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Effect of α-keto acids on the development of flavour in Swiss Gruyere-type cheese
ARTICLE IN PRESS
Lebensm.-Wiss. u.-Technol. 37 (2004) 269–273
Effect of a-keto acids on the development of flavour in Swiss Gruyere-type cheese . . M.G. Casey*, J.O. Bosset, U. Butikofer, Marie-Therese Frohlich-Wyder Microbiology, Swiss Federal Research Station, FAM, Liebefeld, Bern 3003, Switzerland Received 19 June 2003; received in revised form 9 September 2003; accepted 10 September 2003
Abstract The addition of pyruvate to Gruyere-type cheese milk significantly increased cheese aroma. This effect was accompanied by a decrease in the concentration of some amino acids, especially leucine, lysine, methionine, phenylalanine and valine. There was a corresponding increase in aroma components derived from the catabolism of amino acids, such as isobutyrate and isovalerate. Pyruvate also contributed to the aroma of cheese through the production of other compounds since significantly greater amounts of catabolites such as butyrate, and diacetyl were also found in these cheeses. The contribution of pyruvate to the development of cheese aroma is discussed. r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: a-Keto acid; a-Ketoglutarate; Pyruvate; Amino acids; Gruyere-type cheese; Cheese aroma
1. Introduction The characteristic flavour of Swiss cheese was originally attributed to its proline content (Virtanen & Kreula, 1948). Later, a new concept was developed by Kosikowski and Mocquot (1958) who stated that relatively few compounds were responsible for the differences in flavour between the various cheese varieties. Generally, it was considered that proteolysis was the rate-limiting step in flavour development. Nevertheless, when genetically modified lactic acid bacteria with high aminopeptidase activity were used to manufacture cheese, the concentration of free amino acids increased, as expected, but there was no effect on flavour development (McGarry et al., 1994). Until recently, amino acid metabolism was not considered to be the major factor contributing to cheese flavour. The presence of keto acids in Swiss cheese was described by Bassett and Harper (1958); however, later it was considered that the lactic acid bacteria were not active in amino acid degradation (Law & Kolstad, 1983). The recent work by Yvon, Berthelot, and Gripon (1998) showing the importance of amino acid metabolism in contributing to cheese flavour has changed this *Corresponding author. Tel.: +41-31-3238179; fax: +41-313238227. E-mail address: [email protected] (M.G. Casey).
view. When a-ketoglutarate, an a-keto acid acceptor for amino acid transamination, was added to the curd of St. Paulin-type cheese it enhanced the conversion of amino acids to aroma compounds. Similar results were obtained with Cheddar cheese (Banks et al., 2001; Shakeel-Ur-Rehman & Fox, 2002). St. Paulin and Cheddar cheeses are both produced using the mesophilic starter, Lactococcus lactis, whereas Swiss Gruyere cheese is manufactured using the thermophilic starters Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis. The aim of the present study was to determine whether the same enhancement in cheese flavour could be obtained by aketo acids in Swiss Gruyere-type cheese. Since the concentration of the a-keto acid, pyruvate is higher in Swiss cheese than that of a-ketoglutarate (Schormuller . & Langner, 1960), the effect of adding this compound was studied also.
2. Materials and methods Di-sodium-a-ketoglutarate, sodium pyruvate and all standard chemicals were obtained from Merck, Darmstadt. Model Gruyere-type cheeses were produced from 100 l of raw milk. After the addition of 100 ml each of a
0023-6438/$30.00 r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2003.09.002
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fresh (pH 4.9) and an old (pH 4.25) starter culture, the milk was pre-ripened at 31–32 C for 30. Coagulation occurred at 32 C for 40 min. Cutting and stirring at a constant temperature (32 C) was followed by scalding to 56 C for 30 min. Before filling the curd into the moulds, the curds-whey mixture was stirred at 56 C for 20 min. Brining was followed by a ripening in the cold room (14–15 C, 90–96% relative humidity) for 10 days and then in the warm room (16–17 C, 90–96% relative humidity) for 50 days. Final maturation was at 14–15 C (90–96% relative humidity) for 120 days. The a-keto acids were added directly to the milk before the start of manufacture and the pH adjusted, if necessary. Two cheeses were produced to which aketoglutarate was added at a concentration of either 55 or 82 mmol l1. In the cheese, a concentration of 6.94 and 12.79 mmol g1, respectively, was obtained. Five cheeses each were produced with or without the addition of pyruvate at a concentration of 40 mmol l1 milk. Free amino acids were determined in all cheeses by the method of Butikofer . and Ardo. (1999). The concentration of a-ketoglutarate was determined by HPLC, as described by Yvon et al. (1998). Volatile free fatty acids were determined according to Badertscher, Liniger, and Steiger (1993). The volatile aroma components were determined according to Bosset, Butikofer, . Gauch, and Sieber (1997). Sensory analysis of the experimental cheeses was performed at the pilot plant of the FAM and the sensory quality judged individually by a panel of 8 experts from our research station according to a standard protocol. This analysis is used for official grading of cheeses in Switzerland. The following characteristics were judged: overall impression with a score between 1 (lowest) and 6 (highest); aroma intensity with a score between 1 (tasteless) and 7 (very aromatic); aroma quality with a score between 1 (lowest) and 6 (highest); eye number with a score between 0 (no eyes) and 5 (too many); eye quality with a score between 1 (lowest) and 6 (highest); texture quality with a score between 1 (lowest) and 6 (highest) and body elasticity with a score between 1 (very brittle) and 7 (very elastic). For each cheese sample, the average of the scores was calculated and used for the following statistical evaluation. Statistical evaluation (t-test, two-sided, paired) was carried out with the program SYSTAT (SPSS Inc., Chicago, USA).
