Microbial Ecology of the Soppressata of Vallo di Diano, a Traditional Dry Fermented Sausage from Southern Italy, and In Vitro and In Situ Selection of Autochthonous Starter Cultures

The microbial ecology of “soppressata of Vallo di Diano,” a traditional dry fermented sausage from southern Italy, was studied by using both culture-dependent and culture-independent approaches. The ripened fermented sausages were characterized by high microbial loads of both staphylococci and lactobacilli. Using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) targeting the variable V3 and V1 regions of the 16S rRNA gene and direct DNA sequencing, it was possible to identify Staphylococcus xylosus, S. succinus, and S. equorum among the staphylococci and Lactobacillus sakei and L. curvatus within the lactobacilli. Moreover, Debaryomyces hansenii was the main yeast species found by targeting the yeast 26S rRNA gene by PCR-DGGE. Selected strains of S. xylosus, L. sakei, and L. curvatus were characterized for their technological properties in the ripening conditions of the fermented sausages so as to select an autochthonous starter formulation. The selection included the determination of nitrate reductase, lipolytic, and antioxidant activity and proteolysis with myofibrillar and sarcoplasmic protein fractions. Such properties were evaluated in both in vitro and in situ assays; the latter were performed by using each strain as a starter in the laboratory-scale manufacture of soppressata of Vallo di Diano and by monitoring the microbiological and chemical changes at the end of ripening. The results show differences between the in vitro and in situ selection results and indicate that in situ evaluation of the technological performance of specific strains is better suited to selecting autochthonous starter cultures for fermented-meat products than in vitro evaluation.     

Influence of autochthonous starter cultures on microbial dynamics and chemical-physical features of traditional fermented sausages of Basilicata region

In this study, a combination of a Lactobacillus sakei strain and a Staphylococcus equorum strain was used as autochthonous starter for an experimental production of Basilicata fermented sausages. The influence of starter addition on the safety and quality parameters and microbiological and chemical-physical properties of the products was investigated. Microbial counts of safety indicators were lower in the samples with the addition of starter culture, and, at the end of ripening, Enterobacteriaceae and Gram negative bacteria were detected only in samples made without the starter addition. The addition of starter led to a final product with better microbiological and chemical-physical features, and a positive effect on flavor and aroma compounds formation by a good proteolytic and lipolytic activities. The use of autochthonous starter cultures allows to obtain products with the organoleptic characteristics expected and steady in time and to standardize the production process, improving the safety and quality, but preserving the essential character of the product.     

Monitoring of Staphylococcus xylosus DSM 20266 added as starter during fermentation and ripening of soppressata molisana, a typical Italian sausage

AIMS: "Soppressata molisana", a fermented sausage produced in southern Italy, is commonly obtained without starter addition. However, the use of starter cultures is more and more recommended in meat fermentation processes in order to guarantee stable production performance. In this study, the survival of the Staphylococcus xylosus DSM 20266 was evaluated during the ripening of "soppressata molisana" fermented sausage. METHODS AND RESULTS: The fastest method of RAPD-PCR was employed for discrimination of the added strain from those naturally present during the ripening of the "soppressata molisana". The results obtained were confirmed by analysis of the DNA macrorestriction profile by PFGE. The electrophoretic pattern of bacterial total proteins was also studied, but clear differences between the different strains could not be detected. CONCLUSIONS: The RAPD technique was a valid tool for monitoring Staph. xylosus DSM 20266 in "sopressata molisana". SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the possibility of monitoring the presence of Staph. xylosus strains during the ripening of fermented sausages by a reliable and repeatable technique such as RAPD.     

Carriers for starter cultures for the production of garri,a fermented food derived from cassava

Garri is a popular food in Nigeria derived from the fermentation of the mash obtained from the enlarged root of the cassava plant, Manihot esculenta Crantz. As currently produced, the mash used for garri production is spontaneously fermented; on account of this, there is great variability in the organoleptic properties and the quantities of residual cyanide in the garri from Nigeria. The use of starter cultures can help ensure uniformity in these properties if dry carriers can be found on which the fermentative organisms can survive for extended periods so as to facilitate the transportation of their carriers to the many small and scattered garri producers. We therefore studied the survival, singly or mixed, on dry starchy substrates derived from locally available crops, of Lactobacillus coryneformis, Lact. delbruckii, and Saccharomyces sp., which are associated with garri production, as carriers for these organisms. After 16 weeks of storage, between 75% and 85% of the organisms survived on yam, coco-yam, cassava in that order, whereas between 40 and 65% survived on rice and garri. Refrigeration at 4 °C did not improve the survival of the organisms, when compared to room temperature (30 °C) for the organisms stores on yam, coco-yam, and cassava. However where the organisms were stored on rice and garri, refrigeration improved the survivability of the organisms by between 10 and 20%.     

