et al., 2005).

Table 1. Gross composition and free amino acids (FAA) for 3 Cantalet cheeses manufactured without (control) and with addition of yeasts (Kluyveromyces lactis, Pichia fermentans) as adjunct cultures in matured curd (3 d) and during ripening (45 and 95 d)

Item1	Cheese age, d	Control	K. lactis	P. fermentans	P-value2
pH	3	5.16±0.09	5.18±0.01	5.12±0.04	NS
	95	5.42±0.12	5.47±0.04	5.44±0.08	NS
Acidity, Dornic°	3	115±7	110±0	113±4	NS
	95	208±25	180±22	215±15	NS
TS, g/kg	3	523.1±13.9	524.0±7.5	524.4±5.8	NS
	95	613.9±5.8	611.2±9.9	618.8±1.7	NS
Fat, g/kg	3	265.5±3.5	269.0±8.5	264.0±5.7	NS
	95	303.8±8.8	296.3±0.6	296.3±8.8	NS
Fat/TS, %	3	50.8±0.8	51.3±0.9	50.4±0.5	NS
	95	49.5±1.0	48.5±1.0	47.9±1.3	NS
Lactose, g/kg	3	4.7±1.2	4.8±2.7	4.8±1.6	NS
FAA, g/kg	45	5.94±0.18	5.84±0.28	5.28±0.38	NS
	95	14.39±0.55	17.46±1.55	15.86±0.63	NS

Volatile Composition 

Levels of FFA derived from lipolysis (C4:0 to C18:3) did not differ significantly in the control and the experimental cheeses (mean value 1.78±0.07 g/kg at 95 d of ripening) and represented 0.6% of the fat content (Table 2). The 3 Cantalet cheeses also contained VFA derived from carbohydrate (acetic acid) and branched-chain AA (methylpropanoic and methylbutanoic acids) catabolism. Methyl-branched acid concentrations were about 2-fold greater in both experimental cheeses but these differences were not statistically significant (Table 2).

Table 2. Concentrations of selected volatile compounds in Cantalet cheeses manufactured without (control) and with addition of yeasts (Kluyveromyces lactis, Pichia fermentans) as adjunct cultures in matured curd (3 d) and during ripening

Compound1	Cheese age, d	Control	K. lactis	P. fermentans	P-value
Neutral volatiles2
Ethanal, AU	3	ND3	ND	ND	—4
	45	NDa	51±49a	NDa	NS
	95	NDa	61±30b	NDa	NS
Ethanol, μg/g	3	26±1a	207±35b	29±1a	**
	45	47±2a	355±41b	48±5a	**
	95	29±3a	86±97a	19±2a	NS
Ethyl acetate, ng/g	3	8±1a	1,101±299b	9±2a	*
	45	66±37a	2,928±228b	145±67a	***
	95	147±3a	4,448±278b	332±406a	***
2-Methylpropanol, ng/g	3	NDa	101±62a	NDa	NS
	45	NDa	996±348b	NDa	*
	95	42±59a	911±16b	NDa	*
3-Methyl-butanal, ng/g	3	NDa	193±17b	NDa	***
	45	7±0.2a	171±53	9±1a	*
	95	62±23a	183±147a	35±17a	NS
3-Methylbutanol, ng/g	3	NDa	318±135b	NDa	*
	45	70±10a	2,762±781b	36±8a	*
	95	446±50	3,283±199b	143±115a	***
2-Methylbutanol, ng/g	3	ND	ND	ND	—
	45	ND	ND	ND	—
	95	46±65a	432±87b	NDa	*
Ethyl butanoate, ng/g	3	ND	16±1b	ND	***
	45	8±3a	83±0.4b	12±4a	***
	95	19±10a	124±12b	16±2a	**
3-Methylbutyl acetate, ng/g	3	ND	ND	ND	—
	45	NDa	18±2b	NDa	***
	95	NDa	150±13b	NDa	***
Ethyl hexanoate, ng/g	3	ND	ND	ND	—
	45	6±8a	121±25b	27±2a	**
	95	45±25a	182±39b	36±5a	*
VFA, μg/g
Acetic acid, μg/g	95	1,262±78a	1,438±3a	1,025±166a	NS
Methylpropanoic acid, μg/g	95	4.8±3.8a	10.6±5.4a	9.4±2.9a	NS
Methylbutanoic acids,5μg/g	95	6.5±6.4	15.2±9.3a	13.2±6.1a	NS
Butanoic acid, μg/g	95	63.4±15.4a	88.4±18.6a	78.2±6.5a	NS
Hexanoic acid, μg/g	95	18.1±1.1a	18.2±1.5a	19.8±2.6a	NS
FFA (sum C4:0 to C18:3), μg/g	95	1,755±19a	1,775±150a	1,803±6a	NS