3. Results 3.1. Addition of a-ketoglutarate The effect of adding a-ketoglutarate to the cheese milk at either 55 or 82 mmol l2 on the ratio of the concentration of free amino acids to free proline after 90
days of ripening is shown in Table 1. This method of expressing the results was chosen since the concentration of free amino acids in cheese can vary from one batch to another, making interpretation of the results difficult. Proline was chosen as the reference because transamination of proline cannot occur since it is an imino acid. The addition of a-ketoglutarate to cheese milk reduced the level of some amino acids while others were not affected. Higher levels of a-ketoglutarate induced a greater decrease in the levels of the same amino acids. The short chain free fatty acids, a-ketoglutarate and intermediary metabolite content of the cheeses after 90 days ripening are shown in Table 2. The a-keto acid brought about an increase in the level of several metabolites, especially acetate, butyrate, isobutyrate, isovalerate and g-aminobutyrate. The concentration of propionate decreased with increasing level of the a-keto acid. The addition of 55 mmol l2 a-ketoglutarate to cheese milk led to a significantly perceptible increase of the aroma intensity of the Gruyere-type cheese, similarly to Cheddar and St. Paulin. At the age of 10 weeks, the control cheese was given an average score of 1.370.4 and the above-mentioned cheese 3.170.7, which is more than the double. The addition of a higher concentration of the a-ketoglutarate to cheese milk, i.e. 82 mmol l2, did not further influence the sensory characteristics. 3.2. Addition of pyruvate The effect of adding pyruvate on the ratio of concentration of free amino acids to free proline after Table 1 Ratio of the concentration (mmol kg1) of free amino acids to free proline ( 1000) in Gruyere-type cheese, with and without (control) addition of a-ketoglutarate at a concentration of 55 mmol or 82 mmol l1 after 90 days of ripening
Alanine Aspartic acid Glutamine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Serine Threonine Tryptophan Tyrosine Valine
Control (n ¼ 1)
55 mmol l1 (n ¼ 1)
82 mmol l1 (n ¼ 1)
502 151 463 2055 409 356 469 1582 1678 358 618 407 530 37 302 1068
376 210 509 2521 441 341 448 1333 1347 264 516 405 535 32 287 942
324 216 497 2349 401 385 432 1153 1380 227 441 402 497 35 276 844
Systematic differences above or below 10% are in boldface (n=number of cheeses).
ARTICLE IN PRESS M.G. Casey et al. / Lebensm.-Wiss. u.-Technol. 37 (2004) 269–273 Table 2 Volatile acids, a-ketoglutarate and intermediary metabolite content of Gruyere-type cheeses with and without (control) the addition of a-ketoglutarate at a concentration of 55 mmol or 82 mmol l1 after 90 days ripening
a-Ketoglutaratea g-Aminobutyrate Acetate n-Butyrate Caproate Iso-butyrate Iso-valerate Propionate Citrate Pyruvate Succinate
Control (n ¼ 1)
55 mmol l1 (n ¼ 1)
82 mmol l1 (n ¼ 1)
0.00 0.07 3.46 0.67 0.11 0.03 0.04 0.69 8.40 0.04 1.05
0.79 4.50 7.60 1.06 0.14 0.05 0.09 0.50 5.30 0.12 1.50
1.42 8.44 9.78 1.18 0.14 0.07 0.12 0.35 6.20 0.19 1.54
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Table 4 Volatile acid content of Gruyere-type cheeses with and without (control) addition of pyruvate at a concentration of 40 mmol l1 after 90 days ripening. Results are expressed as mmol kg1 cheese water (n=number of cheeses)
Acetate Propionate Butyrate Caproate Iso-butyrate Iso-caproate Iso-valerate
Control (n ¼ 5)
With pyruvate (n ¼ 5)
t-test
4.8172.53 1.4170.72 0.6670.18 0.1170.02 0.0570.02 0.0570.04 0.0470.02
7.9272.69 1.9771.43 1.3870.34 0.1470.02 0.2170.10 0.1070.05 0.2370.13
n.s.=not significant; Po0:05; Po0:01; t-test=two-sided, paired.
Systematic differences above or below 10% are in boldface. Results are expressed as mmol kg1 cheese (n=number of cheeses) a In comparison, after 24 h the following contents of a-ketoglutarate were detected: 0.00; 6.94; and 12.79.
Table 5 Sensory characteristics of Gruyere-type control cheeses (n ¼ 5) and of cheeses with addition of pyruvate (n ¼ 5) at a concentration of 40 mmol l1 (n=number of cheeses) Control
Table 3 Ratio of concentration (mmol kg1) of free amino acids to free proline ( 1000) in Gruyere-type cheeses with and without (control) addition of pyruvate at a concentration of 40 mmol l1 after 90 days ripening (n=number of cheeses)
Alanine Aspartic acid Glutamine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Serine Threonine Tryptophan Tyrosine Valine
n.s.
Control (n ¼ 5)
With pyruvate (n ¼ 5)
t-test
464 214 385 1905 402 300 442 1567 1482 350 609 297 459 40 284 1046
420 207 411 1757 370 298 466 1333 1343 307 550 297 447 35 268 964
n.s.
n.s.
n.s. n.s.
n.s.=not significant; Po0:05; Po0:01; Po0:001; t-test=twosided, paired.
90 days of ripening is shown in Table 3. The addition of pyruvate to cheese milk significantly decreased the content of some free amino acids, especially leucine, lysine, methionine, phenylalanine and valine. The addition of pyruvate to cheese milk brought about an increase in the levels of some volatile acids in cheese (Table 4). The effect of pyruvate on the sensory characteristics are shown in Table 5. The addition of pyruvate increased significantly the overall impression and
Overall impression (1–6) Aroma intensity (1–7) Aroma quality (1–6) Eye number (0–5) Eye quality (1–6) Texture quality (1–6) Body elasticity (1–7)
4.3370.32 2.8170.46 4.2670.42 0.5070.63 5.4370.27 5.1070.33 5.4470.85
With pyruvate
t-test
5.2370.36 4.4770.32 5.1570.34 1.7370.47 5.7770.26 5.3570.26 5.2570.55
n.s. n.s.
