Growth and aroma production by Staphylococcus xylosus, S. carnosus and S. equorum—a comparative study in model systems

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Abstract

A laboratory medium inoculated with 20 different Staphylococcus strains was prepared in accordance with a full factorial experimental design investigating the effect of temperature, pH, NaCl and glucose on growth. The 12 strains most suited to growth in a fermented meat environment were inoculated in sausage minces together with Pediococcus pentosaceus, incubated at 25 °C for 1 week and the produced aroma compounds collected. The data were analysed by multiple linear regression and partial least squares regression analysis.

The results showed that increasing pH and temperature from 4.6 to 6.0 and 10 to 26 °C, respectively, increased growth of all strains with strong synergy between temperature and pH. Increasing salt concentration from 5% to 15% w/v decreased growth of most strains, but the effect of pH and temperature was much stronger than the effect of salt. Strains of S. carnosus were more salt tolerant than strains of S. equorum and S. xylosus, especially at high pH and temperature. Addition of glucose up to 0.5% w/v had no significant influence on growth of any of the strains.

With regard to aroma production, species characteristics were detected. S. carnosus and S. xylosus were quite different regarding the overall aroma profiles, whereas the profiles of S. equorum lied somewhere in-between. Contrary to S. carnosus, S. xylosus and S. equorum did not produce 2-methyl-1-butanol. On the other hand, in particular, S. xylosus produced more 3-methyl-1-butanol. Except for one of the strains of S. equorum, S. xylosus and S. equorum formed more diacetyl, 2-butanone and acetoin and also more of the methyl-branched ketones arising from degradation of leucine, isoleucine and valine. S. carnosus produced more methyl-branched aldehydes, acids and corresponding esters from leucine, isoleucine and valine—compounds that have been correlated with fermented sausage maturity in former studies. S. equorum produced the least of the methyl-branched aldehydes.

Introduction

Staphylococcus species are used as starter cultures for fermented meat products such as sausages and ham. They are mainly used due to their nitrate reductase activity, which accelerates and stabilise colour, but also due to their ability to produce meat products with increased flavour (Montel et al., 1998, Stahnke et al., 2001). During fermented sausage production, the Staphylococcus cultures are exposed to processing conditions adverse to their survival, growth and metabolic activity. But except for the pathogens such as S. aureus, only few investigations have been published on the influence of processing parameters on Staphylococcus.

Sørensen and Jakobsen (1996) and McMeekin et al. (1987) showed that two different strains of S. xylosus had higher growth rate at higher temperatures in yeast broth (growth investigated at, respectively, 10–30 and 5–30 °C). Sørensen and Jakobsen (1996) also found that growth decreased with decreasing pH and that the influence of pH was greatest at low concentrations of salt combined with high temperatures. Increasing the concentration of salt decreased growth, especially at high temperatures and high pH values. The authors concluded that the conditions under which sausage fermentation takes place, in general, are not favourable for growth of S. xylosus (Sørensen and Jakobsen, 1996). Other studies on various non-starter Staphylococcus spp. have shown that the temperature range for growth is 7–48 °C with an optimum of 35–40 °C for most species. For S. aureus, the pH growth range is 4–10 with an optimum at 6–7 (Baird-Parker, 1990).

McMeekin et al. (1987) found that growth rate decreased with increasing salt concentration in the range 3.5–23.4% w/v NaCl (aw 0.976–0.848). The growth rate at aw 0.996 was lower than at aw 0.976, which indicated a growth optimum at relatively high salt concentration (pH=7.0). This was not shown for the strain studied by Sørensen and Jakobsen (1996), but there seem to be strain differences with regards to salt tolerance and growth optima since studies of Hammes et al. (1995) showed that growth of one strain was reduced severely at 10% salt while another strain showed maximum growth at 10% salt. Likewise, Guo et al. (2000) found that one species of S. xylosus had maximum growth at 3% salt, whereas the investigated Micrococcus varians and S. carnosus species grew better at lower salt concentration.

Staphylococcus spp. catabolise glucose via the Embden–Meyerhof pathway and the hexose monophosphate pathway. Investigations have shown that addition of glucose to the substrate reduces the percentage of glucose being oxidised via the hexose monophosphate pathway and the subsequent oxidation via the citric acid cycle. In other words, glucose seems to suppress the citric acid cycle (Blumenthal, 1972). This is in accordance with the findings of Sørensen and Jakobsen (1996), who showed that addition of 1% w/v of glucose to the substrate did not have a positive effect on growth of S. xylosus, but on the contrary, gave a small reduction in growth rate at otherwise optimal conditions of temperature, pH and salt concentration. Stahnke (1999b) also found that the presence of glucose decreased the growth of S. carnosus in sausage minces to some extent.

The aroma of a fermented meat product is influenced by many different factors such as source and type of ingredients, temperature, processing time and choice of starter culture (Lücke, 1998). The biochemical formation of aroma is due to the activity of enzymes present in the meat, to enzymes from the microorganisms present and a combination of both. The starter culture used can be decisive for the aroma of the finished product. Studies of, for example, Berdague et al. (1993), Montel et al. (1996) and Stahnke (1999a) suggest that different species of Staphylococcus produce different aroma compounds in different amounts. However, it has never been studied as to whether there are characteristic species differences with regard to aroma production or with regard to optimal growth conditions for staphylococci used as starter cultures.

The objectives of the present study were as follows: (i) To investigate growth differences on species level for 20 Staphylococcus strains by studying the influence of temperature, pH, NaCl and glucose concentration on growth in a laboratory medium. (ii) To investigate species differences with regard to aroma production in sausage mince models.

Section snippets

Experimental design of growth investigation

Twenty strains (S1–S20) of Staphylococcus were selected from the culture bank of Chr. Hansen (Hørsholm, Denmark) (Table 1). In two separate trials, all 20 strains were identified to species level by the API Staph system (bioMerieux, Marcy-l'Étoile, France) and 12 of those further verified by the 16s rRNA technique (Anon., 1999). The experimental design for each strain was set up as a full four-factorial design at two levels (24 structure) examining the influence of temperature, start pH, the

Strain identification

Table 1 shows the results from the identification trials using the 16s rRNA technique and/or the API Staph system. For the strains only identified by the API system, one should note that S9–S11, S15–S17 and S19 could be S. equorum even if stated as S. xylosus with a high probability—the API Staph test does not differentiate between S. xylosus and S. equorum (bioMerieux, Marcy-l'Étoile, France, January 2001). The two separate API system trials were, in general, in good agreement, though the

Acknowledgements

This study was supported by Chr. Hansen A/S, Hørsholm, Denmark. The authors sincerely thank Lone Andersen, Section Leader of the Meat Cultures Department.

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