Ardö, Ylva Margareta

Bakman, Mette

Jensen, Marie Elisabeth Penderup

Kristensen, Helle Tind

Petersen, Mikael Agerlin

Varming, Camilla

English

Copenhagen University Research Information System

u n i ve r s i t y  o f  co pe n h ag e n  Københavns UniversitetAroma formation in a cheese model system by different lactobacillus helveticus strainsPetersen, Mikael Agerlin; Kristensen, Helle Tind; Bakman, Mette; Varming, Camilla; Jensen,Marie Elisabeth Penderup; Ardö, Ylva MargaretaPublished in:Expression of multidisciplinary flavour sciencePublication date:2010Document versionEarly version, also known as pre-printCitation for published version (APA):Petersen, M. A., Kristensen, H. T., Bakman, M., Varming, C., Jensen, M. E. P., & Ardö, Y. M. (2010). Aromaformation in a cheese model system by different lactobacillus helveticus strains. In I. Blank, M. Wüst, & C.Yeretzian (Eds.), Expression of multidisciplinary flavour science: proceedings of the 12th Weurman Symposium,Switzerland, 2008 (pp. 367-370). Zurich University of Applied Sciences.Download date: 02. Feb. 2020Expression of Multidisciplinary Flavour Science   367AROMA FORMATION IN A CHEESE MODEL SYSTEM BY DIFFERENT LACTOBACILLUS HELVETICUS STRAINS   M.A. PETERSEN1, H.T. Kristensen1, M. Bakman2, C. Varming1, M.P. Jensen1, and Y. Ardö1  1 Department of Food Science, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark 2 Arla Foods Innovation, Rørdrumvej 2, DK-8220 Brabrand, Denmark   Abstract  A model cheese system consisting of mild, young, normal-fat, semi-hard cheese, homogenised with water to a dry matter content of 40% was inoculated with six different Lactobacillus helveticus strains and ripened for four weeks. After ripening, considerable increases were seen in content of free amino acids. Furthermore, the strains effected formation of different aroma patterns, and it is concluded that the model system is sensitive and that the six Lactobacillus helveticus strains resulted in patterns of volatiles that are likely to be associated with different sensory characteristics.  Introduction  Traditionally, Lactobacillus helveticus was not used in semi-hard cheese production, but research has shown that these bacteria may be used to markedly increase amino acid release and to improve the sensory profile of low-fat semi-hard cheese (1). Their impact on aroma production in cheese is suspected to be strain dependent, and in this study six different strains of Lactobacillus helveticus were compared and evaluated using a cheese model system.  Experimental  From a screening using molecularbiological methods of a large number of Lactobacillus helveticus isolates from cheese, cultures and bacteria banks, strains with potentially different properties were selected (See Table 1). The strains were cultivated with 1% inoculation level in 8x20 ml MRS for 19 hours at 37°C. In the beginning of stationary phase the bacteria were harvested by centrifugation for 10 min at 4500 rpm (708 xg), 4°C (Sigma 3-10). The supernatant was removed and the pellet was diluted in 1 ml sterile water before use. The total number of bacteria was determined on MRS plates. A cheese model was made from mild, young (one week) normal-fat, semi-hard cheese, cut into pieces and homogenised with water to a dry matter content of approx. 40%. The cheese paste obtained was heated to 80°C to inactivate enzymes and unwanted bacteria and nisin was added to improve lysis of the Lactobacilllus cells and as a preservative. The mixture was poured into sterile beakers with 100 g in each and the lid was closed immediately. After cooling to 40°C, Lactobacillus helveticus culture was added by stirring (each beaker was stirred app. 60 times) with sterile metal spoons in an aseptic bench, and the samples (100 g) were sprayed with natamysin to avoid growth of fungi. Finally the samples were Expression of Multidisciplinary Flavour Science   368ripened for four weeks at 20°C. Reference samples of the model with no added Lactobacillus were incubated in parallel to assure absence of activities of unwanted microorganisms.  Table 1. Strains of Lb. helveticus used in the study.  Strain Lb. helveticus CNRZ 32 Lb. helveticus CNRZ 303 Lb. helveticus ATCC 15009 (type strain) Lb. helveticus LHC2 Lb. helveticus MI 2275 (Isolate from cheese culture) Lb. helveticus MI 2359 (Isolate from cheese)  After 2 and 4 weeks of ripening, the samples were analysed for volatiles by dynamic headspace sampling and GC-MS (method modified after (2)). For determination of total amount of amino acids and of 12 specific amino acids, cheese samples were extracted with 6.0% 5-sulfosalicyl acid, derivatised with 6-aminoquinolyl-N hydroxysuccinimidyl carbamate (AQC), and analysed using RP-HPLC with scanning fluorescence detection (AccQTag).  