Overall impression: 1 (lowest)–6 (best); aroma intensity: 1(tasteless)–7 (very aromatic); aroma quality: 1 (worst)–6 (best); eye number: 0 (no eyes) and 5 (overloaded); eye quality: 1 (lowest)–6 (best); texture quality: 1 (lowest)–6 (best); body elasticity: 1 (very brittle)–7 (very elastic); n.s.=not significant; Po0:05; Po0:01; Po0:001; ttest=two-sided, paired.
Table 6 Volatile components of Gruyere-type cheeses with and without addition of pyruvate at a concentration of 40 mmol l1 after 90 days of ripening. Only components the value of which changed by more than a factor of 1.5 are listed Volatile substance
Ratio with/without Addition of pyruvate
Possible origin
Diacetyl 2-Methyl-propanal 3-Methyl-butanal 3-Methyl-2-butanone 2-Methyl-butanal Dimethyl-disulphide 1-Butanol
6.7 1.9 2.4 2.1 1.9 10.6 2.5
Pyruvate Valine Leucine Leucine Isoleucine Methionine Pyruvate
especially the aroma of the cheese. The sensory panel estimated in a discussion by consensus that the aroma of cheese, with added pyruvate, was that of a product that had ripened for an additional 60 days. There was also a significant increase in the number of eyes in the cheese.
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Volatile aroma component analysis was performed on cheeses with and without the addition of pyruvate. Since absolute values for the different components were not determined, only the ratio of peak height from both types of cheeses was considered. For the current study it was not necessary to quantify the components analysed. The peak height ratios were sufficient to investigate the effect of the addition of pyruvate on such components. To take into account the uncertainty (710%) of the semi-quantitative determinations of the volatile compounds considered only values which changed by more than a factor of 1.5 were considered significant (Table 6).
4. Discussion Yvon et al. (1998) showed that the first limiting factor for the conversion of amino acids to aroma compounds in cheese was the lack of an a-keto receptor for amino acid transamination. They found that the addition of aketoglutarate to cheese enhanced its aroma by increasing the degradation of aromatic and branched-chain amino acids and methionine. In the present study it was shown that this keto acid significantly increased the aroma of Gruyere-type cheese, however, we found that the pattern of amino acid degradation was slightly different from St. Paulin cheese. Both alanine and lysine were also degraded whereas isoleucine and tyrosine were not affected. These results are not unexpected since the starters used for the manufacture of Gruyere-type cheese contain the thermophilic bacteria S. thermophilus and Lactobacillus delbrueckii ssp. lactis in contrast to the mesophilic starter Lactococcus lactis used for the manufacture of St. Paulin cheese. The above-mentioned authors also found that there was an equivalent increase in the concentration of glutamate in the cheese body and it was suggested that this was due to the transamination of an amino group to a-ketoglutarate. In Gruyere-type cheese we did not observe an increase in the concentration of glutamate but there was a rise in the concentration of gaminobutyrate. Similar high concentrations of g-aminobutyrate were also noted in Cheddar cheese after the addition of a-ketoglutarate (Banks et al., 2001). Since gaminobutyrate can be formed from glutamate by decarboxlyation it is possible that in Gruyere-type cheese a-ketoglutarate is first converted to glutamate by transamination and that this is followed by decarboxylation. The presence of glutamate decarboxylase has been described in lactic acid bacteria (Ueno, Hayakawa, Takahashi, & Oda, 1997; Nomura, Kimoto, Someya, Furukawa, & Suzuki, 1998). The concentration of acetate was found to increase in cheese after the addition of a-ketoglutarate. This could result from the catabolism of alanine by an amino-
transferase leading to the production of pyruvate, which can be converted to acetate by oxidative decarboxylation. Unfortunately, Yvon et al. (1998) did not provide values for the concentration of acetate in St. Paulin cheese. The catabolism of the branched-chain amino acids by an aminotransferase converts leucine and valine to aketoisocaproate and a-ketoisovalerate, respectively. Oxidative decarboxylation of these keto acids leads to the production of isovalerate and isobutyrate. The concentration of the two volatile branched-chain acids were found to increase in cheese after the addition of aketoglutarate. This increase in concentration confirms that amino acid degradation is greater in cheese after the addition of an a-keto acid even if the increase in the concentration of branched chain acids is much lower than the corresponding decrease in branch chain amino acids. Beuvier et al. (1997) also found that Swiss-type cheeses with an higher overall aroma intensity also contained higher concentrations of isovalerate. Butyrate, another potent odorant, also increases in cheese with added a-keto acid. Since g-aminobutyrate is not normally found in Gruyere-type cheese at mmolar concentrations, it was concluded that a-ketoglutarate is not the acceptor for transamination under normal ripening conditions. Normally, a-ketoglutarate is formed from isocitrate by isocitrate dehydrogenase, however, this enzyme has not been detected in lactic acid bacteria (Morishita & Yajima, 1995). a-Keto acid pyruvate seemed to be a more promising candidate since it is possible that lysis of some bacteria could take place during ripening and perhaps release pyruvate into the cheese mass. Our results show that the addition of pyruvate to cheese milk significantly increased the aroma of the Gruye" re-type cheese and that this was accompanied by a decrease in the concentrations of some amino acids, especially leucine, lysine, methionine, phenylalanine and valine. There were also significantly greater amounts of acetate, butyrate, isobutyrate, isovalerate and diacetyl in the cheeses which would explain their greater aroma. The increased concentration of acetate probably arises directly from the oxidative decarboxylation of pyruvate. The high concentration of diacetyl is most likely due to the production of a-acetolactate from pyruvate followed by its decarboxylation. The production of diacetyl from pyruvate, by lactic acid bacteria has been described previously (Ramos, Jordan, Cogan, & Santos, 1994). The higher concentrations of isobutyrate and isovalerate indicate that there was an increase in the degradation of leucine and valine. There was also an increase in other aroma compounds in the cheeses. The majority of these are derived from the metabolism of amino acids. 3-Methyl-butanal and 3-methyl-2-butanone result from the reduction of isovalerate which is derived from leucine. Further
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reduction of 3-methyl-butanal yields 3-methyl-butanol. 2-Methyl-propanal results from the reduction of isobutyrate which is derived from valine. Dimethyldisulphide certainly derive principally from methionine since there are much higher concentrations of this amino acid in cheese than cysteine. The increased levels of butyrate and 1-butanol probably derive from the catabolism of pyruvate and do not involve amino acid metabolism.
