Monitoring the bacterial population dynamics during the ripening of Galician chorizo,a traditional dry fermented Spanish sausage

The dynamics of the bacterial population throughout the ripening of Galician chorizo, a traditional dry fermented sausage produced in the north-west of Spain, were investigated by using classical and molecular approaches.     

Monitoring the lactic acid bacterial diversity during shochu fermentation by PCR-denaturing gradient gel electrophoresis

The presence of lactic acid bacteria (LAB) during shochu fermentation was monitored by PCR-denaturing gradient gel electrophoresis (DGGE) and by bacteriological culturing. No LAB were detected from fermented mashes by PCR-DGGE using a universal bacterial PCR primer set. However, PCR-DGGE using a new primer specific for the 16S rDNA of Lactococcus, Streptococcus, Tetragenococcus, Enterococcus, and Vagococcus and two primers specific for the 16S rDNA of Lactobacillus, Pediococcus, Leuconostoc, and Weissella revealed that Enterococcus faecium, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus nagelii, Lactobacillus plantarum, Lactococcus lactis, Leuconostoc citreum, Leuconostoc mesenteroides, and Weissella cibaria inhabited in shochu mashes. It was also found that the LAB community composition during shochu fermentation changed after the main ingredient and water were added during the fermentation process. Therefore, we confirmed that PCR-DGGE using all three primers specific for groups of LAB together was well suited to the study of the LAB diversity in shochu mashes. The results of DGGE profiles were similar to the results of bacteriological culturing. In conclusion, LAB are present during shochu fermentation but not dominant.     

Microbial population dynamics during spontaneous fermentation of Asparagus officinalis L. young sprouts

The aim of the present study was to assess the spontaneous fermentation of Asparagus officinalis L. young sprouts. For this purpose, asparagus fermentation was performed according to a traditional procedure originating from northern Greece. Young asparagus sprouts were cut, submerged in a brine solution (8 % NaCl) and placed in room temperature for fermentation to occur. Fermentation was monitored by measuring pH and total titratable acidity values as well as by qualitative and quantitative assessment of the microbiota dynamics. The latter was performed by classical microbiological techniques; clustering was performed by SDS-PAGE of whole cell proteins and rep-PCR and identification by sequencing of the 16S-rRNA gene. A culture-independent approach [PCR-denaturing gradient gel electrophoresis (DGGE)] was also applied in order to obtain a more integrated view of the microbiota dynamics. Lactic acid bacteria prevailed the fermentation forming a microbial consortium that was stable at species level; Lactobacillus sakei and Enterococcus faecium dominated until the 5th day while Weissella viridescens and W. cibaria dominated from the 7th day until the end of fermentation. Combination of SDS-PAGE with rep-PCR resulted in a very efficient clustering and differentiation also at sub-species level, revealing a succession at that level of all species throughout fermentation. On the other hand, application of PCR-DGGE was of limited usefulness.     

Molecular analysis of Oenococcus oeni population dynamics and the effect of aeration and temperature during alcoholic fermentation on malolactic fermentation

The effects of aeration and temperature during alcoholic fermentation (AF) on spontaneous and inoculated malolactic fermentation (MLF) of wine have been analysed by following the population dynamics of Oenococcus oeni strains with multiplex random amplified polymorphic DNA‐polymerase chain reaction. It has enabled us to follow precisely the proportion of different autochthonous strains and the starter strain through fermentations. Lack of aeration led to delays in AF, which meant that autochthonous lactic acid bacteria could develop earlier and prevented the starter strain from developing correctly. Temperature was not found to lead to any differences. Two strains were isolated in the same spontaneous MLF, suggesting that, in some cases, multiple strains might be responsible for the degradation of malic acid in wine. It can be concluded that delays in the AF can negatively affect the control of MLF and that this can be studied by following the development of the different strains of O. oeni.     