Selection of starter cultures for the production of kinema,a fermented soybean food of the Himalaya

Kinema, a traditional fermented soybean food, serves as a low-cost source of protein in the diet of the people of the Himalaya. The traditional method of kinema preparation results in a product with inconsistent quality. Forty five strains of spore-forming bacteria were isolated from nine samples of kinema collected from markets in the Darjeeling hills and Sikkim in India. Of these, 10 strains, identified as Bacillus subtilis (Ehrenberg) Cohn, were selected as possible starter cultures on the basis of enzyme activities and the production of slimy material. Protease activity (U/ml), -amylase activity (U/ml) and relative viscosity of the selected strains were 6.5 to 81.5, 0.1 to 9.3, and 1.1 to 20.1, respectively. Kinema produced by these strains showed nitrogen contents (expressed as a percentage of the total nitrogen content) of: water-soluble N, 48.4 to 76.5; TCA-soluble N, 16.0 to 27.6; formal N, 5.0 to 12.5; ammonia-N, 4.4 to 7.8%. Reducing sugar was 1.1 to 2.0% of the wet weight and relative viscosity was from 2.0 to 35.2. Correlation matrices of the biochemical parameters and sensory attributes of the kinema produced by these B. subtilis strains were statistically analysed. Strains KK-2:B10 and GK-2:B10 of B. subtilis were the best starter cultures for improved kinema production.     

Multiphasic approach to study the bacterial ecology of fermented sausages inoculated with a commercial starter culture

In this paper, the ability of a commercial starter culture to perform a sausage fermentation is evaluated. Molecular analysis revealed the presence of several strains of the same species contained in the starter culture with different behavior during the fermentation, and the contribution of Lactobacillus curvatus, which was only marginally isolated during the transformation.     

Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage

The likelihood that products prepared from raw meat and milk may act as vehicles for antibiotic-resistant bacteria is currently of great concern in food safety issues. In this study, a collection of 94 tetracycline-resistant (Tc(r)) lactic acid bacteria recovered from nine different fermented dry sausage types were subjected to a polyphasic molecular study with the aim of characterizing the host organisms and the tet genes, conferring tetracycline resistance, that they carry. With the (GTG)(5)-PCR DNA fingerprinting technique, the Tc(r) lactic acid bacterial isolates were identified as Lactobacillus plantarum, L. sakei subsp. carnosus, L. sakei subsp. sakei, L. curvatus, and L. alimentarius and typed to the intraspecies level. For a selection of 24 Tc(r) lactic acid bacterial isolates displaying unique (GTG)(5)-PCR fingerprints, tet genes were determined by means of PCR, and only tet(M) was detected. Restriction enzyme analysis with AccI and ScaI revealed two different tet(M) allele types. This grouping was confirmed by partial sequencing of the tet(M) open reading frame, which indicated that the two allele types displayed high sequence similarities (>99.6%) with tet(M) genes previously reported in Staphylococcus aureus MRSA 101 and in Neisseria meningitidis, respectively. Southern hybridization with plasmid profiles revealed that the isolates contained tet(M)-carrying plasmids. In addition to the tet(M) gene, one isolate also contained an erm(B) gene on a different plasmid from the one encoding the tetracycline resistance. Furthermore, it was also shown by PCR that the tet(M) genes were not located on transposons of the Tn916/Tn1545 family. To our knowledge, this is the first detailed molecular study demonstrating that taxonomically and genotypically diverse Lactobacillus strains from different types of fermented meat products can be a host for plasmid-borne tet genes.     

