Ganaderia mediterrnea

Sardinian fermented sheep sausage:
Microbial biodiversity resource for quality improvement
N.P. Mangia, M.A. Murgia, G. Garau, R. Merella and P. Deiana
Dipartimento di Scienze Ambientali Agrarie e Biotecnologie Agroalimentari, Sezione di Microbiologia, Università di Sassari, V.le Italia, 39, 07100 Sassari, Italy SUMMARY – With the aim of improving the quality and the value of the Sardinian sheep sausage, the microflora
involved in fermentation and ripening processes of this traditional foodstuff has been studied and characterized.
During the fermentation phase a prominent presence of micrococci and staphylococci has been observed. The
lactic acid bacteria (LAB) that were showing a slow growth rate during the first days were found as the prevalent
microflora during the ripening. The presence of coliforms was observed especial y during the early fermentation
phase. Salmonella and Listeria spp. were never detected. Bacterial species identified were Lactobacillus
, Staphylococcus xylosus, Staphylococcus lentus, Kocuria varians, Micrococcus spp. while yeast
strains belonged to Trichosporon spp. Debaryomyces hansenii and Candida famata. The technological selection
of autochthonous LAB and staphylococci, which is actual y ongoing, will be crucial for the development of starter
cultures suitable for the quality improvement of this typical Mediterranean foodstuff.
Keywords: Sheep sausage, traditional technology, autochthonous microflora, quality.
RESUME – "Saucisse fermentée de brebis de Sardaigne : Ressources en biodiversité microbienne pour
l'amélioration de la qualité". Dans le but d'améliorer la qualité et la valeur de la saucisse de brebis, produite en
Sardaigne, les microflores impliquées dans la fermentation ont été étudiées et caractérisées. Pendant la phase
de la fermentation une présence considérable de microcoques et staphylocoques a été observée. Les résultats
ont montré que les bactéries lactiques, en dépit d'un développement initial lent, se sont avérées être la microflore
prédominante pendant la maturation. La présence de coliformes a été observée pendant la phase de
fermentation. Les espèces bactériennes identifiées étaient
Lactobacillus plantarum, Staphylococcus xylosus,
Staphylococcus lentus, Kocuria varians et Micrococcus spp. parmi les levures les espèces Debaryomyces
hansenii, Candida famata et Trichosporon spp. La sélection technologique de LAB et staphylocoques
autochtones sera cruciale pour le développement des cultures starter pour l'amélioration de la qualité de ce
produit alimentaire méditerranéen typique.

Mots-clés : Saucisse de brebis, technologie traditionnelle, microflore autochtone, qualité.


Different projects have been recently undertaken in Sardinia with the aim to improve the global
value of sheep meat-based food products. Most of the times this was achieved through the recovery
of ancient and/or traditional manufacture procedures. This was the case of the ripened sheep
sausages as well as the "prosciutto" (ham) or "coppa" (air-dried whole shoulder) made from sheep
meat. The production of these kind of traditional foodstuffs constitutes a valuable alternative to jobs
connected with meat-based manufacture and a good opportunity to improve their value possibly
providing higher remunerations.