Selected volatile compounds (see Materials and Methods) were quantified in Cantalet cheeses by DH-GC-MS. Ten of the 21 selected compounds (ethanol, ethanal, 3 ethyl esters, and 5 branched-chain compounds; Table 2) were present at significantly greater concentrations in the Kl cheeses at 95 d compared with the control and Pf cheeses. Ethanol was produced early during manufacture, whereas most of the other compounds increased in concentrations throughout ripening. Esters showed the greatest change in concentration.

Odorous Compounds Analysis 

Thirty-three odors were perceived by using 8W-GC-O. Half of the odors perceived (16 of 33) could be classified in “fruity-floral” and “lactic-cheesy” odor families (9 and 7 occurrences, respectively). In total, 30 of 33 odorous compounds were identified with a high reliability, and their odor attributes, as described in literature and databases, were in agreement with 8W-GC-O data (Table 3). The total olfactory signal (TOS, sum of the 16 individual aromagrams) of the control and the 2 experimental cheeses were compared to investigate the effect of yeast adjuncts on the olfactory characteristics of Cantalet cheese (Figure 2). The Fisher test values revealed that 12 zones of the TOS differed significantly (P<0.01) between the 3 cheeses. In most cases, the Kl cheeses showed a more intense olfactory signal than the other cheeses (Figure 2). Ten of these 12 olfactory signals were related to 3 acids, 4 aldehydes, 2 alcohols, and 1 ester (Table 3). Regarding the 2 remaining olfactory zones, the fruity odor (peak 17) likely corresponded to one or several (coeluted) esters, and the empyreumatical odor (peak 32), described as caramel/toasted bread/roasted, could not be identified. To further confirm the link between the olfactory peaks and the 10 identified volatile compounds, peak areas of the corresponding DH-GC-MS chromatograms were compared for the 3 cheeses (Table 4). Eight of the 10vol.tile compounds were effectively detected at greater concentrations in the Kl cheeses than in the other cheeses (Table 4), as expected from the analysis of olfactometry data (Figure 2).

Table 3. List of the odorous compounds identified by gas chromatography-olfactometry/mass spectrometry (GC-O/MS) of Cantalet cheese at 95 d of ripening

• 
  • View Large Image
  • Download to PowerPoint

• Figure 2. 

  Total olfactory signals obtained by using 8-way GC-olfactometry in 3 Cantalet cheeses without (control) and with addition of yeasts (Kluyveromyces lactis and Pichia fermentans) as adjunct cultures, at 95 d of ripening, and “ANOVAgram” showing the results of the ANOVA.

Table 4. Odorous compounds in the GC-MS profiles corresponding to the olfactory zones that differed significantly between 3 Cantalet cheeses manufactured without (control) and with addition of yeasts Kluyveromyces lactis (Kl) and Pichia fermentans (Pf) adjunct cultures, at 95 d of ripening