5. Conclusion The role of a-keto acids in the production of aromatic substances in cheese, as suggested by Yvon et al. (1998), has been confirmed; however, in Gruyere-type cheese, aketoglutarate is unlikely to be the acceptor for transamination. The addition of pyruvate to cheese milk increased the aroma not only by increasing the catabolism of amino acids but also by intensifiying the production of other aromatic compounds such as diacetyl and butyrate. Thus, the development of flavour in Gruyere-type cheese appears to be a complex process involving not only the degradation of amino acids but also the formation of other aromatic substances. Further work will be necessary to determine which other compounds lead to the production of aroma in cheese.
References Badertscher, R., Liniger, A., & Steiger, G., (1993). Bestimmung der fluchtigen Fettsauren in Kase . . . aus dem Wasserdampfdestillat mit ‘‘Headspace-GC/FID’’. FAM INFO Number 272, Liebefeld-Berne, Switzerland. Banks, J. M., Yvon, M., Grippon, J. C., de la Fuente, M. A., Brechany, E. Y., Williams, A. G., & Muir, D. D. (2001). Enhancement of amino acid catabolism in Cheddar cheese using a-ketoglutarate: amino acid degradation in relation to volatile compounds and aroma character. International Dairy Journal, 11, 235–243. Bassett, E. W., & Harper, W. J. (1958). Isolation and identification of acidic and neutral carbonyl compounds in different cheese varieties of cheese. Journal of Dairy Science, 41, 1206–1217.
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Beuvier, E., Berthaud, K., Cegarra, S., Dasen, A., Pochet, S., Buchin, S., & Duboz, G. (1997). Ripening and quality of Swiss-type cheese made from raw, pasteurized or microfiltrated milk. International Dairy Journal, 7, 311–323. Bosset, J. O., Butikofer, . U., Gauch, R., & Sieber, R. (1997). Reifungsverlauf von in Folien verpacktem Emmentaler k.ase mit und ohne Zutaz von Lactobacillus casei susp. casei. II. Gaschromatographische Untersuchung einiger fluchtiger, . neutraler Verbindungen mit Hilfe einer dynamischen Dampfraumanalyse. Lebensmittelwissenschaft und Technologie, 30, 464–470. . Y. (1999). Quantitative determination of free Butikofer, . U., & Ardo, amino acids in cheese. Bulletin International Dairy Federation, 337, 24–32. Kosikowski, F. V., & Mocquot, G. (1958). Advances in cheese technology. Food and Agricultural Organisation, United Nations Agricultural Study No. 38 (141pp.). Law, B. A., & Kolstad, J. (1983). Proteolytic systems in lactic acid bacteria. Antonie van Leeuwenhoek, 49, 225–245. McGarry, A., Law, J., Coffey, A., Daly, C., Fox, P. F., & Fitzgerald, G. F. (1994). Effect of genetically modifying the lactococcal proteolytic system on ripening and flavour development in Cheddar cheese. Applied and Environmental Microbiology, 60, 4226–4233. Morishita, T., & Yajima, M. (1995). Incomplete operation of biosynthetic and bioenergetic functions of the citric acid cycle in multiple auxotrophic Lactobacilli. Bioscience, Biotechnology and Biochemistry, 59, 251–255. Nomura, M., Kimoto, H., Someya, Y., Furukawa, S., & Suzuki, I. (1998). Production of gamma-aminobutyric acid by cheese starters during cheese ripening. Journal of Dairy Science, 81, 1486–1491. Ramos, A., Jordan, K. N., Cogan, T. M., & Santos, H. 13 (1994). C Nuclear magnetic resonance studies of citrate and glucose cometabolism by Lactococcus lactis. Applied and Environmental Microbiology, 60, 1739–1748. . Schormuller, . J., & Langner, H. (1960). Uber die organischen S.auren verschiedener K.asearten. Zeitschrift fur . Lebensmitteluntersuchung und Forschung, 113, 289–298. Shakeel-Ur-Rehman, & Fox, P. F. (2002) Effect of added alphaketoglutaric acid, pyruvic acid or pyridoxal phosphate on proteolyis and quality of Cheddar cheese. Food Chemistry, 76, 21–26. Ueno, Y., Hayakawa, K., Takahashi, S., & Oda, K. (1997). Purification and characterization of glutamate decarboxylase from Lactobacillus brevis IFO 12005. Bioscience, Biotechnology and Biochemistry, 61, 1168–1171. Virtanen, A. L., & Kreula, M. (1948). On the significance of amino acids for the taste of Emmenthaler cheese. Meijeritieteellinen Aikakauskirja, 10, 13–23. Yvon, M., Berthelot, S., & Gripon, J. C. (1998). Adding aketoglutarate to semi-hard cheese curd highly enhances the conversion of amino acids to aroma compounds. International Dairy Journal, 8, 889–898.
