Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucuk

In this study, the culture-dependent and culture-independent molecular methods were used for the identification of lactic acid bacteria (LAB) in sucuk a Turkish fermented dry sausage. On the one hand, the PCR-DGGE method targetting the V1 and V3 regions of 16S DNA was applied to DNA that was directly extracted from sucuk samples. On the other hand, rep-PCR fingerprinting was performed for the primary differentiation and grouping of the isolates, and the results were confirmed by sequencing of the 16S rDNA and 16S-23S rDNA intergenic spacer region. As a result of the PCR-DGGE analysis of all the samples, total 8 different lactic acid bacteria were identified, and Lactobacillus sakei, Lactobacillus curvatus and Weissella viridescens were the dominant microbiota among these bacteria. The culture-dependent approach indicated that the majority of the strains belonged to the Lactobacillus genera including Lb. sakei, Lactobacillus plantarum, Lb. curvatus, Lactobacillus brevis, Lactobacillus farciminis and Lactobacillus alimentarius. However, Leuconostoc and Weisella were also detected as minor genera. Again, Lactococcus piscium, Weissella halotolerans, Staphylococcus succinus and the comigrated Staphylococcus piscifermentans/Staphylococcus condimenti/Staphylococcus carnosus group were detected only with the culture-independent method while Lb. plantarum, Leuconostoc mesenteroides and Leuconostoc citreum were identified only by using the culture-dependent method. In the results, it was concluded that the combination of culture-dependent and culture-independent methods was necessary for reliable and detailed investigation of LAB communities in fermented food products.     

Ability of meat starter cultures to catabolize leucine and evaluation of the degradation products by using an HPLC method

The degradation of leucine generates aroma compounds involved in the typical flavour of dry fermented sausage. The ability of lactic acid bacteria and staphylococci of meat origin to produce aromatic compounds from leucine was studied. Whole cells were incubated with³H-labelled leucine under different conditions. The radioactive metabolites produced were analysed by high performance liquid chromatography. All the strains studied were able to catabolise leucine but catabolic profiles were quantitatively and qualitatively different. The catabolism of leucine by Lactobacillus sakei,Lactobacillus curvatus and Pediococcus acidilactici was very low and required α -ketoglutarate, which indicates that an aminotransferase was involved in the first step of leucine catabolism. Carnobacterium piscicola produced a large amount of 3-methyl butanal, whereas Staphylococcus carnosus mainly produced 3-methyl butanoic acid. Leucine was mainly degraded into α -ketoisocaproic acid by Lactobacillus plantarum and Carnobacterium divergens. Nitrate affected leucine catabolism only in S. carnosus.     

Lactic acid bacteria as protective cultures against Listeria spp. on cold-smoked salmon

Three bacteriocin producing (Bac⁺) strains of Lactobacillus sakei were used singly and in combination with each other as protective cultures to control the growth of listeria in cold-smoked salmon. Challenge experiments were conducted under practical conditions in a smokehouse. The surface of salmon sides was inoculated with 10⁴ cfu/g of Listeria innocua and 10⁷ cfu/g of Bac⁺ lactic acid bacteria as well as a L. sakei Bac⁻ control. After smoking the counts of listeria and lactic acid bacteria were determined at days 1 and 14. All Bac⁺ L. sakei strains reduced the counts of L. innocua by >2 log units. Strain LTH5754 was an isolate from cold-smoked salmon and achieved even a 5 log reduction of L. innocua within the storage period. In vitro experiments showed, that the Bac⁺ strains were also effective against L. monocytogenes (three strains tested) and L. ivanovii (1 strain). The pH as well the sensorial properties of the smoked salmon were not affected by the L. sakei inocula.     