Dermatological remedies in the traditional pharmacopoeia of Vulture-Alto Bradano, inland southern Italy

This study documents indigenous medicinal plant utilization, management and the threats affecting them. The study was carried out in Mana Angetu district between January 2003 and December 2004. Ethnobotanical data were collected using semi structured interviews, field observations, preference and direct matrix ranking with traditional medicine practitioners. The ethnomedicinal use of 230 plant species was documented in the study area. Most of the plants (78.7%) were reportedly used to treat human diseases. The most frequently used plant part were roots (33.9%), followed by leaves (25.6%). Most of the medicinal species (90.4%) were collected from the wild. Direct matrix analysis showed that Olea europaea L. Subsp. cuspidata (Wall. ex G. Don) was the most important species followed by Acacia tortilis (Forssk.) Hayne (120) indicating high utility value of these species for the local community. The principal threatening factors reported were deforestation (90%), agricultural expansion (85%) and fire (53%). Documenting the eroding plants and associated indigenous knowledge can be used as a basis for developing management plans for conservation and sustainable use of medicinal plants in the area.     

Saccharomyces cerevisiae and Candida tropicalis as starter cultures for the alcoholic fermentation of tchapalo, a traditional sorghum beer

Starter cultures of Candida tropicalis and Saccharomyces cerevisiae isolated from tchapalo were tested in pure culture and co-culture of four ratios [2:1, 25:4, 1:4, 2:3 (cells/cells)] for their ability to ferment sorghum wort to produce tchapalo. All the starters showed means growth rate between 0.043 and 0.101 h^?1. Only C. tropicalis in pure culture showed growth rate lower than that of S. cerevisiae in single culture. During fermentation, according to total soluble solids depletion, yeast starters could be grouped in four different profiles. But in the beer produced, total soluble solids contents were statistically identical. The lowest values were obtained with co-culture C. tropicalis + S. cerevisiae in the ratios of 2:1 and 2:3. Starter cultures with large ratio of C. tropicalis produced a higher organic acids and 2-butanone than S. cerevisiae in pure culture. However, co-culture C. tropicalis + S. cerevisiae (2:1) was the alone starter which produced higher ethanol than S. cerevisiae in pure culture. The beers produced with C. tropicalis + S. cerevisiae (25:4), C. tropicalis + S. cerevisiae (1:4) and C. tropicalis were widely different from those produced with the others starter cultures.     

Mid- and late-Holocene vegetation and fire history at Biviere di Gela, a coastal lake in southern Sicily, Italy

The vegetation and fire history of few coastal sites has been investigated in the Mediterranean region so far. We present the first paleoecological reconstruction from coastal Sicily, the largest island in the Mediterranean Sea. We analysed pollen and charcoal in the sediments of Biviere di Gela, a lake (lagoon) on the south coast of Sicily. Our data suggest that the area became afforested after a marine transgression at ca. 7200 cal b.p. (5250 b.c.). Build-up of forest and shrublands took ca. 200–300 years, mainly with the deciduous trees Quercus, Ostrya and Fraxinus. Juniperus expanded ca. 6900 cal b.p. (4950 b.c.), but declined again 6600 cal b.p. (4650 b.c.). Afterwards, evergreen trees (Q. ilex-type and Olea) became dominant in the forest and Pistacia shrublands were established. Forest and shrubland reached a maximum ca. 7000–5000 cal b.p. (5050–3050 b.c.); subsequently forest declined in response to human impact, which was probably exacerbated by a general trend towards a more arid climate. During the Neolithic, fire was used to open the landscape, significantly reducing several arboreal taxa (Q. ilex, Fraxinus, Juniperus) and promoting herbs and shrubs (Achillea, Cichorioideae, Brassicaceae, Ephedra). Final forest disruption occurred around 2600 cal b.p. (650 b.c.) with the onset of the historically documented Greek colonization. We conclude that the open maquis and garrigue vegetation of today is primarily the consequence of intensive land-use over millennia. Under natural or near-natural conditions arboreal taxa such as Q. ilex, Olea and Pistacia would be far more important than they are today, even under the hot and rather dry coastal conditions of southern Sicily.     