Lebensm.-Wiss. u.-Technol. 37 (2004) 269–273
Effect of a-keto acids on the development of flavour in Swiss Gruyere-type cheese . . M.G. Casey*, J.O. Bosset, U. Butikofer, Marie-Therese Frohlich-Wyder Microbiology, Swiss Federal Research Station, FAM, Liebefeld, Bern 3003, Switzerland Received 19 June 2003; received in revised form 9 September 2003; accepted 10 September 2003
Abstract The addition of pyruvate to Gruyere-type cheese milk significantly increased cheese aroma. This effect was accompanied by a decrease in the concentration of some amino acids, especially leucine, lysine, methionine, phenylalanine and valine. There was a corresponding increase in aroma components derived from the catabolism of amino acids, such as isobutyrate and isovalerate. Pyruvate also contributed to the aroma of cheese through the production of other compounds since significantly greater amounts of catabolites such as butyrate, and diacetyl were also found in these cheeses. The contribution of pyruvate to the development of cheese aroma is discussed. r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: a-Keto acid; a-Ketoglutarate; Pyruvate; Amino acids; Gruyere-type cheese; Cheese aroma
1. Introduction The characteristic flavour of Swiss cheese was originally attributed to its proline content (Virtanen & Kreula, 1948). Later, a new concept was developed by Kosikowski and Mocquot (1958) who stated that relatively few compounds were responsible for the differences in flavour between the various cheese varieties. Generally, it was considered that proteolysis was the rate-limiting step in flavour development. Nevertheless, when genetically modified lactic acid bacteria with high aminopeptidase activity were used to manufacture cheese, the concentration of free amino acids increased, as expected, but there was no effect on flavour development (McGarry et al., 1994). Until recently, amino acid metabolism was not considered to be the major factor contributing to cheese flavour. The presence of keto acids in Swiss cheese was described by Bassett and Harper (1958); however, later it was considered that the lactic acid bacteria were not active in amino acid degradation (Law & Kolstad, 1983). The recent work by Yvon, Berthelot, and Gripon (1998) showing the importance of amino acid metabolism in contributing to cheese flavour has changed this *Corresponding author. Tel.: +41-31-3238179; fax: +41-313238227. E-mail address: [email protected] (M.G. Casey).
view. When a-ketoglutarate, an a-keto acid acceptor for amino acid transamination, was added to the curd of St. Paulin-type cheese it enhanced the conversion of amino acids to aroma compounds. Similar results were obtained with Cheddar cheese (Banks et al., 2001; Shakeel-Ur-Rehman & Fox, 2002). St. Paulin and Cheddar cheeses are both produced using the mesophilic starter, Lactococcus lactis, whereas Swiss Gruyere cheese is manufactured using the thermophilic starters Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis. The aim of the present study was to determine whether the same enhancement in cheese flavour could be obtained by aketo acids in Swiss Gruyere-type cheese. Since the concentration of the a-keto acid, pyruvate is higher in Swiss cheese than that of a-ketoglutarate (Schormuller . & Langner, 1960), the effect of adding this compound was studied also.
2. Materials and methods Di-sodium-a-ketoglutarate, sodium pyruvate and all standard chemicals were obtained from Merck, Darmstadt. Model Gruyere-type cheeses were produced from 100 l of raw milk. After the addition of 100 ml each of a
0023-6438/$30.00 r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2003.09.002
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fresh (pH 4.9) and an old (pH 4.25) starter culture, the milk was pre-ripened at 31–32 C for 30. Coagulation occurred at 32 C for 40 min. Cutting and stirring at a constant temperature (32 C) was followed by scalding to 56 C for 30 min. Before filling the curd into the moulds, the curds-whey mixture was stirred at 56 C for 20 min. Brining was followed by a ripening in the cold room (14–15 C, 90–96% relative humidity) for 10 days and then in the warm room (16–17 C, 90–96% relative humidity) for 50 days. Final maturation was at 14–15 C (90–96% relative humidity) for 120 days. The a-keto acids were added directly to the milk before the start of manufacture and the pH adjusted, if necessary. Two cheeses were produced to which aketoglutarate was added at a concentration of either 55 or 82 mmol l1. In the cheese, a concentration of 6.94 and 12.79 mmol g1, respectively, was obtained. Five cheeses each were produced with or without the addition of pyruvate at a concentration of 40 mmol l1 milk. Free amino acids were determined in all cheeses by the method of Butikofer . and Ardo. (1999). The concentration of a-ketoglutarate was determined by HPLC, as described by Yvon et al. (1998). Volatile free fatty acids were determined according to Badertscher, Liniger, and Steiger (1993). The volatile aroma components were determined according to Bosset, Butikofer, . Gauch, and Sieber (1997). Sensory analysis of the experimental cheeses was performed at the pilot plant of the FAM and the sensory quality judged individually by a panel of 8 experts from our research station according to a standard protocol. This analysis is used for official grading of cheeses in Switzerland. The following characteristics were judged: overall impression with a score between 1 (lowest) and 6 (highest); aroma intensity with a score between 1 (tasteless) and 7 (very aromatic); aroma quality with a score between 1 (lowest) and 6 (highest); eye number with a score between 0 (no eyes) and 5 (too many); eye quality with a score between 1 (lowest) and 6 (highest); texture quality with a score between 1 (lowest) and 6 (highest) and body elasticity with a score between 1 (very brittle) and 7 (very elastic). For each cheese sample, the average of the scores was calculated and used for the following statistical evaluation. Statistical evaluation (t-test, two-sided, paired) was carried out with the program SYSTAT (SPSS Inc., Chicago, USA).