Among the stuffed meat-based fermented products manufactured in Sardinia (Italy) ripened sheep
sausage (actually a kind of salami) plays a special role because of its typical features. The production
technology, that is currently very similar to the traditional one, is comparable to the manufacturing
process employed for pork sausages. Few differences can be identified with respect to this latter and
they are mainly related to the extra-care for sheep meat selection and preparation such as the
elimination of the connective tissues as well as the fat, both responsible for unpleasant taste. The
technological procedures employed for sheep sausage production are as follows.
Meat preparation: different cuts of sheep’s meat from healthy animals are commonly used, in Options Méditerranéennes, Series A, No. 78 Preparation of the mix: traditionally, sheep meat is minced in small pieces (approx. 1 cm diameter) and then mixed with pork fat (cut in small pieces) that soften the typical taste of ovine meat. Variable amounts of salt (2.8-3%) and spices (mostly pepper and garlic) are added. In the traditional manufacturing process, that was made without any starter culture, the fermentation process is carried out by natural microflora associated to the ingredients. In some cases vinegar or white wine, undergoing malolactic fermentation, are used to obtain a superior quality product. The mix is left to set down overnight in a fresh and dry environment. Only rarely sugar, a microbial starter and/or other addictives are added. Stuffing sausage and ripening: after stuffing the sausages into the casings, these are smoked for up to 10-15 days and then ripened for up to 20 or 30 days. The Sardinian sheep sausage is currently a niche foodstuff product that, even if manufactured following a traditional technology, is often inadequate and too much diversified in order to obtain a qualitatively stable product with constant features. Moreover, and most importantly, the final quality of the sheep sausage is strictly dependent on the knowledge of all those factors influencing the microbial presence and contamination in the production chain and hence the safety of the product. Ovine meat, particularly deep tissues, presents a less extent of microbial contamination with respect to other meats. Microbiological studies on sheep meat revealed the presence, almost exclusive, of staphylococci while clostridia, Escherichia coli, Pseudomonas, Enterobacter and Yersinia were not found (Newton et al., 1978). More recently the presence of Lactobacillus plantarum, Staphylococcus xylosus and a significant number of yeasts and micrococci was also assessed (Mangia et al., 2002). Research investigations on meat-based Sardinian foodstuffs such as sheep sausages are globally few and do not offer a global picture of the microbial and chemicophysical status of these products (Mazzette et al., 1996). Deepening our knowledge on the microbial species responsible for fermentative and ripening
processes in traditional Sardinian sausages can be certainly a suitable way to improve the intrinsic
value and the quality of these foodstuffs.
The aim of this work was to isolate, characterize and select from a physiological, biochemical and
technological point of view the natural microflora present in sheep sausages manufactured with the
traditional technology. This will finally offer the possibility to make up an autochthonous and specific
starter culture for this typical Mediterranean production.
Materials and methods
Sampling procedures
Mince sheep meat and sausage samples (made from this meat) where colleted at different
ripening times and particularly at 1, 2, 3, 5, 7, 9, 13, 18 and 23 days of ripening. Samples were
collected from a factory that ordinary employs a traditional manufacturing technology. Meat samples
were collected before and after spice and wine addition. Before starting microbiological analysis,
sheep sausages were cleaned with a brush to avoid microbial contamination from the outside.
pH measurements
Potentiometric measurements of the pH were made using a pin electrode of a pH-meter (KNICK Portamess 911) inserted directly into the sample.

Microbiological analysis

Aliquots of 25 g form each sample were transferred into a sterile stomacher bag and 225 ml of
peptone water (Oxoid) were added and mixed for 1.5 min in a Stomacher Lab-Blender 80 (PBI).
Further, ten-fold dilutions were made and aliquots used to inoculate the following solid media in
Options Méditerranéennes, Series A, No. 78 order to detect and quantify different microbial groups: aerobic mesophilic bacteria were determined on PCA (Oxoid) incubated at 30°C for 48-72 h; lactic acid bacteria (LAB) were counted on a double layer MRS agar (Oxoid) after incubation for 48 h at 30°C; staphylococci and micrococci were quantified on Mannitol Salt Agar (Oxoid) incubated for 48 h at 30°C; BGBB (Oxoid) was used for the enumeration of total and faecal coliforms. Numbers were determined after incubation on a double layer BGBB at 37 and 44°C for 24-48 h; yeasts and moulds were enumerated on Malt Extract Agar (Oxoid) incubated at 25°C for 48-72 h; the presence of Salmonella spp. was tested using a Salmonella Rapid Test (Oxoid) while the presence of Listeria spp. was verified after enrichment in Fraser Broth Base (Oxoid) plus supplement Fraser SR 156 (Oxoid). From each counting media approximately 10 colonies were randomly selected for further identification. The morphology, Gram-stain and catalase activity of these isolates were preliminary tested. Gram positive and catalase-positive cocci were purified on MSA and maintained on slant tubes with the same medium at 4°C. Purified strains were classified according to Kloos et al. (1991). They were further subjected to the following tests to differentiate micrococci and staphylococci: sensitivity to furazolidone (Von Rheinbaben and Hadlok 1981) and lysostaphin (Schleifer and Kloos 1975). Staphylococci were assayed for coagulase activity, and finally identified using API STAPH and ID 32 STAPH Systems (bioMerieÁux, Marcy l'Etoile, France). LAB identification was carried out according to Bergey’s Manual of Systematic Bacteriology (Sneath et al., 1986), Bridge and Sneath (1983) and carbohydrate fermentation patterns determined using API 50 CH and API 20 Strep test galleries (API System bioMerieux, Marcy l’Etoile, France). Yeast identification was carried out after Kurtzman and Fell (2000).
Results and discussion

