Aroma Compounds In Cheese Research Articles

Biochemistry of aroma compounds in cheese

Cheese is one of the most popular dairy products, with an incredible diversity of varieties, shapes, textures, and flavours available locally and globally. Extensive research has been conducted on the aroma compounds present in cheese. Numerous studies have identified over 3000 volatile and non-volatile components in cheese. The volatile components, which consist of fatty acids, alcohols, aldehydes, esters, ketones, lactones, sulphur compounds, amines, and other flavour-active compounds, are primarily responsible for the aromatic characteristics (aroma) of cheese. This paper thoroughly discusses the biochemistry behind the formation of significant aroma compounds that affect overall cheese quality and sensory characteristics. It provides an extensive review of the most recent methodologies for the extraction and determination of aroma compounds in cheese.

The effect of using microencapsulated pro and prebiotics on the aromatic compounds and sensorial properties of synbiotic goat cheese

In this study, synbiotic goat cheese was obtained by adding free and microencapsulated probiotic bacteria and prebiotics to goat milk. Three types of microcapsules containing Lacticaseibacillus casei and Bifidobacterium longum, fructooligosaccharide and inulin were obtained and these microcapsules were added during the cheese production process. In synbiotic microcapsule added goat cheese, changes in volatile aroma substances and sensory properties during storage (180 days at +4 °C) and the effect of microencapsulation on these changes were determined. Most of the aroma compounds determined during storage in goat cheeses are alcohols and acids, and it is seen that the use of probiotic supplement culture increases the number of aroma compounds in cheese. It was determined that sensory properties of goat cheese are not affected by the use of probiotic supplements, prebiotics, and microencapsulation and was appreciated by the panelists.

Aroma formation in retentostat co-cultures of Lactococcus lactis and Leuconostoc mesenteroides

Lactococcus lactis subsp. lactis biovar diacetylactis and Leuconostoc mesenteroides are considered to be the main aroma producers in Dutch-type cheeses. Both species of lactic acid bacteria were grown in retentostat mono- and co-cultures to investigate their interaction at near-zero growth rates and to determine if co-cultivation enhances the aroma complexity compared to single species performance. During retentostat mono-cultures, the growth rates of both species decreased to less than 0.001 h−1 and a large fraction of the cells became viable but not culturable. Compared to Lc. mesenteroides, L. lactis reached a 3.4-fold higher biomass concentration caused by i) a higher ATP yield on substrate, ii) a higher biomass yield on ATP and iii) a lower maintenance requirement (mATP). Dynamic models estimated that the mATP of both species decreased approximately 7-fold at near-zero growth rates compared to high growth rates. Extension of these models by assuming equal substrate distribution resulted in excellent prediction of the biomass accumulation in retentostat co-cultures with L. lactis dominating (100:1) as observed in ripened cheese. Despite its low abundance (∼1%), Lc. mesenteroides contributed to aroma production in co-cultures as indicated by the presence of all 5 specific Lc. mesenteroides compounds. This study provides insights in the production of cheese aroma compounds outside the cheese matrix by co-cultures of L. lactis and Lc. mesenteroides, which could be used as food supplements in dairy or non-dairy products.

Aroma formation during cheese ripening is best resembled by Lactococcus lactis retentostat cultures

BackgroundCheese ripening is a complex, time consuming and expensive process, which involves the generation of precursors from carbohydrates, proteins and fats and their subsequent conversion into a wide range of compounds responsible for the flavour and texture of the cheese. This study aims to investigate production of cheese aroma compounds outside the cheese matrix that could be applied for instance as food supplements in dairy or non-dairy products.ResultsIn this study, aroma formation by a dairy Lactococcus lactis was analysed as a function of the growth medium [milk, hydrolysed micellar casein isolate (MCI) and chemically defined medium (CDM)] and the cultivation conditions (batch culture, retentostat culture and a milli-cheese model system). In the retentostat cultures, the nutrient supply was severely restricted resulting in low growth rates (~ 0.001 h−1), thereby mimicking cheese ripening conditions in which nutrients are scarce and bacteria hardly grow. In total 82 volatile organic compounds were produced by the bacteria. Despite the use of a chemically defined medium, retentostat cultures had the biggest qualitative overlap in aroma production with the milli-cheese model system (36 out of 54 compounds). In the retentostat cultures, 52 known cheese compounds were produced and several important cheese aroma compounds and/or compounds with a buttery or cheese-like aroma increased in retentostat cultures compared to batch cultures and milli-cheeses, such as esters, methyl ketones, diketones and unsaturated ketones. In cultures on CDM and MCI, free fatty acids and their corresponding degradation products were underrepresented compared to what was found in the milli-cheeses. Addition of a mixture of free fatty acids to CDM and MCI could help to enhance flavour formation in these media, thereby even better resembling flavour formation in cheese.ConclusionsThis study demonstrates that retentostat cultivation is the preferred method to produce cheese flavours outside the cheese matrix by mimicking the slow growth of bacteria during cheese ripening.