3. Results 3.1. Addition of a-ketoglutarate The effect of adding a-ketoglutarate to the cheese milk at either 55 or 82 mmol l2 on the ratio of the concentration of free amino acids to free proline after 90
days of ripening is shown in Table 1. This method of expressing the results was chosen since the concentration of free amino acids in cheese can vary from one batch to another, making interpretation of the results difficult. Proline was chosen as the reference because transamination of proline cannot occur since it is an imino acid. The addition of a-ketoglutarate to cheese milk reduced the level of some amino acids while others were not affected. Higher levels of a-ketoglutarate induced a greater decrease in the levels of the same amino acids. The short chain free fatty acids, a-ketoglutarate and intermediary metabolite content of the cheeses after 90 days ripening are shown in Table 2. The a-keto acid brought about an increase in the level of several metabolites, especially acetate, butyrate, isobutyrate, isovalerate and g-aminobutyrate. The concentration of propionate decreased with increasing level of the a-keto acid. The addition of 55 mmol l2 a-ketoglutarate to cheese milk led to a significantly perceptible increase of the aroma intensity of the Gruyere-type cheese, similarly to Cheddar and St. Paulin. At the age of 10 weeks, the control cheese was given an average score of 1.370.4 and the above-mentioned cheese 3.170.7, which is more than the double. The addition of a higher concentration of the a-ketoglutarate to cheese milk, i.e. 82 mmol l2, did not further influence the sensory characteristics. 3.2. Addition of pyruvate The effect of adding pyruvate on the ratio of concentration of free amino acids to free proline after Table 1 Ratio of the concentration (mmol kg1) of free amino acids to free proline ( 1000) in Gruyere-type cheese, with and without (control) addition of a-ketoglutarate at a concentration of 55 mmol or 82 mmol l1 after 90 days of ripening
Alanine Aspartic acid Glutamine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Serine Threonine Tryptophan Tyrosine Valine
Control (n ¼ 1)
55 mmol l1 (n ¼ 1)
82 mmol l1 (n ¼ 1)
502 151 463 2055 409 356 469 1582 1678 358 618 407 530 37 302 1068
376 210 509 2521 441 341 448 1333 1347 264 516 405 535 32 287 942
324 216 497 2349 401 385 432 1153 1380 227 441 402 497 35 276 844
Systematic differences above or below 10% are in boldface (n=number of cheeses).
ARTICLE IN PRESS M.G. Casey et al. / Lebensm.-Wiss. u.-Technol. 37 (2004) 269–273 Table 2 Volatile acids, a-ketoglutarate and intermediary metabolite content of Gruyere-type cheeses with and without (control) the addition of a-ketoglutarate at a concentration of 55 mmol or 82 mmol l1 after 90 days ripening
a-Ketoglutaratea g-Aminobutyrate Acetate n-Butyrate Caproate Iso-butyrate Iso-valerate Propionate Citrate Pyruvate Succinate
Control (n ¼ 1)
55 mmol l1 (n ¼ 1)
82 mmol l1 (n ¼ 1)
0.00 0.07 3.46 0.67 0.11 0.03 0.04 0.69 8.40 0.04 1.05
0.79 4.50 7.60 1.06 0.14 0.05 0.09 0.50 5.30 0.12 1.50
1.42 8.44 9.78 1.18 0.14 0.07 0.12 0.35 6.20 0.19 1.54
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Table 4 Volatile acid content of Gruyere-type cheeses with and without (control) addition of pyruvate at a concentration of 40 mmol l1 after 90 days ripening. Results are expressed as mmol kg1 cheese water (n=number of cheeses)
Acetate Propionate Butyrate Caproate Iso-butyrate Iso-caproate Iso-valerate
Control (n ¼ 5)
With pyruvate (n ¼ 5)
t-test
4.8172.53 1.4170.72 0.6670.18 0.1170.02 0.0570.02 0.0570.04 0.0470.02
7.9272.69 1.9771.43 1.3870.34 0.1470.02 0.2170.10 0.1070.05 0.2370.13
n.s.=not significant; Po0:05; Po0:01; t-test=two-sided, paired.
Systematic differences above or below 10% are in boldface. Results are expressed as mmol kg1 cheese (n=number of cheeses) a In comparison, after 24 h the following contents of a-ketoglutarate were detected: 0.00; 6.94; and 12.79.
Table 5 Sensory characteristics of Gruyere-type control cheeses (n ¼ 5) and of cheeses with addition of pyruvate (n ¼ 5) at a concentration of 40 mmol l1 (n=number of cheeses) Control
Table 3 Ratio of concentration (mmol kg1) of free amino acids to free proline ( 1000) in Gruyere-type cheeses with and without (control) addition of pyruvate at a concentration of 40 mmol l1 after 90 days ripening (n=number of cheeses)
Alanine Aspartic acid Glutamine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Serine Threonine Tryptophan Tyrosine Valine
n.s.
Control (n ¼ 5)
With pyruvate (n ¼ 5)
t-test
464 214 385 1905 402 300 442 1567 1482 350 609 297 459 40 284 1046
420 207 411 1757 370 298 466 1333 1343 307 550 297 447 35 268 964
n.s.
n.s.
n.s. n.s.
n.s.=not significant; Po0:05; Po0:01; Po0:001; t-test=twosided, paired.
90 days of ripening is shown in Table 3. The addition of pyruvate to cheese milk significantly decreased the content of some free amino acids, especially leucine, lysine, methionine, phenylalanine and valine. The addition of pyruvate to cheese milk brought about an increase in the levels of some volatile acids in cheese (Table 4). The effect of pyruvate on the sensory characteristics are shown in Table 5. The addition of pyruvate increased significantly the overall impression and
Overall impression (1–6) Aroma intensity (1–7) Aroma quality (1–6) Eye number (0–5) Eye quality (1–6) Texture quality (1–6) Body elasticity (1–7)
4.3370.32 2.8170.46 4.2670.42 0.5070.63 5.4370.27 5.1070.33 5.4470.85
With pyruvate
t-test
5.2370.36 4.4770.32 5.1570.34 1.7370.47 5.7770.26 5.3570.26 5.2570.55
n.s. n.s.