Sensory Analyses 

Cheeses were first evaluated by 2 industrial graders experienced in Cantal/Cantalet cheese tasting, who considered that all the Cantalet cheeses had typical Cantal characteristics. Cantalet cheeses were also evaluated by the trained assessors of Les Maisons du Goût to be further characterized. The average intensities of odor and flavor did not significantly vary between the 3 cheeses (Table 5), but more attributes were used to described the odor of Kl cheeses (P=0.060), and the flavor of both Kl and Pf cheeses (P=0.053; Table 5). Twenty-five and 26 out of the 53 odor and aroma attributes used to describe the flavor of Cantalet cheeses were cited at a frequency>10% (shown in Figure 3). The attributes used frequently to describe the odor of cheeses were “butter”, “boiled milk”, “whey”, “lemon”, and “fresh milk”, which represented more than 30% of the total citations for at least 1 trial. The attributes used frequently to describe the taste and aroma of cheeses were “acidic”, “boiled milk”, “salty”, “butter”, “bitter”, “whey” and “lemon”, which represented more than 38% of the total citations. Cheeses significantly differed in the frequency of only few attributes (Figure 3). The control cheeses were characterized by “apricot/peach” (P=0.02) odor and “hazel-nut” odor (P=0.02) and aroma (P=0.08). The Kl cheeses were characterized by “acetaldehyde” and “alcohol” odors (P<0.001). At lower levels of significance, Kl cheeses differed from the other cheeses by “yeast” (P=0.12) odor. The Pf cheeses had a higher “acetic” (P=0.13) and “potato” (P=0.12) aromas and a lesser “malty” (P=0.04) aroma than the control and Kl cheeses (Figure 3).

Table 5. Results of ANOVA on the intensity of odor and flavor and of the numbers of attributes cited to describe odor and flavor of 3 Cantalet cheeses manufactured without (control) and with addition of the yeasts Kluyveromyces lactis (Kl) and Pichia fermentans (Pf) as adjunct cultures, evaluated after 95 d of ripening

• 
  • View Large Image
  • Download to PowerPoint

• Figure 3. 

  Results of descriptive analysis of odor and aroma of 3 Cantalet cheeses manufactured without (control) and with addition of yeasts [Kluyveromyces lactis (Kl) and Pichia fermentans (Pf)] as adjunct cultures, evaluated after 95 d of ripening. Values are perception frequencies, expressed as percentage [i.e., the number of “presence” responses divided by the maximal number of responses (36: 18 assessors, duplicate evaluations)]. Values of probability of χ² test<0.05 are marked by an asterisk.

Back to Article Outline

Discussion 

This study shows that K. lactis had a limited but significant influence on some aspects of the ripening process of Cantalet cheese. The presence of viable cells of K. lactis in Cantalet cheese was associated in particular with a longer survival of lactococci, a marked increase in the concentrations of some odorous compounds, and changes in olfactory profiles, but little change in cheese flavor.

The populations of K. lactis and P. fermentans yeasts were stable at approximately 6 log cfu/g during cheese manufacture and showed variations of ∼1.5 log cfu/g over the ripening. The 2 yeast strains used as adjunct cultures in the present study were isolated from the dominant yeast population of a Cantal cheese containing ∼5×10⁴ yeasts/g. Therefore, their lack of growth was unexpected and may result from their inability to adapt their metabolism to cheese conditions after being grown in a glucose-yeast extract medium and directly inoculated in cheese milk. The growth of yeasts in cheese is highly dependent on the environmental conditions and on the species used. In curd slurries, for example, K. lactis grew from 5×10⁵ to 1×10⁸ cfu/g over 5 d of incubation (Arfi et al., 2004), whereas in Raclette cheese, the population of viable cells of 4 species of adjunct yeasts (Galactomyces geotrichum, Pichia jardinii, Y. lipolytica, and D. hansenii) decreased from ∼10⁵ to 10⁶ cfu/g at the molding step to<10² cfu/g after 90 d (Wyder et al., 1999). In contrast, indigenous yeasts grew in the control cheeses during the manufacture and reached 2.7 log cfu/g, as reported in Cheddar cheeses, where populations up to 6 log₁₀ cfu/g in 37-d-old Cheddar were detected (Welthagen and Viljoen, 1999).

Starter cultures may have benefitted from the presence of yeasts at the beginning of ripening, because cultivable lactococcal populations remained greater in both Kl and Pf cheeses than in the control cheeses at 45 d of ripening, in agreement with results reported in Cheddar cheese with D. hansenii or Y. lipolytica added as adjunct cultures (Ferreira and Viljoen, 2003). A synergistic effect between lactic acid bacteria and many species of yeasts including K. lactis, which resulted in a prolonged survival of lactic acid bacteria, has also been observed in cheese slurries (Arfi et al., 2004).