Robustness of Lactobacillus plantarum starters during daily propagation of wheat flour sourdough type I

This study aimed at investigating the robustness of selected sourdough strains of Lactobacillus plantarum. Seven strains were singly used as sourdough type I starters under daily back-slopping propagation (ten days) using wheat flour. Cell numbers of presumptive lactic acid bacteria varied slightly (median values of 9.13–9.46 log cfu g⁻¹) between and within started sourdoughs, as well as the acidifying activity (median values of 1.24–1.33). After three days also the control sourdough (unstarted) had the same values. As shown by RAPD-PCR analysis, five (DB200, 3DM, G10C3, 12H1 and LP20) out of seven strains maintained elevated cell numbers (ca. 9 log cfu g⁻¹) throughout ten days. The other two strains progressively decreased to less than 5 log cfu g⁻¹. As identified by partial sequencing of 16S rRNA and recA genes, L. plantarum (11 isolates), pediococci (7), Lactobacillus casei (3) and Lactobacillus rossiae (2) dominated the flour microbiota. Monitoring of lactic acid bacteria during sourdough propagation was carried out by culture dependent approach and using PCR-DGGE (Denaturing Gradient Gel Electrophoresis). Except for the sourdough started with L. plantarum LP20, in all other sourdoughs at least one autochthonous strain of L. plantarum emerged. All emerging strains of L. plantarum showed different RAPD-PCR profiles. L. rossiae and Pediococcus pentosaceus were only found in the control and sourdough started with strain 12H1. The characterization of the catabolic profiles of sourdoughs (Biolog System) showed that sourdoughs containing persistent starters behaved similarly and their profiles were clearly differentiated from the others. One persistent strain (DB200) of L. plantarum and Lactobacillus sanfranciscensis LS44, previously shown to be persistent ( Siragusa et al., 2009), were used as the mixed starter to produce a wheat flour sourdough. Both strains cohabited and dominated during ten days of propagation.     

The influence of exopolysaccharide-producing lactic acid bacteria on reconstructed ham

The influence of in-situ-formed exopolysaccharides (EPS) by lactic acid bacteria (LAB; Lactobacillus sakeiTMW 1.411 and Lactobacillus plantarumTMW 1.1478) on the yield and texture of reconstructed ham was investigated. No differences in yield (P > 0.05) were observed but weight loss during storage was significantly higher (P < 0.05) for hams produced with L. sakei 1.411. Furthermore, the produced EPS decreased the hardness (P < 0.05; 50.22 N for control compared to 44.81 N) but did not influence the cohesiveness (P > 0.05) of hams. Products with L. plantarum 1.1478 showed no significant differences in comparison to the control. This could be attributed to the lower amount of EPS formed during ripening. L. sakei 1.411 produced 194.49 ± 5.34 mg kg⁻¹ EPS, whereas L. plantarum 1.1478 formed 60.26 ± 2.96 mg kg⁻¹ EPS. Thus, the use of the examined LAB is not recommended in reconstructed ham, since they have no or even a negative influence on the product quality.     

Biogenic amine production by Gram-positive bacteria isolated from Spanish dry-cured "chorizo" sausage treated with high pressure and kept in chilled storage

We studied the production of biogenic amines by 200 strains of lactic acid bacteria and staphylococci isolated during chilled storage from samples of Spanish dry-cured “chorizo” sausage treated with high-pressure. The presence of biogenic amines in a decarboxylase synthetic broth was confirmed by ion-exchange chromatography. β-phenylethylamine was the biogenic amine more frequently produced (22.5%), followed by tyramine (7.5%). In tyramine producer-strains the presence of a tyrosine decarboxylase gene was confirmed by PCR. Among lactic acid bacteria, the production of tyramine was mainly related to the species Lactobacillus curvatus. Most of the L. curvatus strains were also β-phenylethylamine-producers. In relation to staphylococci, tyramine-production was mainly associated to Staphylococcus carnosus strains. The S. carnosus strains analysed in this study produced β-phenylethylamine or β-phenylethylamine and tyramine simultaneously. RAPD-PCR results indicated that the biogenic amine-producer S. carnosus population changes along storage independently of the high-pressure treatment.     