Great interspecies and intraspecies diversity of dairy propionibacteria in the production of cheese aroma compounds

Flavor is an important sensory property of fermented food products, including cheese, and largely results from the production of aroma compounds by microorganisms. Propionibacterium freudenreichii is the most widely used species of dairy propionibacteria; it has been implicated in the production of a wide variety of aroma compounds through multiple metabolic pathways and is associated with the flavor of Swiss cheese. However, the ability of other dairy propionibacteria to produce aroma compounds has not been characterized. This study sought to elucidate the effect of interspecies and intraspecies diversity of dairy propionibacteria on the production of aroma compounds in a cheese context. A total of 76 strains of Propionibacterium freudenreichii, Propionibacterium jensenii, Propionibacterium thoenii, and Propionibacterium acidipropionici were grown for 15days in pure culture in a rich medium derived from cheese curd. In addition, one strain each of two phylogenetically related non-dairy propionibacteria, Propionibacterium cyclohexanicum and Propionibacterium microaerophilum were included. Aroma compounds were analyzed using headspace trap-gas chromatography-mass spectrometry (GC–MS). An analysis of variance performed on GC–MS data showed that the abundance of 36 out of the 45 aroma compounds detected showed significant differences between the cultures. A principal component analysis (PCA) was performed for these 36 compounds. The first two axes of the PCA, accounting for 60% of the variability between cultures, separated P. freudenreichii strains from P. acidipropionici strains and also differentiated P. freudenreichii strains from each other. P. freudenreichii strains were associated with greater concentrations of a variety of compounds, including free fatty acids from lipolysis, ethyl esters derived from these acids, and branched-chain acids and alcohols from amino acid catabolism. P. acidipropionici strains produced less of these compounds but more sulfur-containing compounds from methionine catabolism. Meanwhile, branched-chain aldehydes and benzaldehyde were positively associated with certain strains of P. jensenii and P. thoenii. Moreover, the production of compounds with a common origin was correlated. Compound abundance varied significantly by strain, with fold changes between strains of the same species as high as in the order of 500 for a single compound. This suggests that the diversity of dairy propionibacteria can be exploited to modulate the flavor of mild cheeses.

Inactivation of the panE Gene in Lactococcus lactis Enhances Formation of Cheese Aroma Compounds

Hydroxyacid dehydrogenases limit the conversion of α-keto acids into aroma compounds. Here we report that inactivation of the panE gene, encoding the α-hydroxyacid dehydrogenase activity in Lactococcus lactis, enhanced the formation of 3-methylbutanal and 3-methylbutanol. L. lactis IFPL953ΔpanE was an efficient strain producing volatile compounds related to cheese aroma.

Expression in Lactococcus lactis of functional genes related to amino acid catabolism and cheese aroma formation is influenced by branched chain amino acids

Formation of cheese aroma compounds by Lactococcus lactis from amino acid catabolism depends on a complex network of reactions, which involve enzymes such as aminotransferases, dehydrogenases, lyases, and decarboxylases, among others. Based on the ability of some L. lactis strains to grow with low requirements of amino acids, we have studied in L. lactis IFPL730 the effect of the branched chain amino acid (BCAA) content on the expression of functional genes related to amino acid catabolism and aroma compound formation (araT, bcaT, kivD, ytjE and panE). L. lactis IFPL730 growth rate decreased under leucine, valine or isoleucine starvation but the strain reached similar viable counts at the stationary phase in all culture conditions studied. The level of expression of some genes encoding enzymes involved in amino acid catabolism changed significantly (P<0.05) when those conditions were compared. Specially, α-ketoisovalerate decarboxylase (kivD), BCAA-specific aminotransferase (bcaT) and C-S lyase (yjtE) gene expressions increased markedly by both isoleucine and valine starvation. In addition to gene expression, formation of volatile compounds was determined in all growth conditions. The results showed that BCAA starvation conditions caused a significant increase (P<0.05) in the formation of metabolic end products related to cheese aroma, such as 3-methylbutanal and 3-methylbutanol.