Moulds and ochratoxin A on surfaces of artisanal and industrial dry sausages

The use of moulds as a seasoning for sausage can have both desirable and undesirable consequences. The desirable consequences are the creation of a successful product that appeals to consumers. The undesirable consequences are due to the growth of undesirable moulds that produce highly toxic secondary metabolites referred to as mycotoxins. The aim of the paper was to investigate the presence of moulds producing ochratoxin A (OTA) on the surface of sausages from northern Italy. A total of 757 mould strains were isolated from sausage casings. The most frequently identified species were Penicillium nalgiovense, Penicillium oxalicum, Eurotium amstelodami, Penicillium olsonii, Penicillium chrysogenum, Penicillium verrucosum, Penicillium viridicatum, and Eupenicillium crustaceum. Aspergillus ochraceus was detected in only one production lot. Approximately 45% of these samples were positive for the presence of OTA. On the casings of the investigated sausages, the lowest and highest OTA values were 3 and 18 μg/kg, respectively. The OTA concentration was reduced to below the limit of detection (LOD) by brushing and washing the sausages prior to sale. From these data it appears that the presence of OTA on the surface of sausage (on the casings) is not indicative of any health risk for human consumption of sausage, since OTA was not identified inside the dry meat.     

Lipolytic and microbial changes during the natural fermentation and ripening of Greek dry sausages

The hydrolytic changes in the lipid fraction and the changes in microbial growth during the natural fermentation and ripening of Greek dry sausages were studied. Two batches were manufactured under industrial conditions, without the addition of starter cultures.

The results of the microbiological analyses indicated that, among the micro-organisms well known for their lipolytic activity, micrococci were predominant during the whole fermentation-ripening process. It was shown that the fatty acid composition of the hydrolysed lipid fraction did not remain constant with time. Analysis of the experimental results indicated that, given the length of the aliphatic chain (16 or 18 carbon atoms), the more unsaturated fatty acids were hydrolysed in preference to the saturated homologues. On the other hand, fatty acids having 16 carbon atoms were hydrolysed in preference to the iso-unsaturated ones having 18 carbon atoms.     

Type of bacterial starter culture,aging and fermentation effects on some characteristics of inoculated beef sausages

Beef sausage mixes were inoculated with either Pediococcus acidilactici with Staphylococcus xylosus or P. acidilactici with S. carnosus, subdivided and then held for 0, 24, 48 or 72 h at 8–10 °C prior to fermentation. After aging (pre-fermentation holding), the mixes were fermented for 16 h ending at 41 °C. Moisture, protein and fat contents of all sausage mixes did not differ due to holding effects over all starter cultures. The pH of mixes followed the same pattern for all mixes, declining (p < 0.05) from approximately 5.8 to pH 5.2–5.3 at 72 h aging and to 4.4–4.5 after fermentation. Total acidity of the mixes followed an inverse pattern to pH, increasing (p < 0.05) after fermentation although there was no effect due to type of starter culture. Aging had no effect on nonprotein nitrogen (NPN) content as ΔNPN among all cultures. After fermentation, however, sausages held 72 h and inoculated with S. carnosus had higher NPN contents compared to P. acidilactici alone (p < 0.05) and with S. xylosus (p < 0.10). The same effects of starter cultures on changes in total amino acid concentration were observed. Concentrations of individual amino acids showed increases depending on pre-fermentation aging period (0 h versus 72 h) followed by fermentation.     

High-pressure processing applied to cooked sausages: bacterial populations during chilled storage

Vacuum-packaged cooked sausages were pressurized at 500 MPa for 5 or 15 min at mild temperature (65 degrees C) and later stored at 2 and 8 degrees C for 18 weeks. Counts of aerobic mesophiles and psychrotrophs, lactic acid bacteria, enterobacteria, Baird-Parker microbiota, and Listeria spp. were determined 1 day and 3, 6, 9, 12, 15, and 18 weeks after treatment and compared with those of cooked sausages treated at 80 to 85 degrees C for 40 min. Pressurization generated reductions of about 4 log CFU/g in psychrotrophs and lactic acid bacteria. Enterobacteria and Listeria proved the most pressure sensitive; insignificant or no growth was detected throughout the study. Heat treatment inactivated psychrotrophs and enterobacteria similarly to pressure treatment. Listeria monocytogenes and enterotoxigenic Staphylococcus aureus were not found in treated samples. In general, there was no significant difference in counts of any bacterial populations either among treatments or between storage temperatures. High-pressure processing at mild temperature is an effective preservation method that can replace heat pasteurization applied to some cooked meat and poultry products after packaging.     