The microbial stability of two bakery sourdoughs made from conventionally and organically grown rye

In order to determine the microbial stability of sourdoughs, two bakery sourdoughs made from conventionally and organically grown rye, produced on a large production scale under defined conditions, were studied at regular intervals over a period of 7 months. The sourdoughs contained lactic acid bacteria at levels of 8·43–9·14 log₁₀cfu g⁻¹and yeasts at levels of 6·00–8·04 log₁₀cfu g⁻¹. The number of lactic acid bacteria seemed to increase and the number of yeast to decrease with increasing fermentation time. Four types (A–D) of lactic acid bacteria dominated in both sourdough systems. Their frequencies varied during the sampling period, but seemed to follow the same trend in both systems. Identification based on API 50CH assimilation profiles, classical identification methods and 16S ribosomal DNA sequencing showed that the lactic acid bacteria contained in the two sourdough systems not clearly belonged to any known species of the genus Lactobacillus. Saccharomyces cerevisiae was the only yeast species isolated from the sourdoughs, represented by different phenotypes. In the conventional sourdough system one phenotype dominated throughout the sampling period. In the organic sourdough the phenotype changed after 3 months.     

Effect of the use of autochthonous Lactobacillus curvatus, Lactobacillus plantarum and Staphylococcus xylosus strains on microbiological and biochemical properties of the Sardinian fermented sausage

In this study, a starter culture was tentatively made by testing autochthonous Lactobacillus and Staphylococcus strains, isolated from traditional ‘Salsiccia Sarda’ sausage. Three sausage-making trials were carried out using an association of Staphylococcus xylosus + Lactobacillus curvatus (A), Staphylococcus xylosus + Lactobacillus plantarum (B), and Lactobacillus plantarum alone (C). Microbiological profiles, free amino acids, biogenic amines, and free fatty acids were evaluated after 21 days of ripening. Sausages A and B showed the highest number of lactic acid bacteria that produced a rapid acidification of substrates and reduced the number of spoilage microorganisms. Sausages made with L. plantarum alone or together with S. xylosus had the highest content of free amino acids, whereas the one produced with S. xylosus + L. curvatus showed the lowest content of total biogenic amines and best acceptance score. No significant differences among samples were found as to the free fatty acids concentration.     

Molecular and technological characterization of lactic acid bacteria from traditional fermented sausages of Basilicata region (Southern Italy)

Lactic acid bacteria (LAB) from traditional fermented sausages of the Basilicata region were investigated by ARDRA-PCR and RAPD-PCR for taxonomic identification at species and strain level and characterized on the basis of the growth and acidification at different temperatures, incubation times, levels of NaCl and KNO₂, hydrolysis of sarcoplasmatic and myofibrillar proteins and antimicrobial, peptide/amino acid release and nitrate reductase activities.Lactobacillus sakei was the predominant species (67%) followed by Pediococcus pentosaceus (16%), Leuconostoc carnosum (8%), Lactobacillus plantarum (4%), Lactobacillus brevis (2%) and Leuconostoc pseudomesenteroides (2%). The technological characterization revealed that most of the isolates had good acidifying and proteolytic properties. Moreover, Lb. sakei strains showed antimicrobial ability, while Leuconostoc strains the highest reduction of nitrates.This work was a preliminary study in the formulation of autochthonous starter cultures in order to standardize the production process of sausages, to preserve their typical organoleptic and sensory characteristics and to improve the quality of final product.     

Influence of<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis> on<Emphasis Type="Italic"> Staphylococcus aureus</Emphasis> growth in a fresh vegetable model system

The influence of a Lactobacillus plantarum (B₄) on the growth of Staphylococcus aureus (Sa₄) was verified by impedometric methods in a suitable model reproducing the characteristics of fresh vegetables. The inoculum size of the single strains and their growth temperature were varied according to a Central Composite Design. The results obtained via statistical analysis showed that the temperature affected the growth of both S. aureus and L. plantarum strains. The pathogenic strain, independently of its inoculum size, was inhibited by L. plantarum at all the tested temperatures. A proper combination of specific lactic acid bacteria and storage temperature should improve the safety of the vegetable products.     