In this paper the composition and the evolution of micro-organisms associated to the Sardinian
traditional fermented sheep sausages were investigated at different ripening times. Table 1 is showing
the evolution of the main microbial groups investigated. Total mesophilic count (TMC) revealed higher
values (approx. 3E+10 cfu/g) at the end of ripening with respect of those reported by other authors
(Mazzette et al., 1996). However these numbers can be considered as common for stuffed fermented
products. Micrococci and staphylococci, already present in the meat (MM), showed a very rapid
increase reaching final values of 1.66E+09 cfu/g 23 days after ripening. The significant number of
these micro-organisms is related to the intrinsic features meat as well as to the artisanal technology
employed, that involves an extensive manual treatment of the product. Although LAB showed a slow
growth rate during the first period of ripening, after the 5th day values higher than one billion of cells
per gram were recorded. Our data are globally supporting other findings stating that LAB are the
dominating microflora during the ripening.

Heterofermentors LAB were found absent in all the samples analysed. The microbiological survey showed a significant initial contamination of sheep sausage with faecal coliforms reaching important values most likely due to optimal pH and aw . This can be considered an index of the poor hygienic conditions employed during the preparation steps as well as during the whole manufacturing process. Yeasts were always found present and their number slowly increased during the ripening period while the number of moulds remained stable. Despite suboptimal hygienic conditions Salmonella and Listeria species were never detected. Globally 161 microbial strains belonging to the species Lactobacillus plantarum, Staphylococcus xylosus, Staphylococcus lentus, Kocuria varians, Micrococcus spp., Trichosporon spp., Debaryomyces hansenii and Candida famata were isolated. Table 2 reports the general view of the species isolated and their evolution during the ripening. Options Méditerranéennes, Series A, No. 78 Table 1. Evolution of autochthonous microflora in sheep sausage at different ripening times bacteria staphylococci coliforms (cfu/g) MM=minced meat; MMS= minced meat with spices; S=sausage; d= days of ripening; TMC=total mesophilic count.

Table 2. Number of species isolated from sheep meat and sausage at different ripening times
S 1d S 2d S 3d S 5d S 7d S 9d S 13d S 18d S 23d MM=minced meat; MMS= minced meat with spices; S=sausage; d= days of ripening. The identified LAB were only represented by species belonging to a single genus in contrast to what found for Sardinian pork sausages (Greco et al., 2005). The number of L. plantarum increased until the 13th day then declined in the following period. L. plantarum is the most represented LAB in all the stuffed foodstuffs fermented by natural microflora (Zambonelli et al., 1992). Optimal growth for L. plantarum occurs at temperature higher than 30°C hence the fermentation rate is substantially influenced by this parameter. To this regard temperature of about 25-30°C were recorded during the first phases of drying and smoking of the sheep sausage, most likely maximizing the L. plantarum presence. Strains belonging to the Micrococcus genus have been recovered during the first days of ripening (4.97% frequency); being these micro-organisms obligate aerobes, after this period their number decreased most likely because of a gaseous phase progressively lacking oxygen. During this phase micrococci are replaced by staphylococci that predominated during the last part of the ripening. The most abundant specie was Staphylococcus xylosus with the 12.42% of recovery frequency; this is confirming what reported by other studies investigating the microbiological features of Italian stuffed products (Zambonelli et al., 1992; Aymerich et al., 2003). Staphylococcus lentus and Kocuria varians have been recovered in low number (0.62%). The slow growth rate showed in the beginning by LAB and staphylococci put in evidence the need for adding glucose or lactose in order to support fermentative processes and inhibit undesirable organisms such as faecal coliforms. Debaryomyces hansenii has been the most recovered species (19.25%, possibly because it is more halotolerant than Options Méditerranéennes, Series A, No. 78 other yeasts) confirming previous findings (Dalton et al., 1984). Because of its invariable presence
this species is commonly considered as the typical yeast of stuffed products (Entel, 1961).
Trichosporon spp. that was found highly present in the slaughters has been recovered with a 3.73%
of frequency during the first phase of ripening.
This study allowed to identify the micro-organisms characterizing fermentative and ripening
processes of the Sardinian sheep sausage. This can be considered as a suitable baseline for the
choose of the microbial species that could be employed to make up an autochthonous starter culture
for the manufacturing process. By the way it is essential to deepen our knowledge on strain
metabolism, on their ability to carry out fermentative and ripening processes as well as their suitability
for quality improvement and the maintenance of the typical features of sheep sausage.

