A numerical taxonomic study of lactic acid bacteria isolated from irradiated pork and chicken packaged under various gas atmospheres

Ninety-four Gram-positive, catalase-negative bacteria, isolated from pork and chicken that had been packed in modified atmospheres and irradiated to 1·75 and 2·5 kGy respectively, were studied. The majority of the strains were Lactobacillus saké . Numerical taxonomy, with the group average clustering strategy, revealed the existence of six clusters at the 85% similarity level. The largest, Cluster 1, contained 78 (83%) of the test strains along with three Lact. saké strains. Cluster 2 contained three test strains and the type strains of Carnobacterium piscicola and Carn. divergens . Cluster 3 contained two chicken strains and Lact. curvatus . Cluster 4 contained a pork strain and Leuconostoc dextranicum . Clusters 5 and 6 contained four pork and two chicken strains respectively and no type strains. Four test strains remained unclustered as did the other reference strains included in the study.     

The use of multiplex PCR reactions to characterize populations of lactic acid bacteria associated with meat spoilage

A rapid, systematic and reliable approach for identifying lactic acid bacteria associated with meat was developed, allowing for detection of Carnobacterium spp., Lactobacillus curvatus, Lact. sakei and Leuconostoc spp. Polymerase chain reaction primers specific for Carnobacterium and Leuconostoc were created from 16S rRNA oligonucleotide probes and used in combination with species-specific primers for the 16S/23S rRNA spacer region of Lact. curvatus and Lact. sakei in multiplex PCR reactions. The method was used successfully to characterize lactic acid bacteria isolated from a vacuum-packaged pork loin stored at 2 degrees C. Seventy isolates were selected for identification and 52 were determined to be Lact. sakei, while the remaining 18 isolates were identified as Leuconostoc spp.     

Rapid GC analysis of cellular fatty acids for characterizing Lactobacillus sake and Lact. curvatus strains of meat origin

A rapid procedure based on the gas chromatographic analysis of cellular fatty acids was used to differentiate between strains of Lactobacillus sake and Lact. curvatus isolated from dry salami. All strains had very similar fatty acid profiles except four of them which lacked C19 cycl acid, but neither this feature nor other differences in single fatty acid contents could be successfully correlated with the biochemical discrimination of Lact. sake from Lact. curvatus . When, however, strains were compared on the basis of the total content of fatty acids with 18 carbon atoms divided by that with 16 carbon atoms, a very good correlation with strain characterization by classical methods was achieved. It was concluded that selected cellular fatty acid ratios might be useful for characterizing phylogenetically related strains of lactic acid bacteria.     

Characterization of Lactobacillus sakei by the type of stereoisomers of lactic acid produced

Lactobacillus sakei strains were characterized by the shift of the type of stereoisomers of lactic acid produced in the presence of 50 mM sodium acetate in a medium. Of 27 Lactobacillus sakei strains studied, 20 strains showed high levels of DNA-DNA similarity with L. sakei NRIC 1071(T), and were confirmed as L. sakei. The three remaining strains were identified as Lactobacillus curvatus by DNA-DNA similarity, and three other strains were included in the cluster of Lactobacillus plantarum/Lactobacillus pentosus/Lactobacillus paraplantarum and one strain in the cluster of Lactobacillus paracasei on the basis of 16S rRNA gene sequences. Of the 20 L. sakei strains, 19 strains shifted the type of stereoisomers of lactic acid produced from the DL-type to the L-type in the presence of 50 mM sodium acetate. L. curvatus strains and strains included in the cluster of L. plantarum/L. pentosus/L. paraplantarum and in the cluster of L. paracasei did not shift the type of stereoisomers of lactic acid produced. The change of the type of stereoisomers of lactic acid from the DL-type to the L-type in the presence of sodium acetate was concluded to be species-specific for L. sakei and useful for identification of strains in this species.     