Technological and safety properties of lactic acid bacteria isolated from Spanish dry-cured sausages

Technological and safety-related properties were analyzed in lactic acid bacteria isolated from Spanish dry-cured sausages in order to select them as starter cultures. In relation to technological properties, all the strains showed significative nitrate reductase activity; Lactobacillus plantarum, Lactobacillus paracasei and 52% of the Enterococcus faecium strains showed lipolytic activity and only Lactobacillus sakei strains (43%) were able to form biofilms. Related to safety aspects, E. faecium strains were the most resistant to antibiotics, whereas, L. sakei strains were the most sensitive. In relation to virulence factors, in the E. faecium strains analyzed, only the presence of efaA gene was detected. The analysis of biogenic amine production showed that most E. faecium strains and L. sakei Al-142 produced tyramine. In conclusion, L. paracasei Al-128 and L. sakei Al-143 strains possess the best properties to be selected as adequate and safe meat starter cultures.     

Nonstarter Lactobacillus strains as adjunct cultures for cheese making: in vitro characterization and performance in two model cheeses

Nonstarter lactic acid bacteria are the main uncontrolled factor in today's industrial cheese making and may be the cause of quality inconsistencies and defects in cheeses. In this context, adjunct cultures of selected lactobacilli from nonstarter lactic acid bacteria origin appear as the best alternative to indirectly control cheese biota. The objective of the present work was to study the technological properties of Lactobacillus strains isolated from cheese by in vitro and in situ assays. Milk acidification kinetics and proteolytic and acidifying activities were assessed, and peptide mapping of trichloroacetic acid 8% soluble fraction of milk cultures was performed by liquid chromatography. In addition, the tolerance to salts (NaCl and KCl) and the phage-resistance were investigated. Four strains were selected for testing as adjunct cultures in cheese making experiments at pilot plant scale. In in vitro assays, most strains acidified milk slowly and showed weak to moderate proteolytic activity. Fast strains decreased milk pH to 4.5 in 8 h, and continued acidification to 3.5 in 12 h or more. This group consisted mostly of Lactobacillus plantarum and Lactobacillus rhamnosus strains. Approximately one-third of the slow strains, which comprised mainly Lactobacillus casei, Lactobacillus fermentum, and Lactobacillus curvatus, were capable to grow when milk was supplemented with glucose and casein hydrolysate. Peptide maps were similar to those of lactic acid bacteria considered to have a moderate proteolytic activity. Most strains showed salt tolerance and resistance to specific phages. The Lactobacillus strains selected as adjunct cultures for cheese making experiments reached 10⁸ cfu/g in soft cheeses at 7 d of ripening, whereas they reached 10⁹ cfu/g in semihard cheeses after 15 d of ripening. In both cheese varieties, the adjunct culture population remained at high counts during all ripening, in some cases overcoming or equaling primary starter. Overall, proximate composition of cheeses with and without added lactobacilli did not differ; however, some of the tested strains continued acidifying during ripening, which was mainly noticed in soft cheeses and affected overall quality of the products. The lactobacilli strains with low acidifying activity showed appropriate technological characteristics for their use as adjunct cultures in soft and semihard cheeses.     

Taxonomic structure of the yeasts and lactic acid bacteria microbiota of pineapple (Ananas comosus L. Merr.) and use of autochthonous starters for minimally processing