Results  All the examined strains of Lactobacillus helveticus effected considerable increases in total concentrations of free amino acids (Table 2). Mainly the same amino acids increased in all the cheese models (data not shown), and for half of the 12 analysed amino acids (Leu, Ile Val, Phe, Pro and Lys) the increase was highest for cheese models with Lactobacillus helveticus ATCC 15009 and MI 2275, indicating higher lysis The presence of the same aminopeptidase activities in all the examined strains was revealed. Amino acids are important flavours, but since they also are intermediates in aroma production, differences in overall flavour of the cheese models were still expected.  Table 2. Total content of amino acids.   Lactobacillus helveticus strain mmol/kg Control 4.8 CNRZ 32 17.9 LHC2 18.3 MI 2359 (from cheese) 18.8 CNRZ 303 19.3 ATCC 15009 (type strain) 19.8 MI 2275 (from cheese culture) 25.9  This was confirmed by (Figure 1). When type of strain was related to aroma pattern using PLS regression, a clear separation of most of the cheese models was obtained, thus demonstrating considerable impact of Lactobacillus helveticus on volatiles normally considered potentially important aroma compounds in cheese (3). This was obtained after 2 and 4 weeks of ripening in the model system used.   Expression of Multidisciplinary Flavour Science   369  Figure 1.  Loadings plot from a dummy PLS relating aroma data to strain. A red circle indicates a volatile compound where the concentration is significantly influenced by the culture (ANOVA). The boxes list compounds having higher concentration in the model and asterisks indicate significant influence of culture (ANOVA, *: p<0.05; **: p<0.01; *** p<0.001).  Most compounds increased in concentration as compared to the control (however, phenylacetaldehyde, hexanal and 3-methylbutanal were lower than the control for all Lactobacillus helveticus strains except MI 2275 (data not shown). The results indicate that MI 2275 has high capacity of amino acid catabolism since all the volatiles found in significantly higher amounts than in the control were possibly amino acid derived (Table 2) (3). The other strains had increased levels of mainly alcohols and also ketones derived from lipids (except 3-methylbutanol which is amino acid derived), indicating significant lipase/esterase activities. It should be noted that there was a characteristic difference for MI 2359, being the only strain with increased levels of several methyl ketones. Free fatty acids are the principal methyl ketone precursors through β-oxidation to β-keto acids (4) and the increased formation in the MI 2359 model could be a result of increased lipase activity. It is concluded that the model system has a potential in screening experiments by achieving useful results about processes going on during cheese ripening within the short time of only 4 weeks. Furthermore, the six Lactobacillus helveticus strains tested had a clear effect on release of free amino acids and of volatiles that are likely Expression of Multidisciplinary Flavour Science   370to be associated with different sensory characteristics. Cheese making experiments are needed to confirm the interesting effects in different cheese varieties.  Table 2. Volatiles present in significantly higher amounts in models with Lb. helveticus strains than in controls (ANOVA, p<0.05). The strain CNRZ32 did not significantly increase the amount of any volatile component.  MI 2275 LHC2 ATCC CNRZ 303 MI 2359 3-Methylbutanol  3-Methylbutanol   2-Methylbutanal  Butanol Butanol Butanol 3-Methylbutanal Pentanol Pentanol  Pentanol  Hexanol Hexanol  Hexanol    2-Butanone  Decanal Benzaldehyde     2-Heptanone Acetoin     2-Octanone       2-Nonanone  Acknowledgment  The work was financially supported by the Danish Dairy Research Foundation and the Danish Directorate for Food, Fisheries and Agri Business  References  6. Ardö Y., Larsson P.O., Månsson H.L., Hedenberg A. (1989) Milchwissenschaft 44: 485-490. 7. Andersen C.M., Andersen L.T., Hansen A.M., Skibsted L.H., Petersen M.A. (2008) J. Agric. Food Chem. 56: 1611–1618. 8. Ardö Y (2006) Biotechnology Advances 24: 238-242. 9. Collins Y.F., McSweeney P.L.H., Wilkinson M.G. (2003) Int. Dairy J. 13: 841-866. 

Aroma formation in a cheese model system by different lactobacillus helveticus strains

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Aroma formation in a cheese model system by different lactobacillus helveticus strains

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