Safety properties and molecular strain typing of lactic acid bacteria from slightly fermented sausages

AIM: To evaluate the biodiversity of lactobacilli from slightly fermented sausages (chorizo, fuet and salchichon) by molecular typing, while considering their safety aspects. METHODS AND RESULTS: Species-specific PCR, plasmid profiling and randomly amplified polymorphic DNA (RAPD)-PCR were used to characterize 250 lactic acid bacteria (LAB) isolated from 21 low acid Spanish fermented sausages. Lactobacillus sakei was the predominant species (74%) followed by Lactobacillus curvatus (21.2%) and Leuconostoc mesenteroides (4.8%). By plasmid profiling and RAPD-PCR 144 different strains could be differentiated, 112 belonging to Lact. sakei, 23 to Lact. curvatus and 9 to Leuc. mesenteroides. Ion-pair high performance liquid chromatography was used to detect biogenic amine production. Tyramine and phenylethylamine were produced by 14.4 and 12.4% of the isolates, respectively, all belonging to the species Lact. curvatus. The production of tyramine was stronger than that of phenylethylamine. Partial sequencing of the tyrosine decarboxylase gene from Lact. curvatus was achieved. A specific PCR assay to detect the Lact. curvatus tyramine-producers was designed. The disc diffusion test was used to detect antibiotic resistance among the isolates. Most isolates displayed resistance to vancomycin and gentamicin. Only four strains were resistant to most of the antibiotics tested. None of the isolates were resistant to erythromycin. CONCLUSIONS: Lactobacillus sakei would be the species of choice for further use as starter culture in fermented sausage production. Strain typing and characterization of biogenic amine production together with antibiotic susceptibility testing for the selection of starter cultures could help to increase the quality and safety of the products. SIGNIFICANCE AND IMPACT OF THE STUDY: Species-specific PCR, RAPD and plasmid profiling proved to be efficient at typing LAB at species and strain level. Information on biogenic amine production and transferable antibiotic resistance is important in order to avoid selection of strains with undesirable properties as starter cultures.     

A survey of lactic acid bacteria in Italian silage

G razia , L. & S uzzi , G. 1984. A survey of lactic acid bacteria in Italian silage. Journal of Applied Bacteriology 56 , 373–379.
Lactic acid bacteria, isolated from Italian ensiled products, were represented by strains of the genera Lactobacillus and Leuconostoc . The predominant strains were heterofermentative lactobacilli, with Lactobacillus buchneri being the most frequent. Among homofermentative lactic acid bacteria, strains of Lact. plantarum and Lact. casei were recovered. Almost all strains utilized malic acid and showed good acid-tolerance, but only some of them were able to metabolize malic acid at extremely low pH; these were five homofermentative lactobacilli (4 Lact. plantarum and 1 Lacr. casei var. casei ) and two heterofermentative lactobacilli ( Lact. cellobiosus and Lactobacillus sp.).     

Phenotypic diversity of lactic acid bacteria isolated from fermented sausages produced in Basilicata (Southern Italy)

AIMS: to evaluate the evolution of lactic acid bacteria (LAB) populations in traditional fermented sausages (salsiccia and soppressata) produced in artisanal and industrial plants in Basilicata (Southern Italy). METHODS AND RESULTS: Four hundred and fourteen lactic acid bacteria (LAB) cultures were isolated from samples of sausages at different stages of ripening. A phenotypic characterization of the isolates was carried out using a set of 28 tests, and 34 clusters were identified at the 80% similarity level using hierarchical cluster analysis. Of the isolates 50% were identified as Lactobacillus sakei (with several biotypes), 22% as Pediococcus spp. (mainly Ped. pentosaceus), 7% as Leuconostoc (Leuc. carnosum, Leuc. gelidum, Leuc. pseudomesenteroides), 6% as Lact. plantarum, 1% as Lact. curvatus. Other lactobacilli, including unidentified species, were present in lower numbers. CONCLUSION: The phenotypic diversity and composition of the LAB flora varied as a function of the production plant, product type and ripening time. SIGNIFICANCE AND IMPACT OT THE STUDY: A new procedure based on bootstrapping and Multidimensional Scaling was successfully used to obtain a graphical representation of the evolution of the LAB populations.     

Polyphasic screening, homopolysaccharide composition, and viscoelastic behavior of wheat Sourdough from a Leuconostoc lactis and Lactobacillus curvatus exopolysaccharide-producing starter culture