Overall impression: 1 (lowest)–6 (best); aroma intensity: 1(tasteless)–7 (very aromatic); aroma quality: 1 (worst)–6 (best); eye number: 0 (no eyes) and 5 (overloaded); eye quality: 1 (lowest)–6 (best); texture quality: 1 (lowest)–6 (best); body elasticity: 1 (very brittle)–7 (very elastic); n.s.=not significant; Po0:05; Po0:01; Po0:001; ttest=two-sided, paired.
Table 6 Volatile components of Gruyere-type cheeses with and without addition of pyruvate at a concentration of 40 mmol l1 after 90 days of ripening. Only components the value of which changed by more than a factor of 1.5 are listed Volatile substance
Ratio with/without Addition of pyruvate
Possible origin
Diacetyl 2-Methyl-propanal 3-Methyl-butanal 3-Methyl-2-butanone 2-Methyl-butanal Dimethyl-disulphide 1-Butanol
6.7 1.9 2.4 2.1 1.9 10.6 2.5
Pyruvate Valine Leucine Leucine Isoleucine Methionine Pyruvate
especially the aroma of the cheese. The sensory panel estimated in a discussion by consensus that the aroma of cheese, with added pyruvate, was that of a product that had ripened for an additional 60 days. There was also a significant increase in the number of eyes in the cheese.
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Volatile aroma component analysis was performed on cheeses with and without the addition of pyruvate. Since absolute values for the different components were not determined, only the ratio of peak height from both types of cheeses was considered. For the current study it was not necessary to quantify the components analysed. The peak height ratios were sufficient to investigate the effect of the addition of pyruvate on such components. To take into account the uncertainty (710%) of the semi-quantitative determinations of the volatile compounds considered only values which changed by more than a factor of 1.5 were considered significant (Table 6).
4. Discussion Yvon et al. (1998) showed that the first limiting factor for the conversion of amino acids to aroma compounds in cheese was the lack of an a-keto receptor for amino acid transamination. They found that the addition of aketoglutarate to cheese enhanced its aroma by increasing the degradation of aromatic and branched-chain amino acids and methionine. In the present study it was shown that this keto acid significantly increased the aroma of Gruyere-type cheese, however, we found that the pattern of amino acid degradation was slightly different from St. Paulin cheese. Both alanine and lysine were also degraded whereas isoleucine and tyrosine were not affected. These results are not unexpected since the starters used for the manufacture of Gruyere-type cheese contain the thermophilic bacteria S. thermophilus and Lactobacillus delbrueckii ssp. lactis in contrast to the mesophilic starter Lactococcus lactis used for the manufacture of St. Paulin cheese. The above-mentioned authors also found that there was an equivalent increase in the concentration of glutamate in the cheese body and it was suggested that this was due to the transamination of an amino group to a-ketoglutarate. In Gruyere-type cheese we did not observe an increase in the concentration of glutamate but there was a rise in the concentration of gaminobutyrate. Similar high concentrations of g-aminobutyrate were also noted in Cheddar cheese after the addition of a-ketoglutarate (Banks et al., 2001). Since gaminobutyrate can be formed from glutamate by decarboxlyation it is possible that in Gruyere-type cheese a-ketoglutarate is first converted to glutamate by transamination and that this is followed by decarboxylation. The presence of glutamate decarboxylase has been described in lactic acid bacteria (Ueno, Hayakawa, Takahashi, & Oda, 1997; Nomura, Kimoto, Someya, Furukawa, & Suzuki, 1998). The concentration of acetate was found to increase in cheese after the addition of a-ketoglutarate. This could result from the catabolism of alanine by an amino-
transferase leading to the production of pyruvate, which can be converted to acetate by oxidative decarboxylation. Unfortunately, Yvon et al. (1998) did not provide values for the concentration of acetate in St. Paulin cheese. The catabolism of the branched-chain amino acids by an aminotransferase converts leucine and valine to aketoisocaproate and a-ketoisovalerate, respectively. Oxidative decarboxylation of these keto acids leads to the production of isovalerate and isobutyrate. The concentration of the two volatile branched-chain acids were found to increase in cheese after the addition of aketoglutarate. This increase in concentration confirms that amino acid degradation is greater in cheese after the addition of an a-keto acid even if the increase in the concentration of branched chain acids is much lower than the corresponding decrease in branch chain amino acids. Beuvier et al. (1997) also found that Swiss-type cheeses with an higher overall aroma intensity also contained higher concentrations of isovalerate. Butyrate, another potent odorant, also increases in cheese with added a-keto acid. Since g-aminobutyrate is not normally found in Gruyere-type cheese at mmolar concentrations, it was concluded that a-ketoglutarate is not the acceptor for transamination under normal ripening conditions. Normally, a-ketoglutarate is formed from isocitrate by isocitrate dehydrogenase, however, this enzyme has not been detected in lactic acid bacteria (Morishita & Yajima, 1995). a-Keto acid pyruvate seemed to be a more promising candidate since it is possible that lysis of some bacteria could take place during ripening and perhaps release pyruvate into the cheese mass. Our results show that the addition of pyruvate to cheese milk significantly increased the aroma of the Gruye" re-type cheese and that this was accompanied by a decrease in the concentrations of some amino acids, especially leucine, lysine, methionine, phenylalanine and valine. There were also significantly greater amounts of acetate, butyrate, isobutyrate, isovalerate and diacetyl in the cheeses which would explain their greater aroma. The increased concentration of acetate probably arises directly from the oxidative decarboxylation of pyruvate. The high concentration of diacetyl is most likely due to the production of a-acetolactate from pyruvate followed by its decarboxylation. The production of diacetyl from pyruvate, by lactic acid bacteria has been described previously (Ramos, Jordan, Cogan, & Santos, 1994). The higher concentrations of isobutyrate and isovalerate indicate that there was an increase in the degradation of leucine and valine. There was also an increase in other aroma compounds in the cheeses. The majority of these are derived from the metabolism of amino acids. 3-Methyl-butanal and 3-methyl-2-butanone result from the reduction of isovalerate which is derived from leucine. Further
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reduction of 3-methyl-butanal yields 3-methyl-butanol. 2-Methyl-propanal results from the reduction of isobutyrate which is derived from valine. Dimethyldisulphide certainly derive principally from methionine since there are much higher concentrations of this amino acid in cheese than cysteine. The increased levels of butyrate and 1-butanol probably derive from the catabolism of pyruvate and do not involve amino acid metabolism.