Pichia guilliermondii was the only identified yeast in pineapple fruits. Lactobacillus plantarum and Lactobacillus rossiae were the main identified species of lactic acid bacteria. Typing of lactic acid bacteria differentiated isolates depending on the layers. L. plantarum 1OR12 and L. rossiae 2MR10 were selected within the lactic acid bacteria isolates based on the kinetics of growth and acidification. Five technological options, including minimal processing, were considered for pineapple: heating at 72 °C for 15 s (HP); spontaneous fermentation without (FP) or followed by heating (FHP), and fermentation by selected autochthonous L. plantarum 1OR12 and L. rossiae 2MR10 without (SP) or preceded by heating (HSP). After 30 days of storage at 4 °C, HSP and SP had a number of lactic acid bacteria 1000 to 1,000,000 times higher than the other processed pineapples. The number of yeasts was the lowest in HSP and SP. The Community Level Catabolic Profiles of processed pineapples indirectly confirmed the capacity of autochthonous starters to dominate during fermentation. HSP and SP also showed the highest antioxidant activity and firmness, the better preservation of the natural colours and were preferred for odour and overall acceptability.     

The population change of yeasts in commercial salami

The survival of yeast during a pilot scale production of commercial salami was investigated. A total of 108 distinctive yeast strains were isolated and identified during the processing of the salami. Initially, the number of yeasts remained below 10³cfu g⁻¹but their numbers increased after the 12^thday of maturation, reaching a maximum of 2·0×10⁵cfu g⁻¹at day 20. During maturation, the pH declined from 5·72 to 4·36, water content from 58% to 43%, while the salt content increased by 1%. The number of lactic acid bacteria remained above 10⁵cfu g⁻¹throughout processing and maturation. Of the 108 yeast strains isolated, 22 strains were identified as members of the species Debaryomyces hansenii, being present in all samples taken. In descending numbers, Rhodotorula mucilaginosa, Yarrowia lipolytica and Cryptococcus albidus, were also frequently isolated during processing and maturation.     

Characterization of Enterobacteriaceae strains isolated from spoiled dry-cured hams

Microbiological and physicochemical aspects of spoiled specimens of dry-cured hams affected by deep putrefaction were studied. Total aerobe (10³–10⁴cfu g⁻¹), Micrococcaceae(10⁴cfu g⁻¹) and lactic acid bacteria (<10²cfu g⁻¹) were present at the same levels as in unspoiled control hams. The overall hygienic quality of the hams was good, even when spoiled. Only Enterobacteriaceae counts (10²–10³cfu g⁻¹) were higher in spoiled dry-cured hams. pH (5·85–6·09) and A_w(0·888–0·909) were similar in spoiled and unspoiled hams. Thirty strains of the family Enterobacteriaceae were isolated and characterized. The strains were identified as Serratia liquefaciens and Proteus vulgaris. The strains of S. liquefaciens were slightly lipolytic, proteolytic, and psychrotrophic. Only two strains of this species were able to grow at an A_wlevel of 0·949. The strains of P. vulgaris were not lipolytic, were strongly proteolytic and only slightly psychrotrophic, and were able to grow at an A_wlevel of 0·949. None of them were able to grow at an A_wlevel of 0·929. The results indicate that the isolated strains could have caused deep putrefaction of dry-cured hams, growing during the first non-refrigerated steps of the curing process before the decrease of A_w. Enterobacteriaceae ought therefore to be considered a microbial quality-related hazard in the development of HACCP systems for dry-cured ham.     

Survival of Lactobacillus casei,Lactobacillus plantarum and Bifidobacterium bifidum in free and microencapsulated forms on Iranian white cheese produced by ultrafiltration

Survival of probiotic strains Lactobacillus casei ( ATCC 39392 ), Lactobacillus plantarum ( ATCC 8014 ) and Bifidobacterium bifidum ( ATCC 29521 ) was investigated either in microencapsulated or in free form in the Iranian white cheese produced by ultrafiltration technique. The results indicated that the survival of encapsulated probiotic bacteria was higher than free cells. Both free and microencapsulated forms were successful in keeping counts of L. casei, L. plantarum and B. bifidum in the cheese high enough for the therapeutic minimum (10⁶–10⁷ cfu/g) after 60 days. Addition of probiotic adjunct also did not alter the chemical composition, but pH was lower in probiotic cheeses.     