Characterization and technological properties of Oenococcus oeni strains from wine spontaneous malolactic fermentations: a framework for selection of new starter cultures

Aims:  To characterize the genetic and phenotypic diversity of 135 lactic acid bacteria (LAB) strains isolated from Italian wines that undergone spontaneous malolactic fermentation (MLF) and propose a multiphasic selection of new Oenococcus oeni malolactic starters.
Methods and Results:  One hundred and thirty-five LAB strains were isolated from 12 different wines. On the basis of 16S amplified ribosomal DNA restriction analysis (ARDRA) with three restriction enzymes and 16S rRNA gene sequencing, 120 O. oeni strains were identified. M13-based RAPD analysis was employed to investigate the molecular diversity of O. oeni population. Technological properties of different O. oeni genotypes were evaluated in synthetic medium at increasing selective pressure, such as low pH (3·5, 3·2 and 3·0) and high ethanol values (10, 11 and 13% v/v). Finally, the malolactic activity of one selected strain was assessed in wine by malolactic trial in winery.
Conclusions:  The research explores the genomic diversity of wine bacteria in Italian wines and characterizes their malolactic metabolism, providing an efficient strategy to select O. oeni strains with desirable malolactic performances and able to survive in conditions simulating the harsh wine environment.
Significance and Impact of the Study:  This article contributes to a better understanding of microbial diversity of O. oeni population in Italian wines and reports a framework to select new potentially O. oeni starters from Italian wines during MLF.     

Fate of Escherichia coli O157:H7 as affected by pH or sodium chloride and in fermented, dry sausage.

The influence of pH adjusted with lactic acid or HCl or sodium chloride concentration on survival or growth of Escherichia coli O157:H7 in Trypticase soy broth (TSB) was determined. Studies also determined the fate of E. coli O157:H7 during the production and storage of fermented, dry sausage. The organism grew in TSB containing less than or equal to 6.5% NaCl or at a pH of 4.5 to 9.0, adjusted with HCl. When TSB was acidified with lactic acid, the organism grew at pH 4.6 but not at pH 4.5. A commercial sausage batter inoculated with 4.8 x 10(4) E. coli O157:H7 per g was fermented to pH 4.8 and dried until the moisture/protein ratio was less than or equal to 1.9:1. The sausage chubs were then vacuum packaged and stored at 4 degrees C for 2 months. The organism survived but did not grow during fermentation, drying, or subsequent storage at 4 degrees C and decreased by about 2 log10 CFU/g by the end of storage. These studies reveal the importance of using beef containing low populations or no E. coli O157:H7 in sausage batter, because when initially present at 10(4) CFU/g, this organism can survive fermentation, drying, and storage of fermented sausage regardless of whether an added starter culture was used.     

Activity of plantaricin SA6, a bacteriocin produced by Lactobacillus plantarum SA6 isolated from fermented sausage

N. REKHIF, A ATRIH AND G. LEFEBVRE. 1995. Plantaricin SA6, a bacteriocin produced by Lactobacillus plantarum SA6, exhibited an inhibitory action against several mesophilic lactobacilli. It was stable at 90–100°C at pH 2–4 and it remained stable in the presence of several organic solvents, urea or β-mercaptoethanol. Plantaricin SA6 bound specifically to the cell surface of only plantaricin SA6-sensitive bacteria. The putative receptors are not destroyed by different hydrolytic enzymes added to the phosphate buffer. Plantaricin SA6 acted as a bactericidal agent lysing sensitive strains, that became more permeable to ortho-nitro-phenol-β-galactoside and lost their intracellular K⁺ ions and u.v.-absorbing materials. Both the adsorption and lethal action of plantaricin SA6 were maximal between pH 4 and 7, but the range of temperature tested (5–37βC) had no effect. Ions (of several salts such as MgCl₂) inhibited the binding of plantaricin SA6 and protected cells against bacteriocin action.     

Effect of sausage ingredients and additives on the production of enterocin A and B by Enterococcus faecium CTC492. Optimization of in vitro production and anti-listerial effect in dry fermented sausages

Enterocin A and B in Enterococcus faecium CTC492 were co-induced by the different factors assayed in this study (r = 0.93) and followed primary metabolic kinetics. Enterocin production was significantly inhibited by sausage ingredients and additives, with the exception of nitrate. The addition of sodium chloride and pepper decreased production 16-fold. The temperature and pH influenced enterocin production, with optima between 25 and 35 degrees C, and from 6.0 to 7.5 of initial pH. The maximum activity was achieved, under favourable growth conditions, with MRS supplemented with sucrose (2%) plus glucose (0.25%) and Tween-80 (1%). MRS concentration, NaCl plus pepper addition, absence of Tween-80 in the growth medium, incubation at 45 degrees C and an initial pH under 5.5 were detrimental to bacteriocin production. Stress conditions did not favour enterocin production. Desadsorption was Tween-dependent. Enterocin A activity in the crude extracts stored at -80 degrees C was better preserved than enterocin B (when tested against their specific indicator strain), but anti-listerial activity remained intact. Applied as anti-listerial additives in dry fermented sausages, enterocins significantly diminished Listeria counts by 1. 13 log (P < 0.001), while Enterococcus faecium CTC492 added as starter culture did not significantly reduce Listeria counts (P > 0. 1) compared with the standard starter culture (Bac-). Enterocins A and B could be considered as extra biopreservative hurdles for listeria prevention in dry fermented sausages.     