After isolation from different doughs and sourdoughs, 177 strains of lactic acid bacteria were screened at the phenotypic level for exopolysaccharide production on media containing different carbohydrate sources. Two exopolysaccharide-producing lactic acid bacteria (Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A) were selected through quantitative analysis on solid media containing sucrose and yeast extract. The PCR detection of homopolysaccharide (gtf and lev) and heteropolysaccharide (epsA, epsB, epsD and epsE, and epsEFG) genes showed different distributions within species and strains of the lactic acid bacteria studied. Moreover, in some strains both homopolysaccharide and heteropolysaccharide genes were detected. Proton nuclear magnetic resonance spectra suggest that Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A produced the same exopolysaccharide, which was constituted by a single repeating glucopyranosyl unit linked by an α-(1→6) glycosidic bond in a dextran-type carbohydrate. Microbial growth, acidification, and viscoelastic properties of sourdoughs obtained by exopolysaccharide-producing and nonproducing lactic acid bacterial strains were evaluated. Sourdough obtained after 15 h at 30°C with exopolysaccharide-producing lactic acid bacteria reached higher total titratable acidity as well as elastic and dissipative modulus curves with respect to the starter not producing exopolysaccharide, but they showed similar levels of pH and microbial growth. On increasing the fermentation time, no difference in the viscoelastic properties of exopolysaccharide-producing and nonproducing samples was observed. This study suggests that dextran-producing Leuconostoc lactis 95A and Lactobacillus curvatus 69B2 can be employed to prepare sourdough, and this would be particularly useful to improve the quality of baked goods while avoiding the use of commercially available hydrocolloids as texturizing additives.     

Molecular characterization of Lactobacillus curvatus and Lact. sake isolated from sauerkraut and their application in sausage fermentations

Lactobacillus curvatus and Lact. sake are best adapted to meat fermentations and dominate the flora during the whole process. In fermenting sauerkraut, Leuconostoc mesenteroides subsp. mesenteroides is the major organism only during the early phase. In this environment Lact. curvatus and Lact. sake provide up to 50% of the microbial flora especially of the later phase, depending on the process conditions. Strains of Lact. curvatus and Lact. sake isolated from fermenting sauerkraut were identified by hybridization with species specific 23S rRNA-targeted oligonucleotide probes and further characterized. In 59 of 72 strains, plasmid DNA was detected. Small cryptic plasmids of 20 strains were found to be homologous with pLc2, a 2·6 kb plasmid from Lact. curvatus LTH683, which was originally isolated from meat. The ability to compete was investigated in fermenting sausages of two strains each of Lact. curvatus and Lact. sake isolated from sauerkraut. One strain each of Lact. curvatus and Lact sake was found to outnumber the meat-borne flora and govern the process.     

Characterization and distribution of lactic acid bacteria in cassava fermentation during fufu production

One hundred and thirty four lactic acid bacterial strains isolated during the 96-h period of cassava fermentation for fufu production were identified. The spectrum and proportion of the strains include Lactobacillus plantarum , 81%; Leuconostoc mesenteroides , 16%; Lact. cellobiosus , 15%; Lact. brevis , 9%; Lact, coprophilus , 5%; Lact. lactis , 4%; Leuc. lactis , 3% and Lact. bulgaricus , 1%. The isolates were characterized into strains. The succession among the lactic isolates was established. Lactobacillus plantarum was identified as the most dominant lactic acid bacterial strain involved in the fermentation.     

Effect of Sodium Nitrite and Sodium Chloride on Growth of Lactic Acid Bacteria

The effect of NaNO2 and NaCl on the growth of 24 lactic acid bacteria strains isolated from vacuum-packed cooked ring sausages were examined by analyzing different growth parameters with Bioscreen. NaNO2 had a very limited effect on the growth of lactic acid bacteria at 50 and 100 mg/l but at 400 mg/l a more pronounced inhibitory effect was found. Bacterial growth was enhanced by 1–2% (w/v) of added NaCl, while NaCl concentrations above 3% (w/v) had a clear inhibitory effect. Leuconostoc isolates seemed to be more sensitive to sodium nitrite and sodium chloride than homofermentative lactobacilli strains. Among homofermentative lactobacilli, the strains resembling Lactobacillus curvatus were more sensitive to NaCl than those resembling Lactobacillus sake.     

Use of unsupervised and supervised artificial neural networks for the identification of lactic acid bacteria on the basis of SDS-PAGE patterns of whole cell proteins