Aymerich, T., Martin, B., Garriga, M. and Hugas, M. (2003). Microbial quality and direct PCR
identification of lactic acid bacteria and non pathogenic Staphilococci from artisanal low-acid sausage. Appl. Environ. Microbiol., 69(8): 4583-94. Bridge, P.D. and Sneath, P.H.A. (1983). Numerical taxonomy of Streptococcus. J. Gen. Microbiol., Dalton, H.K. Board, R.G. and Davemport, R.R. (1984). The yeast of British fresh sausage and minced beef. A. van Leeuwenhoek, 50: 227. Entel, H.J. (1961). Die in Rohwursten vorkomenden Hefen. Fleischwirtschaft, 13. Atti A.I.V.I., 5: 183. Greco, M., Mazzette, R., De Santis, E.P.L., Corona, A. and Cosseddu, A.M. (2005). Evolution and identification of lactic acid bacteria isolated during the ripening of Sardinian sausages. Meat Science, 69: 733-739. Kloos, W.E., Schleifer, K.H. and Gotz, F. (1991). The genus Staphylococcus. In: The Prokaryotes. 2nd edn. Balows, A., Truper, H.G., Dworkin, M., Harder, W. and Schleifer, K. (eds). Springer Verlag, New York, pp. 1369-1420. Kurtzman, C.P. and Fell, J.W. (2000). The Yeasts: A Taxonomy Study. Elsevier, Amsterdam. Newton, K.G., Harrison, J.C.L. and Wauters, A.M. (1978). Sources of psychrotrophic bacteria on meat at the abattoir. J. Applied Bacteriol., 45: 75-82. Mangia, N.P., Sanna, M.G., Murgia, M.A., Madrau, M., Fresi, A., Farris, G.A. and Deiana, P. (2002). Microbiologia lattica e lievitiforme della salsiccia sarda di pecora. Atti XV Congresso Nazionale S.I.P.A.O.C., Cagliari (Italy), 11-14 September 2002, p.253. Mazzette, R., De Santis, E.L.P., Coppa, G., Serra, P.G., Bean, V. and Cosseddu, A.M. (1996). Valorizzazione delle carni ovine: Utilizzazione per la produzione dei prodotti di salumeria. Atti A.I.V.I., 4: 181. Schleifer, K.H. and Kloos, W.E. (1975). A simple test for separation of staphylococci from micrococci. Journal of Clinical Microbiology, 1: 337-338. Sneath, P.H.A., Mair, N.S., Sharpe, M.E. and Holt, J.G. (1986). Bergey’s Manual of Sistematic Bacteriology, Vol.2. Williams & Wilkins, Baltimore, MD, USA. Von Rheinbaben, K.E. and Hadlok, R.M. (1981). Rapid distinction between micrococci and staphylococci with furazolidone agar. Antonie Van Leeuwenhoek, 47: 41-51. Zambonelli, C., Papa, F., Romano, P., Suzzi, G. and Grazia, L. (1992). Microbiologia dei Salumi, Options Méditerranéennes, Series A, No. 78


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