5. Conclusion The role of a-keto acids in the production of aromatic substances in cheese, as suggested by Yvon et al. (1998), has been confirmed; however, in Gruyere-type cheese, aketoglutarate is unlikely to be the acceptor for transamination. The addition of pyruvate to cheese milk increased the aroma not only by increasing the catabolism of amino acids but also by intensifiying the production of other aromatic compounds such as diacetyl and butyrate. Thus, the development of flavour in Gruyere-type cheese appears to be a complex process involving not only the degradation of amino acids but also the formation of other aromatic substances. Further work will be necessary to determine which other compounds lead to the production of aroma in cheese.
References Badertscher, R., Liniger, A., & Steiger, G., (1993). Bestimmung der fluchtigen Fettsauren in Kase . . . aus dem Wasserdampfdestillat mit ‘‘Headspace-GC/FID’’. FAM INFO Number 272, Liebefeld-Berne, Switzerland. Banks, J. M., Yvon, M., Grippon, J. C., de la Fuente, M. A., Brechany, E. Y., Williams, A. G., & Muir, D. D. (2001). Enhancement of amino acid catabolism in Cheddar cheese using a-ketoglutarate: amino acid degradation in relation to volatile compounds and aroma character. International Dairy Journal, 11, 235–243. Bassett, E. W., & Harper, W. J. (1958). Isolation and identification of acidic and neutral carbonyl compounds in different cheese varieties of cheese. Journal of Dairy Science, 41, 1206–1217.
273
Beuvier, E., Berthaud, K., Cegarra, S., Dasen, A., Pochet, S., Buchin, S., & Duboz, G. (1997). Ripening and quality of Swiss-type cheese made from raw, pasteurized or microfiltrated milk. International Dairy Journal, 7, 311–323. Bosset, J. O., Butikofer, . U., Gauch, R., & Sieber, R. (1997). Reifungsverlauf von in Folien verpacktem Emmentaler k.ase mit und ohne Zutaz von Lactobacillus casei susp. casei. II. Gaschromatographische Untersuchung einiger fluchtiger, . neutraler Verbindungen mit Hilfe einer dynamischen Dampfraumanalyse. Lebensmittelwissenschaft und Technologie, 30, 464–470. . Y. (1999). Quantitative determination of free Butikofer, . U., & Ardo, amino acids in cheese. Bulletin International Dairy Federation, 337, 24–32. Kosikowski, F. V., & Mocquot, G. (1958). Advances in cheese technology. Food and Agricultural Organisation, United Nations Agricultural Study No. 38 (141pp.). Law, B. A., & Kolstad, J. (1983). Proteolytic systems in lactic acid bacteria. Antonie van Leeuwenhoek, 49, 225–245. McGarry, A., Law, J., Coffey, A., Daly, C., Fox, P. F., & Fitzgerald, G. F. (1994). Effect of genetically modifying the lactococcal proteolytic system on ripening and flavour development in Cheddar cheese. Applied and Environmental Microbiology, 60, 4226–4233. Morishita, T., & Yajima, M. (1995). Incomplete operation of biosynthetic and bioenergetic functions of the citric acid cycle in multiple auxotrophic Lactobacilli. Bioscience, Biotechnology and Biochemistry, 59, 251–255. Nomura, M., Kimoto, H., Someya, Y., Furukawa, S., & Suzuki, I. (1998). Production of gamma-aminobutyric acid by cheese starters during cheese ripening. Journal of Dairy Science, 81, 1486–1491. Ramos, A., Jordan, K. N., Cogan, T. M., & Santos, H. 13 (1994). C Nuclear magnetic resonance studies of citrate and glucose cometabolism by Lactococcus lactis. Applied and Environmental Microbiology, 60, 1739–1748. . Schormuller, . J., & Langner, H. (1960). Uber die organischen S.auren verschiedener K.asearten. Zeitschrift fur . Lebensmitteluntersuchung und Forschung, 113, 289–298. Shakeel-Ur-Rehman, & Fox, P. F. (2002) Effect of added alphaketoglutaric acid, pyruvic acid or pyridoxal phosphate on proteolyis and quality of Cheddar cheese. Food Chemistry, 76, 21–26. Ueno, Y., Hayakawa, K., Takahashi, S., & Oda, K. (1997). Purification and characterization of glutamate decarboxylase from Lactobacillus brevis IFO 12005. Bioscience, Biotechnology and Biochemistry, 61, 1168–1171. Virtanen, A. L., & Kreula, M. (1948). On the significance of amino acids for the taste of Emmenthaler cheese. Meijeritieteellinen Aikakauskirja, 10, 13–23. Yvon, M., Berthelot, S., & Gripon, J. C. (1998). Adding aketoglutarate to semi-hard cheese curd highly enhances the conversion of amino acids to aroma compounds. International Dairy Journal, 8, 889–898.