Conventional multivariate statistical techniques (hierarchical cluster analysis, linear discriminant analysis) and unsupervised (Kohonen Self Organizing Map) and supervised (Bayesian network) artificial neural networks were compared for as tools for the classification and identification of 352 SDS-PAGE patterns of whole cell proteins of lactic acid bacteria belonging to 22 species of the genera Lactobacillus, Leuconostoc, Enterococcus, Lactococcus and Streptococcus including 47 reference strains. Electrophoretic data were pre-treated using the logistic weighting function described by Piraino et al. [Piraino, P., Ricciardi, A., Lanorte, M. T., Malkhazova, I., Parente, E., 2002. A new procedure for data reduction in electrophoretic fingerprints of whole-cell proteins. Biotechnol. Lett. 24, 1477-1482]. Hierarchical cluster analysis provided a satisfactory classification of the patterns but was unable to discriminate some species (Leuconostoc, Lb. sakei/Lb. curvatus, Lb. acidophilus/Lb. helveticus, Lb. plantarum/Lb. paraplantarum, Lc. lactis/Lc. raffinolactis). A 7x7 Kohonen self-organizing map (KSOM), trained with the patterns of the reference strains, provided a satisfactory classification of the patterns and was able to discriminate more species than hierarchical cluster analysis. The map was used in predictive mode to identify unknown strains and provided results which in 85.5% of cases matched the classification obtained by hierarchical cluster analysis. Two supervised tools, linear discriminant analysis and a 23:5:2 Bayesian network were proven to be highly effective in the discrimination of SDS-PAGE patterns of Lc. lactis from those of other species. We conclude that data reduction by logistic weighting coupled to traditional multivariate statistical analysis or artificial neural networks provide an effective tool for the classification and identification of lactic acid bacteria on the basis of SDS-PAGE patterns of whole cell proteins.     

青海湖裸鲤肠道乳酸菌多样性与抑菌活性

【目的】通过生理生化特性,结合16S r RNA基因序列分析研究青海湖裸鲤肠道乳酸菌分离株的多样性,并对这些代表株的抑菌活性进行初步探讨,以期筛选具有高效抑菌活性的鱼源益生菌。【方法】对分离的47株乳酸菌代表株进行p H、温度生长范围、耐盐性等生理生化特征检测,结合16S r RNA基因序列对已分离到的乳酸菌进行基因分型和菌种鉴定,采用牛津杯双层平板法检测乳酸菌代表株的抑菌活性。【结果】鉴定结果显示:23株为Lactobacillus fuchuensis(48.94%),12株为Lactobacillus curvatus(25.53%),3株为Leuconostoc fallax(6.38%),2株为Lactobacillus sakei(4.26%),2株为Weissella ceti(4.26%);2株为Lactococcus cremoris(4.26%),1株为Leuconostoc lactis(2.13%),1株为Weissella minor(2.13%),1株为Enterococcus devriesei(2.13%)。qz1217、qz1196、qz1220所在的A、B、C三组乳酸菌在5-50°C的温度范围内生长良好,qz1196、qz1220所在的B、C组在pH 3.0-10.0的范围内生长良好,几乎所有乳酸菌都具有耐6.5%盐浓度特性。13株乳酸菌菌株对6种病原菌都具有抑制作用。通过排除酸、过氧化氢实验,发现上清液仍然具有抑菌活性。对qz1251发酵液进行蛋白酶处理,抑菌活性消失,确定其抑菌物质属于蛋白类物质,是一种细菌素。【结论】青海湖裸鲤肠道附着乳酸菌的多样性为益生性乳酸菌的筛选提供优质资源及数据参考。     

Identification of Lactobacillus sakei and Lactobacillus curvatus by multiplex PCR-based restriction enzyme analysis

Two closely related lactic acid bacteria, Lactobacillus sakei and Lactobacillus curvatus, are very difficult to be rapidly differentiated. Here we report multiplex polymerase chain reaction (PCR)-based restriction enzyme analysis that is useful for rapid and reliable identification of these two species. This method employs both polymerase chain reaction (PCR) and restriction enzyme analysis (REA). First, multiplex-PCR using three primers that were designed from 16S rDNA sequence produces two bands, a 433-bp and a 623-bp band. A 433-bp band represents only L. sakei and L. curvatus among lactobacilli and genetically related bacteria, and a 623-bp band is used for further identification by restriction analysis. Second, restriction analysis of 623-bp band using Hind III restriction enzyme discriminates L. sakei from L. curvatus. This method could identify 28 strains as L. sakei or L. curvatus, which were frequently isolated from kimchi, a traditional fermented cabbage product in South Korea. Therefore, these results suggest that this method is simple, rapid, and reliable for the identification of L. sakei and L. curvatus species.     