Yeast dynamics during spontaneous wine fermentation of the Catalanesca grape

Although the use of starter cultures in winemaking has improved the reproducibility and predictability of wine quality, the main drawback to this practice is the lack of the typical traits of wines produced by spontaneous fermentation. In this study, we identified for the first time the yeast population occurring during spontaneous fermentation of the Catalanesca white grape, a variety from Campania (Italy). Yeasts were identified using molecular tools: PCR-DGGE and partial sequence analysis of the 26S rRNA gene from isolates. Eighteen different species belonging to 11 different genera were identified. Hanseniaspora spp., Issatchenkia spp. and Candida spp. were the dominant yeasts during the early stages of fermentation, while the middle and end phases were dominated by Saccharomyces cerevisiae. Four species of Issatchenkia spp., rarely isolated from wine fermentation, were found in this study accounting for the 33.5% of the total isolates. The RAPD-PCR screening of the isolates followed by partial rRNA gene sequencing proved to be a very effective approach to first differentiate the isolates and then identify yeast species involved in a wine making procedure. The results show very high yeast diversity in this natural wine fermentation and also highlight the possibility of considering interesting autochthonous strains for starter selection.     

Physical and chemical changes of silver carp sausages during fermentation with Pediococcus pentosaceus

Changes in protein composition and physicochemical properties of silver carp sausages during fermentation were investigated. As fermentation progressed, the amount of salt-soluble and water-soluble proteins decreased gradually with a concomitant increase in insoluble proteins and non-protein constituents. The rapid reduction in pH to 4.5 within 48 h of fermentation coincided with a progressive increase in titratable acidity. The increasing content of TCA-soluble peptides during fermentation indicated intensive degradation of muscle proteins. SDS–PAGE showed that muscle proteins tended to aggregate into large polymers through disulphide bonds and non-disulphide covalent bonds during fermentation, which may be responsible for the formation of superior textural properties of silver carp sausages.     

Microbial community dynamics during the Scamorza Altamurana cheese natural fermentation

The growth dynamics of the natural microbial community responsible for the fermentation of Scamorza Altamurana, a typical Southern Italian cheese made using backslopping, was investigated applying a polyphasic approach combining 1) microbial enumeration with culture media, 2) randomly amplified polymorphic DNA (RAPD) fingerprinting of microbial communities, 3) sequencing of partial 16S ribosomal DNA (rDNA) genes, and 4) physiological tests. Viable cell counts on different culture media showed that the cocci community prevailed during the 18 h of curd fermentation and the 6 d of cheese ripening. RAPD fingerprinting made it possible to isolate 25 different strains identified by 16S rDNA sequencing as belonging to five species of Lactobacillus, three species of Streptococcus, one species of Weissella, and one species of Enterococcus. The physiological analyses of all lactic acid bacteria strains revealed that the isolates belonging to Streptococcus genus were the most acidifying, whereas lactobacilli were most proteolytic. Streptococcus thermophilus C48W and Lactobacillus delbrueckii subsp. bulgaricus B15Z dominated all through the fermentation process. Furthermore, they seemed to be stable in a subsequent whey sample analyzed after 7 mo. The recovery of strains endowed with interesting technological features, such as acidifying and proteolytic activities, and surviving in natural whey could allow the upscaling of cheese processing safeguarding the organoleptic characteristics of Scamorza Altamurana and could possibly improve other fermented dairy products.     

Effect of pasteurization and fermentation on residues of sulphonamides in sausages

The different effects of pasteurization and fermentation on sulphamethazine (SMZ) and sulphachlorpyridazine (SCP) were investigated in two common German meat products, luncheon meat and raw fermented sausages. There was no decrease in sulphonamide concentration (5 μg g^-1) in luncheon meat during the different stages of processing and storage (4°C up to 27 days). Less than 40% of the initial concentration remained in fermented sausages after ripening for 10 days in a climatized room. The sulphonamide content was reduced only if fermentation was involved in contrast with meat contaminated with other chemotherapeutic residues.     

