Spontaneously fermented meats: Isolation and characterization of lactic acid bacteria as possible starter cultures

Nowadays, fermented meat producers are interested much more consumer perception than meat preservation. Fermentation of meats is an ancient and excellent preservation technique which results in stable and safe end products. In addition, as a result of many chemical, biochemical reactions involving proteins, carbohydrates, and lipids and microbiological changes occur during fermentation characteristic taste, odor, aroma, texture and color developments of the product occur. These phenomena are due to both endogenous and microbial enzymes, the contribution of which depend mainly on the type of process. The need for process standardization and quality assurance strategies has increased the use of starter cultures and thus overcoming the need to rely on the " in - house" flora or " back-sloping" for the fermentation process. Studies on the ecology of fermented sausages showed that lactic acid bacteria, mainly Lactobacillus and coagulase-negative cocci represented by Micrococcaceae, are the two main bacterial groups technologically important in the fermentation and ripening of sausage. The most common lactic acid bacteria species identified are Lactobacillus sakei, Lactobacillus curvatus, and Lactobacillus plantarum, with L. Sakei prevailing. Among coagulase-negative cocci, Staphylococcus xylosus and Staphylococcus carnosum are the most common species identifi ed from traditional products. Pediococci and enterococci have also been often identified from fermented sausages. Fast acidification and lower pH values can be ensured by Pediococcus in semidry sausages in which they grow and metabolize carbohydrates at higher temperatures, while Lactobacillus are mostly used in dry sausage production. Despite their traditional origins, Italian fermented sausage production processes have been adapted to advanced technological standards and adapted to processed meat sensation. Today, using of starter culture and temperature and humidity conditions during ripening are generally the same in most fermented italian meat products. This could determine a standardisation of the traditional features of the different products, even if the wild ripening microbiota continues to play a crucial role in flavor formation in many products. In this respect, the aims of this study are as follows: the characterization of the microbiological, physiological and characteristic properties of two different traditional Italian sausage (salami) produced in different regions without the use of starter cultures and identification and characterization of isolated strains in order to select the most suitable strain that preserves typical sensory quality and provide safe and standardized products as a starter culture. Strains used as starter cultures must be "generally regarded as safe" (GRAS) because they are considered to be food additives. Laws regulating the market of starter cultures may vary depending on the country but, overall, there are some requirements for the starter cultures; they must be neither pathogenic, toxic, nor allergenic. They must to have phenotypical and genotypical stability. They have to be competitive in the typical conditions of the process (tolerance to salt, nitrite, low pH and water activity, considerable growth at manufacturing temperatures, etc.). Provide some technological benefits; for instance, on acidification, preservation, flavor formation, quality assurance, etc. They have to resist phage infection. They have to be identifiable by specific methods. For this study, two different types of italian sausage (Bresciano and Romagnolo) which are non-industrial and produce without starter culture and 23 isolated strains which are mostly L.sakei are used. This study has been carried out in two parts. In the first part the microbial flora, water activity, pH, biogenic amine content and aromatic profile of sausages were examined. In the second part, antimicrobial activitiy, growth and acidification tendencies at different salt concentrations, production of organic acid and biogenic amine and aromatic profile of isolated strains were investigated. The water activity is particularly low in salame Romagnolo (0.853), while in salame Bresciano is higher (0.908). the concentration of lactic acid of the two sausage is similar, while in the sample R there is a higher concentration of acetic acid (0.37 g/kg) differently from sample B, in which there is a lower concentration of this acid (0.08 g/kg). The microbiological parameters showed a high concentration of lactic acid bacteria (9.09 log CFU/g) in salame Bresciano, while a lower concentration of the same bacteria characterized the salame Romagnolo. In general, the values of all amines in salame Bresciano were slightly higher than salame Romagnolo, but still not dangerous. In particular, in salami Brasciano (sample B), the most important aroma compound is benzeacetaldehyde, which derives from the metabolism of aromatic aminoacids. Also in salame romagnolo, the most representative compound was benzeneacetaldehyde, followed by ethanol. Differently from salame bresciano, in this sausage a remarkable amount of esters was found. After clustering analysis from 50 isolated from Bresciano sausage, subsequently 11 strains were sequenced that are 9 Lactobacillus sakei and 2 Lactobacillus curvatus. After clustering analysis from 100 isolated from Romagnolo sausage, subsequently 20 strains were sequenced that are 16 Lactobacillus sakei and 4 Lactobacillus curvatus. After 24 hours of incubation, all strains acidify the medium compared to MRS broth containing 0%, 2%, 4%, 6% NaCl. It was observed that all strains except E3B, E7B, E8G and E18G showed acidification activity in inverse proportion with increasing salt concentration. No of the isolates presented ability to accumulate biogenic amine with the exception of the strain C26G, identified as Lactobacillus curvatus, isolated from Romagnolo sausage. It confirmed its capacity to accumulate great amount of putrescine that is came from decarboxylation of ornitidin. Variability was observed among the volatile compound profile of all strains. Given the complexity of this medium in which any aroma compounds are already present, this screening took into consideration the differences between the concentration of each compounds and the (eventual) concentration of the same compound in the not-inoculated medium. As it is possible to observe seven of the main compounds detected are common with the isolates from Bresciano and Romagnolo. They are the namely 2,3-butanedione, 3-hydroxy-2-butanone (acetonile), 2-methyl-butanal, 3-methly-butanal,acetic acid, benzaldehyde, benzenacetaldehyde and 3-methyl-1-butanol. The PCA was able to group separately the isolates of salami bresciano, by contrast the major part of the isolates from the salami romagnolo were grouped, well separated from three strains (E26G, E1G, E28G). All strains isolated from the Bresciano strain were examined spot agar test against L. monocytogenes, L.innocua, Salmonella enterica ssp. Typhimurium and E. coli and all strains has antimicrobial resistance against indicator microorganisms with the clear zone between 0.75 and 1.4 cm. Cell-free supernatant test results showed that the use of mMRS broth was not suitable and that for MRS broth, incubation conditions of 25 ° C for 18 hours could be selected. This preliminary study has clarified some aspects regarding the differences between Bresciano and Romagnolo sausage with respect to their microbiolagical, physiological parameters and aromatic profile and also their isolates with respect to their growth behaviour, acidification profile, aromatic profile, antimicrobial activity against pathogens. And also owing to this study, we could eliminate a lot of strains which have not good technological properties as a starter culture. Further investigations are also needed to better elucidate aromatic profile and antimicrobial activity of strains in real systems. Antibiotic resistance of these strains should be tested.