Rerouting citrate metabolism in Lactococcus lactis to citrate-driven transamination.

Oxaloacetate is an intermediate of the citrate fermentation pathway that accumulates in the cytoplasm of Lactococcus lactis ILCitM(pFL3) at a high concentration due to the inactivation of oxaloacetate decarboxylase. An excess of toxic oxaloacetate is excreted into the medium in exchange for citrate by the citrate transporter CitP (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:4049-4056, 2011). In this study, transamination of amino acids with oxaloacetate as the keto donor is described as an additional mechanism to relieve toxic stress. Redirection of the citrate metabolic pathway into the transamination route in the presence of the branched-chain amino acids Ile, Leu, and Val; the aromatic amino acids Phe, Trp, and Tyr; and Met resulted in the formation of aspartate and the corresponding α-keto acids. Cells grown in the presence of citrate showed 3.5 to 7 times higher transaminase activity in the cytoplasm than cells grown in the absence of citrate. The study demonstrates that transaminases of L. lactis accept oxaloacetate as a keto donor. A significant fraction of 2-keto-4-methylthiobutyrate formed from methionine by citrate-driven transamination in vivo was further metabolized, yielding the cheese aroma compounds 2-hydroxy-4-methylthiobutyrate and methyl-3-methylthiopropionate. Reducing equivalents required for the former compound were produced in the citrate fermentation pathway as NADH. Similarly, phenylpyruvate, the transamination product of phenylalanine, was reduced to phenyllactate, while the dehydrogenase activity was not observed for the branched-chain keto acids. Both α-keto acids and α-hydroxy acids are known substrates of CitP and may be excreted from the cell in exchange for citrate or oxaloacetate.

Characterization of aromatic properties of old-style cheese starters

Old-style cheese starters were evaluated to determine their ability to produce cheese aroma compounds. Detailed analyses of the aroma-producing potential of 13 old-style starter cultures were undertaken. The proteolytic profile of the starters was established by an accelerated ripening study using a model cheese slurry and compared with those of a commercial aromatic starter and commercial Cheddar cheeses. To evaluate the aromatic potential of the starter cultures, quantification of free amino acids liberated and volatile compounds after 15 d of ripening at 30°C as well as sensory analysis were carried out. Results showed that proteolysis patterns of all 13 starter cultures in the curd model were comparable to those of commercial Cheddar cheeses. All tested cultures demonstrated the ability to produce high amounts of amino acids recognized as precursors of aroma compounds. Several differences were observed between the starters and commercial Cheddar cheeses regarding some amino acids such as glutamate, leucine, phenylalanine, proline, and ornithine, reflecting the various enzymatic systems present in the starters. Starters Bt (control) and ULAAC-E exhibited various significant differences regarding their free amino acid profiles, as confirmed by sensory analysis. In addition, identification of volatile compounds confirmed the presence of several key molecules related to aroma, such as 3-methylbutanal and diacetyl. Besides the aroma-producing aspect, 2 starters (ULAAC-A and ULAAC-H) seem to possess an important ability to generate large amounts of γ-aminobutyric acid, which contributed up to 15% of the total amino acids present in the model curd after 15 d ripening. γ-Aminobutyric acid is an amine well-known for its antihypertensive and calming effects.

Comparison of purge and trap and solid phase microextraction techniques for studying the volatile aroma compounds of three European PDO hard cheeses

Abstract Solid phase microextraction (SPME) and purge and trap (P&T) methods were compared to establish their effectiveness for the extraction of cheese aroma compounds from selected raw milk protected designation of origin (PDO) cheeses. The profiles of the volatile fraction from each cheese variety obtained by using the two extraction methods were significantly different. SPME fibres were more effective for extracting medium and high boiling compounds, P&T was better for extracting the highly volatile compounds. Gruyere Switzerland cheeses showed high concentrations of alkenes, aldehydes, methyl ketones, butane-2,3-dione, unsaturated alcohols, branched chain acids and 2,6-dimethyl pyrazine. Manchego cheeses contained high concentrations of alkanes, alkanols, prop-2-en-1-ol, propan-2-one and butan-2-one and their corresponding reduction products propan-2-ol and butan-2-ol, propyl esters and aromatic compounds. Ragusano cheeses contained high concentrations of fatty acids and ethyl and butyl esters. Discriminant analyses, performed separately for each extraction method, correctly classified all the samples by their PDO origin.