Effect of the lactic acid bacteria on the control of listerial activity and shelf life of smoked salmon scraps

The potential protective activity of lactic acid bacteria (LAB) from cold smoked salmon scraps was evaluated towards Listeria monocytogenes. Seventy‐three LAB strains were isolated and identified by biomolecular methods; Lactobacillus curvatus and Lactobacillus sakei prevailed. Three of the strains tested, identified as L. sakei, profile O, had a significant inhibitory activity against L. monocytogenes ATCC 19115 and clones DUP‐1042 and DUP‐18596. The evolution of microbial populations and chemical parameters were determined at time intervals to verify the shelf life. Listeria monocytogenes was isolated in half the packages also exceeding the legal limit. The shelf life of scraps was set at 30 days. Clonal characterisation of L. monocytogenes was performed by ribotyping. DUP‐1042, one of the human pathogen clones, was the most represented pattern. The results suggest further studies aimed at the selection of autochthonous nonspoilage LAB strains as bioprotective agents for cold smoked salmon.     

Effect of encapsulated Lactobacillus plantarum as probiotic on dry-sausages during chilled storage

The impact of free and encapsulated Lactobacillus plantarum addition on the physicochemical and microbiological properties during chilled storage (60 days) of dry-fermented sausages have been studied. Control (C) treatment was performed without probiotic incorporation, and the reformulation was comprised of L. plantarum as free cells (F) in alginate spheres (EA), in water-in-oil simple emulsion (ESM) and in water-in-oil-in-water double emulsion (EDM). After 60 days of storage, lower (P < 0.05) lipid oxidation was observed in F and EA (0.602–0.625 mg MDA kg⁻¹) while it was higher (P < 0.05) in EDM (1.949 mg MDA kg⁻¹). Treatments C and ESM presented intermediate TBARS levels compared to other treatments at the end of storage. All dry-fermented sausages presented high levels of lactic acid bacteria during the whole chilled storage (8.06–9.29 log CFU g⁻¹ at day 0 and 8.02–9.35 log CFU g⁻¹); however, EA treatment presented the highest L. plantarum viability even at 60 days of storage (8.34 log CFU g⁻¹). Therefore, the strategy of L. plantarum inoculation in alginate spheres seems to be the best strategy for the delivery of probiotics during chilled storage of dry-fermented sausages.     

Characterization of Proteolytic Effect of Lactic Acid Bacteria Starter Cultures on Thai Fermented Sausages

The objective of this study was to investigate the effect of starter culture addition on proteolysis of Thai fermented sausages. Sausages inoculated with six different external starter cultures—Pediococcus pentosaceous, Pediococcus acidilactici, Weissella cibaria, Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus sakei—were compared with naturally fermented sausages. The results of microbiological analysis indicated that the dominance of lactic acid bacteria (LAB) could inhibit the growth of pathogens and spoilage. Proteolysis was observed during fermentation by the reduction of myofibrillar and sarcoplasmic proteins and the increase in nonprotein nitrogen (NPN) and total free amino acids. The highest increase in concentration of NPN and free amino acids was obtained from sausages inoculated with LAB. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed a similar pattern of proteolysis of sarcoplasmic proteins in all sausages, while that of the inoculated sausages with L. plantarum, L. pentsus, and L. sakei exhibited increased degradation of myofibrillar protein bands at 200 and 45 kDa.     

Characterization of the triterpenoid 4,4′-diaponeurosporene and its isomers in food-associated bacteria

4,4'-Diaponeurosporene isomers were identified by LC/MS in extracts obtained from bacteria isolated from food (three strains of Staphylococcus carnosus, Lactobacillus plantarum LTH 4936, and Enterococcus sulfureus DSM 6905^T) as well as from Enterococcus mundtii DSM 4838^T. In addition to the predominant all-trans-isomer, seven cis-isomers of the C30-carotenoid were detected. Triterpenoid carotenoids have not been found before neither in Staphylococci other than Staphylococcus aureus nor in any species of the genus Lactobacillus.