The presence of yeasts did not modify gross cheese composition (Table 1), which can be explained by the fact that the adjunct yeasts did not grow in Cantalet cheeses, and thus did not modify the rate of acidification. Moreover, the presence of yeasts did not modify the level and the specificity of lipolysis, and had only a limited impact on the concentrations of FAA. In contrast, the presence of K. lactis in Cantalet cheese induced large variations in the concentrations of some volatile odorous compounds. This effect was particularly marked for ethanol, ethyl esters, some aldehydes, branched-chain alcohols (Table 2), branched-chain acids, and 1-octen-3-ol (Figure 2, Table 4). Ethanol was formed at concentrations ∼200μg/g in Kl cheeses [vs. ∼40μg/g in the control cheeses and in raw milk Cantal cheese (De Freitas et al., 2005)]. Such high values are observed in Emmental cheese containing obligatory heterofermentative lactobacilli as adjuncts (Chamba, 2000). Ethyl esters were formed at concentrations approximately 5-fold greater in the presence of K. lactis, and up to 30-fold greater for ethyl acetate (Table 2). Moreover, a branched-chain ester (3-methylbutyl acetate) was also detected in Kl cheeses only. Only one strain of K. lactis was tested in the present study, but the results observed were in general agreement with previous reports regarding the use of K. lactis as adjunct. Hence, high levels of ethyl acetate were detected in cheese slurries containing K. lactis (Arfi et al., 2004). Various esters including ethyl acetate, ethyl butanoate, and 3-methylbutyl acetate were also produced by K. marxianus in cheese slurries (Leclercq-Perlat et al., 2004). The formation of esters in Kl cheeses can result directly from the ester-forming activity of K. lactis, because yeasts, and particularly K. lactis, are known to produce esters by the acylation of an alcohol by an acylCoA catalyzed by alcohol acyltransferases (Mason and Dufour, 1985). Alternatively, the formation of greater levels of ethyl and 3-methylbutyl esters in Kl cheeses could have simply resulted from the presence of greater concentrations of the corresponding alcohols (ethanol and 3-methylbutanol), because the concentration of alcohols can be a limiting factor of ester synthesis in cheese (Thierry et al., 2006). Branched-chain compounds were detected at concentrations from 2- (acids) to 20-fold (alcohols) greater in Kl cheeses than in the 2 other cheeses, in agreement with previously reported results for K. lactis or D. hansenii added in model cheese media (Arfi et al., 2004; Leclercq-Perlat et al., 2004). Branched-chain compounds likely originate from the catabolism of branched-chain AA. Several saturated (hexanal, octanal) and unsaturated (2-pentenal, 2-hexenal) aldehydes were also detected in the present study at approximately 4-fold-greater concentrations in the presence of K. lactis, in agreement with previous observations in cheese slurries inoculated with D. hansenii (Leclercq-Perlat et al., 2004). These results were unexpected because K. lactis possess alcohol dehydrogenase activity (Bozzi et al., 1997), which could have enhanced the rate of reduction of aldehydes in Kl cheeses, as generally observed in raw milk cheeses compared with pasteurized or microfiltered milk cheeses (Beuvier and Buchin, 2004). Regarding the formation of sulfur compounds, only dimethyl disulfide was detected in the present study, and its concentration did not vary with the presence of yeasts (data not shown), and no sulfur odors were detected by GC-O. This result is in contrast with previous studies reporting that many yeast species including K. lactis are able to produce volatile sulfur compounds from Met (Spinnler et al., 2001). The ability to produce volatile compounds can also depend on the strain, although most of the effects of K. lactis on cheese volatiles shown in the present study are in agreement with previously reported results regarding the species K. lactis. The effects observed were largely dependent on the species. Hence, at the same populations of viable K. lactis and P. fermentans over ripening, only the former had an effect on cheese flavor under the experimental conditions used.