Pathways for -ketoglutarate formation by Lactococcus lactis and their role in amino acid catabolism

Abstract The production of α -ketoglutarate, which is required for amino acid transamination, by lactic acid bacteria is often a limiting factor in the conversion of amino acids to aroma compounds in cheese. In theory, there are three main pathways for its formation. The direct pathway, involving a glutamate dehydrogenase (GDH), has already been demonstrated to be functional for certain Lactococcus lactis strains. In this work, we explored two other pathways for the formation of α -ketoglutarate by L. lactis and evaluated their impact on amino acid catabolism. The pathway that requires citrate permease (CitP), citrate lyase (CitL) and aspartate aminotransferase was operative for L. lactis subsp. diacetylactis and stimulated the conversion of amino acids. However, the pathway requiring aconitase and isocitrate dehydrogenase activities was never operative; none of the 13 strains tested had detectable aconitase and only one had detectable isocitrate dehydrogenase. Therefore, only the GDH and CitP/CitL pathways can stimulate flavour development during cheese ripening.

Autolysis of Lactococcus lactis AM2 stimulates the formation of certain aroma compounds from amino acids in a cheese model

The autolysis of Lactococcus lactis starters accelerates the formation of free amino acids during cheese ripening. The purpose of this study was to evaluate the impact of L. lactis autolysis on the conversion of free amino acids to aroma compounds. The study was conducted with two L. lactis isogenic strains differing by their autolytic behaviour in cheese: the highly autolytic strain AM2 and a non-autolytic derivative. The effect of bacterial lysis on phenylalanine catabolism was first evaluated in buffer solution. L. lactis cells were also included in a cheese paste model with or without α-ketoglutarate, which stimulates the first step of amino acid degradation by lactococci. Amino acid catabolism and volatile aroma compound formation were analysed after 28 days of ripening. When α-ketoglutarate concentration was not limiting, autolysis of L. lactis cells stimulated the catabolism of aromatic amino acids and methionine and enhanced the formation of sulphur aroma compounds and benzaldehyde, which are desirable aroma compounds in cheese.

Solid phase microextraction (SPME) combined with gas-chromatography and olfactometry-mass spectrometry for characterization of cheese aroma compounds

The applicability of solid phase microextraction (SPME) as a technique for the concentration of cheese aroma for analysis by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry (GC-O) was assessed in this preliminary study. Three each of the following cheese varieties were examined: cheddar, hard grating and mold-ripened blue. Volatile components were concentrated by Carboxen-PDMS SPME fibres for 16 h (overnight) and analysed by GC-MS and GC-O. Odor compounds, which could be perceived at the olfactory port (OP), were matched with electron impact (EI) and methanol chemical ionization (CI) mass spectra. The volatile compounds identified were compared to previously reported cheese aroma compounds. Of the components identified via olfactometry, methanethiol, methional, dimethyl trisulfide and butanoic acid were present in all of the cheeses implying their essential role in the formation of basic cheese aroma. A number of alkyl-pyrazines were also found to impart roasted nutty, raw potato and savoury broth-like notes in some of the cheeses. In all cases, the aroma active compounds identified via olfactometry were in agreement with those reported in the literature. In a separate study, it was demonstrated on a number of cheeses that the adsorption of most important aroma volatiles increased consistently up to 16 h; i.e. sulfur compounds, lactones, pyrazines, phenolic compounds and benzene derivatives. For the strong-smelling pecorino and blue cheeses, however, some analyte displacement effects were observed. Data indicated that a sampling time between 9 and 16 h was appropriate and displacement/competition reactions tended to occur in strong cheeses, i.e. those which have undergone extensive lipolysis e.g. pecorino, very high concentration of butanoic and hexanoic acid, and/or β-oxidation e.g. blue cheese, with high concentrations of 2-heptanone and 2-nonanone.