Nitrate reduction during fermentation by Gram-negative bacterial activity in carrots

Carrots were subjected to lactic acid fermentation through the action of a starter culture, Lactobacillus plantarum, and changes in the amount of both the naturally present and added nitrate were recorded. The nitrate content in the carrots decreased to about 10% of the original amount during the initial stage of the fermentation process. By using irradiation-sterilized carrots it was shown that the decrease in the nitrate content is a result of the activity of the Gram-negative bacteria, which dominate the flora during the initial stage of the fermentation process, and that the lactic acid bacteria present were unable to affect the nitrate content. The nitrite concentration was also determined and was found not to exceed 0.2 mg/kg on any occasion. The conclusion is that if the nitrate content of carrots is to be lowered in a fermentation process, this process must be controlled in such a way as to allow the original Gram-negative flora to reduce the nitrate amount before the starter organism takes over.     

Monitoring the dynamic density of dough during fermentation using digital imaging method

A simple and new method was developed for monitoring the dynamic density of dough during fermentation process. In this method digital imaging was applied to determine volume of dough sample in actual proofing conditions, i.e., temperature and relative humidity of the fermentation oven. The method resulted that the volume increasing profile affected by temperature and relative humidity conditions of the fermentation oven. As when temperature and relative humidity was increased, volume expansion rate was higher. The data also demonstrated that dough density decrease with the investigated proofing temperatures of 25, 30 and 35 °C more significantly (p < 0.01) than proofing relative humidity of 65%, 75% and 85% (p < 0.05). The new imaging method have the advantage of being low cost and measuring dough density in actual proofing conditions as used in bread making.     

Lactic acid bacteria isolated from artisanal dry sausages: characterization of antibacterial compounds and study of the factors affecting bacteriocin production

Lactic acid bacteria (LAB) were isolated from artisanal dry sausages sampled from north-eastern region of Chaco, Argentina. Among 141 isolates, 27 showed antimicrobial activity against Listeria innocua, Staphyloccus aureus or Brochothrix spp. One isolate, identified as Lb. curvatus/sakei, produced bacteriocin like substances (BLIS). These BLIS were heat stable, effective after refrigerated storage and freeze/thaw cycles and even active against pathogens when produced under refrigeration at 3% NaCl concentration. The influence of several factors on production of BLIS was assessed in MRS broth added with: EDTA, ascorbic acid, KCl, potassium sorbate, sodium citrate, 3 and 6% NaCl, Tween 20 or Brij 35. These additives showed different effects towards the effectiveness of the bacteriocin produced by Lb. sakei/curvatus against L. innocua and S. aureus. Conditions that provided high cell density favored high bacteriocin production. BLIS production by this LAB strain was greatly influenced by NaCl concentration and the presence of surfactants.     

Molecular analysis of bacterial community dynamics during the fermentation of <Emphasis Type="Italic">soy-daddawa</Emphasis> condiment

Bacterial community dynamics during soy-daddawa fermentation was investigated using culture-dependent and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) molecular methods. The total titratable acidity (TTA), pH, and bacterial counts (BCs) were monitored daily during a 72-h fermentation period. Bacteria were characterized based on 16S rRNA gene sequencing. TTA ranged from 0.08 to 0.26 mg lactic acid/g, whereas pH ranged from 7.01 to 8.19. BCs increased from 3.9 to 10.61 log CFU/g. Fifty-eight isolates were obtained by culture method and clustered into seven operational taxonomic units (OTUs) at 97% sequence similarity, whereas four OTUs were obtained from the PCR-DGGE method. Taxonomic identification revealed that bacteria belonged to the genera Bacillus, Enterobacter, Enterococcus, and Staphylococcus with B. subtilis being present throughout fermentation. Medically significant isolates, including B. anthracis, Enterococcus casseliflavus, and Enterobacter hormaechei were detected. These results emphasize the need for starter culture utilization and offer a platform for starter culture screening and selection.     