Technological properties of Lactobacillus fermentum involved in the processing of dolo and pito, West African sorghum beers, for the selection of starter cultures

Aim: Technological properties of Lactobacillus fermentum isolates involved in spontaneous fermentation of dolo and pito wort were examined to select starter cultures. Methods and Results: 264 isolates were screened for antimicrobial activity, acidifying activity, exopolysaccharides (EPSs) and amylase production. An antimicrobial activity was detected for 33·3%, 31·8%, 22·7% and 15·9% of the isolates towards Staphylococcus aureus enterotoxin A producer, Staphylococcus aureus enterotoxin A and B producer, Escherichia coli and Listeria innocua, respectively. A similarity was found between the isolates which were clustered in four groups according to their rates of acidification of sorghum malt broth. The faster acidifying group of isolates (43·48%) had a rate of acidification evaluated as ΔpH of 1·14 ± 0·15 pH unit after 6 h of fermentation, followed by a second group of isolates (38·08%) with a similar rate of acidification after 9 h of fermentation. From the isolates endowed with an antimicrobial activity, 5·76% belonged to the faster acidifying group and 40·38% belonged to the second group. 88·7% of the isolates had the ability for producing EPSs but not amylase. Conclusion: Lactobacillus fermentum ferments dolo and pito wort by lowering the pH and providing organic acids, EPSs and antimicrobial compounds. Significance and Impact of the Study: Strains with a rapid rate of acidification, an antimicrobial activity and producing EPSs are suggested to have potential for starter cultures.     

Isolation and characterization of lactic acid bacteria from suan-tsai (fermented mustard), a traditional fermented food in Taiwan

AIMS: To isolate, characterize, and identify lactic acid bacteria (LAB) in suan-tsai (fermented mustard), a traditional fermented food in Taiwan. METHODS AND RESULTS: Suan-tsai samples were collected at five time points from a fixed fermenting bucket. Fifty cultures were isolated from suan-tsai samples, and isolates were divided into classes by phenotype and then into groups by restriction-fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Phenotypic and biochemical characteristics identified two different bacterial groups (A and B), and the results showed that Pediococcus pentosaceus was the most abundant LAB during the initial fermentation time. However, the more NaCl-tolerant species Tetragenococcus halophilus took the place of P. pentosaceus and became the most abundant LAB later. All isolates were grown in de Man, Rogosa, and Sharpe (MRS) broth containing 6% NaCl, but T. halophilus could grow only in MRS broth containing 10% NaCl. CONCLUSIONS: These results suggest that the LAB P. pentosaceus and T. halophilus play roles in the fermentation of suan-tsai. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the distribution and varieties of LAB that exist in the suan-tsai fermentation process.     

Isolation and characterization of lactic acid bacteria from pobuzihi (fermented cummingcordia), a traditional fermented food in Taiwan

Lactobacillus pobuzihii is a novel species which has been previously found in pobuzihi (fermented cummingcordia), a traditional fermented food in Taiwan. However, the lactic acid bacteria (LAB) microflora in pobuzihi has not been studied in detail. In this study, LAB from pobuzihi were isolated, identified, and characterized. A total of 196 LAB were isolated; 79 cultures were isolated from the sample collected from a manufacturing factory, 38 from pobuzihi samples collected from 4 different markets, and 79 from 2 fresh cummingcordia samples. These isolates were characterized phenotypically and then divided into eight groups (A to H) by restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Lactobacillus plantarum was the most abundant LAB found in most samples during the fermentation of pobuzihi. On the other hand, Enterococcus casseliflavus and Weissella cibaria were, respectively, the major species found in the two fresh cummingcordia samples. A potential novel species or subspecies of lactococcal strain was found. In addition, seven L. plantarum and five W. cibaria strains showed inhibitory activity against the indicator strain Lactobacillus sakei JCM 1157^T. This is the first report describing the distribution and varieties of LAB existing in the pobuzihi during its fermentation process and the final product on the market.