The use of the recently developed multicanal olfactometry device (8W-GC-O), in parallel with GC-MS, allowed us to identify most (29 of 33) of the odorous compounds responsible for the olfactory zones detected in Cantalet cheese. Most of the compounds are common in cheese (Maarse et al., 1994) and were previously identified in cheeses by GC-O/MS (Curioni and Bosset, 2002). Three original compounds, however, were detected: 2-pentenal, 2-hexenal, and 2-ethyl-3-methoxypyrazine. 2-Pentenal and 2-hexenal, associated with the fruity odors perceived in peaks 12 and 18, were previously detected by GC/MS in cheese and in butter, respectively (Maarse et al., 1994), but their role in cheese flavor was not previously investigated, to our knowledge. 2-Ethyl-3-methoxypyra-zine was tentatively identified as being responsible for the empyreumatic odor perceived in peak 28. Various alkyl-pyrazines and alkyl-methoxy-pyrazines have been detected in cheese, including Cheddar cheese (Curioni and Bosset, 2002; Suriyaphan et al., 2001), but not 2-ethyl-3-methoxypyrazine, to the authors’ knowledge.

The main notes of Cantalet cheese flavor appeared to be “fruity” and “lactic-cheesy”, from the results of both olfactory profiles and cheese qualitative sensory evaluations. Hence, 16 of the 33 olfactory zones identified in 8W-GC-O profiles were classified in 1 of these 2 families (Table 3), whereas the main attributes of Cantalet cheese odor and aroma were “boiled milk”, “butter”, “lemon”, and “whey” (Figure 3). Regarding the impact of K. lactis as an adjunct culture, divergent results were given by 8W-GC-O profiles and sensory evaluations of cheeses. Hence, the 8W-GC-O profiles of Kl cheeses differed very significantly from those of the control and Pf cheeses by a greater intensity of perception of 8 olfactory zones: 1 alcohol odor (ethanol), 4 fruity odors associated with esters and unsaturated aldehydes, 2 cheesy odors associated with branched-chain VFA, and 1 empyreumatical odor (peak 32; Figure 2). The ability of K. lactis to develop strong fruity olfactory notes was previously reported (Martin et al., 2002). Regarding sensory evaluations, however, K. lactis had only a low impact on Cantalet cheese flavor in the present study. The Kl cheeses differed from the other cheeses by the perception of only 2 odor attributes, “acetaldehyde” and “alcohol” (Figure 3), in agreement with the data of concentrations of ethanol and acetaldehyde (ethanal). The other differences detected by DH-GC-MS or GC-O between Kl cheeses and the other cheeses did not result in detectable differences in cheese odor and flavor. This result could be because instrumental analyses such as DH-GC-MS have, in most cases, better sensitivity and accuracy than sensory evaluations. Regarding GC-O, this method gives only a first indication of the potent flavor-active compounds of cheese, because it does not make allowance for the effects of combinations of odorous compounds (Delahunty and Piggott, 1995). Moreover, the volatility of compounds is modulated by their interactions with cheese fat and proteins, which differ in the mouth during mastication and in the headspace device. Regarding VFA, their volatility and thus their perception depends on the pH. It could explain why VFA, which were the most potent odorous compounds and differed in CG-O profiles (Figure 2), did not play a role in differentiating the flavor profiles of the whole cheeses. These results also highlight the fact that sensory evaluations are required to characterize the characteristics of cheese flavor.

In conclusion, yeasts added as adjunct cultures can influence the formation of odorous compounds and, even if the overall effects on cheese flavor were quite small in the present study, yeasts could have the potential to impact cheese flavor.

Back to Article Outline

Acknowledgements 

The authors thank M.-C. Montel for making the pilot plant available; R. Lavigne, J. Fauquant, A. Vigier, and P. Serre for tangential filtration and technical assistance in cheese manufacture; N. Kondjoyan for technical assistance in GC-O analyses; and J.-L. Maubois and M. Place for fruitful discussions.