Lactococcal aminotransferases AraT and BcaT are key enzymes for the formation of aroma compounds from amino acids in cheese

Amino acid catabolism plays a major role in cheese aroma development. Previously, we showed that the lactococcal aminotransferases AraT and BcaT initiate the conversion of aromatic amino acids, branched-chain amino acids and methionine to aroma compounds. In this study, we evaluated the importance of these two enzymes in the formation of aroma compounds in a cheese model by using single araT and bcaT mutants and a double araT/bcaT mutant. We confirmed that addition of α-ketoglutarate, a co-substrate of aminotransferases, stimulates the conversion of amino acids to aroma compounds in cheese. The results demonstrated that AraT and BcaT are essential for conversion of aromatic and branched-chain amino acids to aroma compounds by Lactococcus lactis in the cheese model and that they also play a major role in the formation of volatile sulphur compounds from methionine. However, another pathway or another aminotransferase appears also to be weakly involved in the formation of these sulphur compounds.

Key odorants in various cheese types as determined by gas chromatography-olfactometry

Cheese odour-active compounds identified by gas chromatography-olfactometry (GC-O) are compiled in nine tables dealing with alcohols, aldehydes, ketones, esters, lactones, furans, nitrogen-containing compounds together with pyrazines and sulphur-containing compounds, terpenes and miscellaneous compounds, and, finally, aromatic compounds and free fatty acids. For each component, the odour descriptors, the cheese variety in which it was evaluated, the concentration (or an indication of the odour intensity), the extraction method, the GC-O type of analysis, and, if available, the perception threshold values are also provided. The sensory properties and metabolic origin of major cheese aroma compounds are discussed in the text, explaining to a certain extent the discrepancies between different authors.

Volatile Fraction of Ewe's Milk Semi-Hard Cheese Manufactured with and without the Addition of a Cysteine Proteinase

A dynamic headspace technique (purge and trap) coupled to gas chromatography-mass spectrometry was used for the study of the volatile fraction of pasteurized ewe's milk cheese. The effect of the addition of the cysteine proteinase of Micrococcus sp. INIA 528 to milk on the formation of volatile aroma compounds in cheese was also evaluated. Forty-five compounds, in total, were identified, including hydrocarbons, alcohols, ketones, aldehydes, esters, terpenes and sulfur compounds. The abundance of most volatile compounds increased significantly (P &lt; 0.05) with ripening time, except those of ethanol and 2,3-butanedione which decreased. Acetaldehyde and some minor components did not vary remarkably during ripening. Acetaldehyde, 2-methyl-I-butanal, 3-methyl-I-butanal, 2-propanol, 2-pentanone and 3-methyl-3-buten-1-ol were the only compounds affected by the addition of cysteine proteinase. The more extensive proteolysis in cheese with cysteine proteinase might have enhanced the formation of volatile compounds derived from amino acids, such as acetaldehyde, 2-methyl-1-butanal and 3-methyl-I-butanal, formed from threonine, isoleucine and leucine breakdown, respectively.

Inactivation of lactococcal aromatic aminotransferase prevents the formation of floral aroma compounds from aromatic amino acids in semi-hard cheese

The enzymatic conversion of aromatic amino acids to aroma compounds plays a role in the formation of an undesirable floral aroma in Cheddar-like cheeses. In lactococci, the first step of aromatic amino acid degradation is a transamination, catalysed by an aromatic aminotransferase (AraT). We observed previously that in vitro, araT inactivation prevented degradation of aromatic amino acids and decreased degradation of Met and Leu. In this study we evaluated the effect of araT inactivation in Lactococcus lactis on flavour development in St. Paulin-type cheese. The degradation of amino acids was monitored by using radiolabelled amino acids and the volatile compounds formed were analysed by GC-MS. The development of cheese odour was also evaluated by sniffing. We confirmed that the availability of an α-ketoacid acceptor for transamination is the first limiting factor for amino acid conversion to aroma compounds in cheese. In the presence of α-ketoglutarate, araT inactivation greatly prevented formation of floral aroma compounds from aromatic amino acids while it did not affect the formation of volatile aroma compounds from branched-chain amino acids and methionine. However, the sensory analysis by sniffing did not reveal any significant effect of the gene inactivation although the odour of cheese made with the mutant tended to be less floral than that of cheese made with the wild type strain.