Article pp.150-153 du Vol.23 n°1 (2003)

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150-153 Sci. Aliments 23(1), 2003 J.B. Gros et al

FOCUS : JSMTV

Selection of mould strains from the surface flora of French saucissons and study of their

biocatalytic behaviour

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J.B. Gros¹, M.F. Roquebert², J.L. Berdagué³, C. Larroche¹¹, P. Fontanille¹, J. Sirami⁴

INTRODUCTION

The aromatic qualities of processed meat products and in particular of French saucissons (French dried mould fermented salamis ) are a major element in consu- mer choice. Aroma is mainly of microbiological origin, it is produced by both the surface and the internal flora of the saucisson. The former seems to play a predo- minant role, even if the proportion of each flora remains unknown. This work concerns the study of mould strains isolated from the surface flora of saucissons.

MATERIALS AND METHODS

Method

The work was divided into 6 steps with the aim to evidence specific biocatalytic properties of the surface fungal strains:

1) gathering traditionally-produced saucissons from different parts of France. Selection of a reference set of saucissons, based on the quality and intensity of their aromas.

2) isolating and identifying fungi on the surface of saucissons.

3) manufacturing saucissons using separately each of the isolated and selected strains.

1. Laboratoire de génie chimique et biochimique, Université B Pascal, CUST, BP 206, 63174 Aubière cedex.

2. MNHN, Laboratoire de mycologie, 12, rue Buffon, 75005 Paris.

3. INRA Clermont-Ferrand-Theix, Station de Recherche sur la Viande, Theix, 63122 Saint-Genés-Champa- nelle.

4. Adiv, 2, rue Chappe, 63039 Clermont-Ferrand cedex 2.

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Selection of mould strains from the surface flora of French saucissons… 151

4) analysing the volatile compounds produced by the microbial flora present on the surface of the reference set.

5) analysing the volatile compounds produced by the surface flora of saucissons inoculated with a single pure isolated strain.

6) culture in instrumented reactors of each pure isolated strain. Determina- tion of growing conditions of strains; analysis of their capability to pro- duce saucissons volatile compounds from given known precursors.

Materials

Growth media used to isolate fungi were MA (Malt-Agar 2%w/w), PDA (Potato Dextrose Agar) and DRBC (Dichloran Rose Bengal Chloramphenicol agar). Incubation temperature was 25˚C.

The ingredients of the "model" saucissons were the following:

– meat ingredients: pork shoulder 4D: 80%w/w, pork back fat ("bardière"):

20%w/w;

– non meat ingredients: salt (NaCl): 3%w/w, sucrose: 0.6%, saltpetre (potas- sium nitrate): 400 ppm, and pepper: 0.1%;

– internal flora: starter culture Lyoflore 2M (Texel, France)

Meat was ground to 6 mm particles, kneaded for 2.5 min., and stuffed into natural casings.

Processing conditions were usual:

– surface seeding with fungi by individual soaking for approx. 2 min. An uni- noculated standard was also manufactured. Development of spoliage organisms on the standard was inhibited by soaking it for c.a. 2 min. in a pimaricine solution.

– drainage during a 12 h period at 12˚C in a cabinet drier previously disinfec- ted.

– ripening at 24˚C maximum temperature for 26 hours and "fleurissement"

(surface development of moulds) between 19 and 15˚C for 3 days.

– drying in the same chamber for 21 days at 12˚C, humidity maintained between 75% and 85% RH (Figure 1).

After extraction and trapping by dynamic headspace chromatography, the volatile compounds fraction was injected at 220˚C in a Hewlett-Packard 5890 series A gas chromatograph coupled to a Hewlett-Packard 5971A mass spec- trometer. Identification of compounds was realised by comparing experimental spectra with known spectra in compiled libraries (NIST/EPA/MSDC (1996) and NIST/EPA/NIH Mass Spectral Library version 1.5 (1996)), and by comparing their relative retention indexes (Kovats index) with the home-made compilation of Kondjoyan and Berdagué (Inra, Theix).

Statistical analysis was performed using STATISTICA (1997).

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152 Sci. Aliments 23(1), 2003 J.B. Gros et al

Figure 1

Saucissons at the end of the controlled air drying period

Results

The selection of traditional saucissons, based on their aromatic properties, and the identification of their surface flora has established a collection of 59 fungal strains, essentially Penicillium (Laboratoire de Cryptogamie, MNHN). Table 1 summarises the main fungal species isolated and identified.

The sensory analysis and the GC-MS analysis of the volatile fraction produced by the surface flora have confirmed its active role. All the visual descriptors (implementation, homogeneity, colour) have been influenced in a highly significant way by the strain selected for inoculating and colonising the surface. Olfac- tory verbal descriptors such as "saucisson-like ", "cellar-like", "mushroom-like",

"humus" have also been influenced in a highly significant way by surface inocu- lations. The volatile fraction constituents are similar to those commonly identified inside saucissons but at very different concentrations. We found alcohols, aldehydes, ketones, alkanes, aromatic compounds, terpenes and sesquiterpe- nes. Chromatographic profiles of saucissons cultured by pure selected strains were richer in compounds with a benzenic structure and in C8 molecules (compounds with 8 atoms of carbon), and "poor" in substances usually found in the saucissons mix.

The behaviour of selected strains on a solid substrate has been examined and the protocol to propagate them is now defined. It has been possible to cul- tivate them in a liquid medium using a bioreactor, in conditions far from their natural environment. Their ability to carry out biosynthesis and biotransforma- tion processes was maintained. They have been shown to be able to synthetise various metabolites such as ketones, alcohols (from fatty acids), aldehydes, esters, volatile fatty acids, alkanes, benzenic compounds and terpenoïds.

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Selection of mould strains from the surface flora of French saucissons… 153

Table 1

Summary of mould species isolated and identified from the surface of the reference set of traditional saucissons

CONCLUSION

Cultures of pure isolated strains from the surface confirms the aromatic potentials revealed in analyses conducted on saucissons. The study did not evi- denced any specific molecules produced by the surface flora, compared to the internal flora; but it showed the not inconsiderable role of certain compounds, such as 3-octanol, 6-méthyl-3-heptanone and an isomer of 1-octène-3-ol, in the perception of the quality and intensity of saucisson aroma. It therefore would be of interest to continue this research by selecting and studying the strains capable of producing these compounds with 8 carbon atoms and by looking for the conditions of overproduction.

Mould flora Number of strains

identified Penicillium

Penicillim aurantiogriseum 17

Penicillium jensenii 7

Penicillium expansum 4

Penicillium verrucosum 4

Penicillium brevicompactum 3

Penicillium crustosum 2

Penicillium puberulum 2

Penicillium variabile 2

Penicillium camembertii 1

Penicillium chrysogenum 1

Penicillium decumbens 1

Penicillium implicatum 1

Penicillium roquefortii 1

Penicillium simplicissimum 1

Penicillium viridicatum 1

Mucorales

Mucor racemosus 8

Mucor plumbeus 2

Rhizopus stolonifer 2

Others

Cladosporium phaerospermum 4

Aspergillus claviforme 1

Aspergillus niger 1

Epicoccum purpurascens 1