Back to Article Outline

Supplementary data 

Back to Article Outline

References 

1. Addis E, Fleet GH, Cox JM, Kolak D, Leung T. The growth, properties and interactions of yeasts and bacteria associated with the maturation of Camembert and blue-veined cheeses. Int. J. Food Microbiol. 2001;69:25–36
   • View In Article
   • MEDLINE
   • CrossRef
2. Arfi K, Leclercq-Perlat MN, Baucher A, Tache R, Delettre J, Bonnarme P. Contribution of several cheese-ripening microbial associations to aroma compound production. Lait. 2004;84:435–447
   • View In Article
   • CrossRef
3. Berdagué JL, Tournayre P. The VIDEO-Sniff^® method, a new approach for the “Vocabulary-Intensity-Duration” study of “Elementary Odours” perceived by gas chromatography-olfaction. Flavour Research at the Dawn of the Twenty-First Century. Paris, France: Lavoisier; 2004;514–519
   • View In Article
4. Berdagué JL, Tournayre P, Cambou S. Novel multi-gas chromatography-olfactometry device and software for the identification of odour-active compounds. J. Chromatogr. A. 2007;1146:85–92
   • View In Article
   • MEDLINE
   • CrossRef
5. Beuvier E, Buchin S. Raw milk cheeses. In:  Fox PF,  McSweeney PLH,  Cogan TM,  Guinee TP editor. Cheese. Chemistry, Physics and Microbiology. London, UK: Elsevier; 2004;p. 319–345
   • View In Article
6. Bozzi A, Saliola M, Falcone C, Bossa F, Martini F. Structural and biochemical studies of alcohol dehydrogenase isozymes from Kluyveromyces lactis. Biochim. Biophys. Acta. 1997;1339:133–142
   • View In Article
   • MEDLINE
7. Callon C, Millet L, Montel MC. Diversity of lactic acid bacteria isolated from AOC Salers cheese. J. Dairy Res. 2004;71:231–244
   • View In Article
   • MEDLINE
   • CrossRef
8. Chamba JF. L’emmental, un écosystème complexe. Conséquences sur la sélection et l’utilisation des ferments. Sci. Aliments. 2000;20:37–54
   • View In Article
9. Chamba JF, Irlinger F. Secondary and adjunct cultures. In:  Fox PF,  McSweeney PLH,  Cogan TM,  Guinee TP editor. Cheese. Chemistry, Physics and Microbiology. London, UK: Elsevier; 2004;p. 191–206
   • View In Article
10. Curioni PMG, Bosset JO. Key odorants in various cheese types as determined by gas chromatography-olfactometry. Int. Dairy J. 2002;12:959–984
    • View In Article
11. De Freitas I, Pinon N, Lopez C, Thierry A, Maubois J-L, Lortal S. Microstructure, physicochemistry, microbial populations and aroma compounds of ripened Cantal cheeses. Lait. 2005;85:453–468
    • View In Article
    • CrossRef
12. De Freitas I, Pinon N, Thierry A, Lopez C, Maubois J-L, Lortal S. In-depth dynamic characterisation of French PDO Cantal cheese made from raw milk. Lait. 2007;87:97–117
    • View In Article
    • CrossRef
13. De Jong C, Badings HT. Determination of free fatty acids in milk and cheese. Procedures for extraction clean up and capillary gas chromatographic analysis. J. High Resol. Chromatogr. 1990;13:94–98
    • View In Article
14. Delahunty CM, Piggott JR. Current methods to evaluate contribution and interactions of components to flavour of solid foods using hard cheese as an example– Review. Int. J. Food Sci. Technol. 1995;30:555–570
    • View In Article
15. Ferreira AD, Viljoen BC. Yeasts as adjunct starters in matured Cheddar cheese. Int. J. Food Microbiol. 2003;86:131–140
    • View In Article
    • MEDLINE
    • CrossRef
16. FIL-IDF. Standard 005:2004 /ISO 1735–Cheese and processed cheese products – Determination of fat content. Brussels, Belgium: Fédération Internationale de Laiterie-International Dairy Federation; 1987;
    • View In Article
17. FIL-IDF. Standard 021B:1987: Milk, cream and evaporated milk – Determination of total solids content. Brussels, Belgium: Fédération Internationale de Laiterie-International Dairy Federation; 1987;