Changes in sugars during storage of sausages

The shelf-life of fresh British sausages is determined by the production of off-odours and colour changes due to microflora in the sausage. From work on meat, and in pure culture, it is known that the carbon source of the principal spoilage microorganism (Brochothrix thermosphacta) can affect its metabolism and change the nature of the secondary metabolic products. The sugar content of sausages was determined immediately after manufacture and during storage at 4°C. Reducing-sugar content (measured by dinitrophenol phenolate, or DNP) increased in the first few days after manufacture, then decreased. The apparent increase in reducing sugar was due to hydrolysis of rusk components (oligo- or polysaccharides) either by meat or microbial enzymes. Thus, the amounts and types of sugar available for microbial growth change during storage. The effect of sulphite on the sugar assay and on sugar utilisation was also measured. Sulphite affected the DNP reducing sugar assay and caused a small increase in the absorbance at 510 nm. Addition of sulphite to the sausage formulation also caused some changes in the reducing-sugar content during storage at 4°C.     

Culture independent methods to assess the diversity and dynamics of microbiota during food fermentation

Culture independent methods first appeared in the food microbiology field at the end of the 90s and since then they have been applied extensively. These methods do not rely on cultivation and target nucleic acids (DNA and RNA) to identify and follow the changes that occur in the main populations present in a specific ecosystem. The method that has most often been used as a culture independent method in food microbiology is denaturing gradient gel electrophoresis (DGGE). The number of papers dealing with DGGE grew exponentially in the late nineties and, by analysing the studies available in the literature, it is possible to describe a trend in the subjects that have been investigated. DGGE was first used as a tool to monitor the ecology of fermented food, such as fermented sausage, cheese and sourdough, and later it also showed its potential in microbial spoilage process. In the last few years, the main application of DGGE has been to study fermented food from Asia, Africa and South America.     

Rapid differentiation of lactic acid bacteria from autochthonous fermentation of Iberian dry-fermented sausages

The populations of lactic acid bacteria (LAB) in different types of Iberian dry-fermented sausages from central-west Spain were identified. A simple and rapid electrophoretic method of whole-cell protein profiles was evaluated, correlating it with 16S rRNA gene sequence analysis and biochemical identification by API 50 CHL. A total of 96 isolates were identified by SDS-PAGE showing stable profiles corresponding to 30-45 polypeptides in the range 95-8kDa that were clearly different for the different species and were grouped with those of the 9 reference strains used in this study. The SDS-PAGE method showed that the predominant species were Pediococcus acidilactici (48%) followed by Lactobacillus plantarum (23%) and Lactobacillus brevis (18%). The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database. However, biochemical identifications by API 50 CHL showed different errors at the genus and species level. In sum, the SDS-PAGE analysis showed itself to be a rapid and accurate differentiation method for the most commonly encountered LAB isolates in dry-fermented sausages.     

Changes in the components of dry-fermented sausages during ripening.

Several chemical changes occur during the ripening of dry-fermented sausages that determine the flavor and odor of the end product. The phenomena that take place during fermentation, that is, both acidification of the sugars by lactic acid bacteria and reduction of nitrates and nitrites to nitric oxide by micrococci have been known for several years. However, the chemical changes involved in this process, and, particularly, the agents responsible have not yet been established, although they have been attributed to changes in the majority components (proteins and lipids) and to the ingredients added (spices and condiments) in the preparation of the original mixture. The typical flavor and odor of dry-fermented sausages cannot be attributed to volatile substances alone, but to a large number of volatile and nonvolatile compounds present in the product in suitable proportions. Microbial growth in the sausage together with activity of the meat endogenous enzymes are undoubtedly partially responsible for the development of a number of aromatic and sapid compounds. However, lipid autooxidation reactions are also an important source of these substances, and it is not yet known which of these processes is more important in sausage ripening. Much research has focused on the break up of triglycerides into free fatty acids, diglycerides, and monoglycerides during ripening and the progressive increase in the amounts of different carbonyl oxidation products. Carbonyl compounds probably play a significant role in determining the flavor because, in general, these have very low perception thresholds, in the ppm and ppb range. Similarly, the protein breakdown to yield peptides and amino acids has been studied extensively, the latter being substrates of several microbial and chemical reactions that generate many flavor compounds.