    • View In Article
18. FIL-IDF. Standard 149A:1997: Dairy starter cultures of lactic acid bacteria (LAB)– Standard of identity. Brussels, Belgium: Fédération Internationale de Laiterie-International Dairy Federation; 1997;
    • View In Article
19. Fleet GH. Yeasts in Dairy Products. J. Appl. Bacteriol. 1990;68:199–211
    • View In Article
    • MEDLINE
20. Jakobsen M, Narvhus J. Yeasts and their possible beneficial and negative effects on the quality of dairy products. Int. Dairy J. 1996;6:755–768
    • View In Article
21. Leclercq-Perlat MN, Corrieu G, Spinnler HE. Comparison of volatile compounds produced in model cheese medium deacidified by Debaryomyces hansenii or Kluyveromyces marxianus. J. Dairy Sci. 2004;87:1545–1550
    • View In Article
    • Abstract
    • Full Text
    • Full-Text PDF (146 KB)
    • CrossRef
22. Lenoir J. The surface flora and its role in the ripening of cheese. IDF Bull. 1984;171:3–19
    • View In Article
23. Maarse H, Visscher CA, Willemsens LC, Nijssen LM, Boelens MH. Volatile Compounds in Food: Qualitative and Quantitative Data. 6th ed. Zeist, the Netherlands: 5th suppl. TNO Nutrition and Food Research; 1994;
    • View In Article
24. Martin N, Berger C, Spinnler HE. Sensory and instrumental flavor analyses of cheese curd cocultured with selected yeast and bacteria. J. Sens. Stud. 2002;17:1–17
    • View In Article
25. Mason AB, Dufour JP. Alcohol acetyltransferases and the significance of ester synthesis in yeast. Yeast. 1985;16:1287–1298
    • View In Article
    • MEDLINE
    • CrossRef
26. Millet L, Melcion D, Devoyod JJ. La flore microbienne du fromage de Cantal fabriqué à partir de lait cru. I. Techniques d’études et résultats préliminaires. Lait. 1974;537:397–407
    • View In Article
27. Spinnler HE, Berger C, Lapadatescu C, Bonnarme P. Production of sulfur compounds by several yeasts of technological interest for cheese ripening. Int. Dairy J. 2001;11:245–252
    • View In Article
28. Suriyaphan O, Drake MA, Chen XQ, Cadwallader KR. Characteristic aroma components of British Farmhouse Cheddar cheese. J. Agric. Food Chem. 2001;49:1382–1387
    • View In Article
    • MEDLINE
    • CrossRef
29. Thierry A, Maillard MB, Bonnarme P, Roussel E. The addition of Propionibacterium freudenreichii to Raclette cheese induces biochemical changes and enhances flavor development. J. Agric. Food Chem. 2005;53:4157–4165
    • View In Article
    • MEDLINE
    • CrossRef
30. Thierry A, Maillard MB, Richoux R, Kerjean JR, Lortal S. Propionibacterium freudenreichii strains quantitatively affect production of volatile compounds in Swiss cheese. Lait. 2005;85:57–74
    • View In Article
    • CrossRef
31. Thierry A, Maillard MB, Richoux R, Lortal S. Ethyl ester formation is enhanced by ethanol addition in mini Swiss cheese with and without added propionibacteria. J. Agric. Food Chem. 2006;54:6819–6824
    • View In Article
    • MEDLINE
    • CrossRef
32. Viljoen BC. The interaction between yeasts and bacteria in dairy environments. Int. J. Food Microbiol. 2001;69:37–44
    • View In Article
    • MEDLINE
    • CrossRef
33. Viljoen BC. Yeast ecological interactionS Yeast-yeast F s.yeast-bacteria, yeast-fungi interactions and yeasts as biocontrol agents. In:  Querol A,  Fleet GH editor. Yeasts in Food and Beverages. Berlin, Germany: Springer-Verlag; 2006;p. 83–110
    • View In Article
34. Welthagen JJ, Viljoen BC. The isolation and identification of yeasts obtained during the manufacture and ripening of Cheddar cheese. Food Microbiol. 1999;16:63–73
    • View In Article
    • CrossRef
35. Wyder MT, Bachmann HP, Puhan Z. Role of selected yeasts in cheese ripening: An evaluation in foil-wrapped Raclette cheese. Lebensm. Wiss. Technol. 1999;32:333–343
    • View In Article