The effect of prebiotics on production of antimicrobial compounds, resistance to growth at low pH and in the presence of bile, and adhesion of probiotic cells to intestinal mucus

AIMS: Screening of five bile salt-resistant and low pH-tolerant lactic acid bacteria for inhibitory activity against lactic acid bacteria and bacterial strains isolated from the faeces of children with HIV/AIDS. Determining the effect of prebiotics and soy milk-base on cell viability and adhesion of cells to intestinal mucus. METHODS AND RESULTS: Lactobacillus plantarum 423, Lactobacillus casei LHS, Lactobacillus salivarius 241, Lactobacillus curvatus DF 38 and Pediococcus pentosaceus 34 produced the highest level of antimicrobial activity (12,800 AU ml(-1)) when grown in MRS broth supplemented with 2% (m/v) dextrose. Growth in the presence of Raftilose Synergy1, Raftilose L95 and Raftiline GR did not lead to increased levels of antimicrobial activity. Cells grown in the presence of Raftilose Synergy1 took longer to adhere to intestinal mucus, whilst cells grown in the absence of prebiotics showed a linear rate of binding. CONCLUSIONS: A broad range of gram-positive and gram-negative bacteria were inhibited. Dextrose stimulated the production of antimicrobial compounds. Adhesion to intestinal mucus did not increase with the addition of prebiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: The strains may be incorporated in food supplements for HIV/AIDS patients suffering from gastro-intestinal disorders.     

A numerical taxonomic study of lactic acid bacteria isolated from irradiated pork and chicken packaged under various gas atmospheres

Ninety-four Gram-positive, catalase-negative bacteria, isolated from pork and chicken that had been packed in modified atmospheres and irradiated to 1.75 and 2.5 kGy respectively, were studied. The majority of the strains were Lactobacillus saké. Numerical taxonomy, with the group average clustering strategy, revealed the existence of six clusters at the 85% similarity level. The largest, Cluster 1, contained 78 (83%) of the test strains along with three Lact. sak'e strains. Cluster 2 contained three test strains and the type strains of Carnobacterium piscicola and Carn. divergens. Cluster 3 contained two chicken strains and Lact. curvatus. Cluster 4 contained a pork strain and Leuconostoc dextranicum. Clusters 5 and 6 contained four pork and two chicken strains respectively and no type strains. Four test strains remained unclustered as did the other reference strains included in the study.     

Screening of lactic acid bacteria from fermented vegetables by carbohydrate profiling and PCR-ELISA

AIMS: The aim of this study was to identify potential souring agents, isolated from fermented plant material, by API 50 CHL assay and a molecular method based on polymerase chain reaction and colorimetric hybridization (PCR-ELISA). METHODS AND RESULTS: Forty-two strains of lactic acid bacteria derived from plant material were screened by taking advantage of API 50 CHL and PCR-ELISA. Oligonucleotide probes used for hybridization in PCR-ELISA were specific for lactobacilli, the Leuconostoc family, Lactobacillus pentosus/plantarum and Lactobacillus brevis. The hybrides were detected by a colour-developing reaction. Bacteria isolated from fermented cucumbers were identified as Lact. plantarum-related (Lact. plantarum and Lact. pentosus) and Leuconostoc species. Most of the strains isolated from sauerkraut were identified as Lact. pentosus/plantarum. CONCLUSIONS: Complementary results were obtained in the identification of bacterial strains, isolated from fermented cucumbers and sauerkraut, by API 50 CHL and PCR-ELISA. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR-ELISA proved to be suitable for the screening of large numbers of bacterial isolates from fermented vegetables. This will be useful for the identification of strains suitable for the design of starter cultures for the fermentation of plant material.     

The antimicrobial effect of organic acids, sour dough and nisin against Bacillus subtilis and B. licheniformis isolated from wheat bread

Prevention of growth in wheat bread for more than 6 d of approximately 10⁶ rope-producing Bacillus subtilis spores per gram of dough was achieved by addition of propionic or acetic acids at levels of 0·10% v/w (based on flour weight), or by addition of 15% sour dough fermented with Lactobacillus plantarum C11, Lact. brevis L62, Lact. plantarum ('vege-start 60'), Lact. plantarum (ch 20), Lact. maltaromicus (ch 15), or the commercial sour dough starter culture, Lact. sanfrancisco L99. These cultures resulted in an amount of total titratable acids above 10 in the sour dough and a pH value below 4·8 in the final bread. Bacteriocin-producing lactic acid bacteria added as starter cultures in wheat dough and nisin (Nisaplin) at levels up to 100 p.p.m. g⁻¹ flour had no effect against B. subtilis and B. licheniformis strains, despite the fact that nisin-producing strains of Lactococcus lactis ssp. lactis among 186 strains of lactic acid bacteria had demonstrated inhibitory activity against B. subtilis and B. licheniformis in an agar spot assay.     

Arabinose fermentation by Lactobacillus plantarum in sourdough with added pentosans and alphaalpha-L-arabinofuranosidase: a tool to increase the production of acetic acid

Sixty-five strains of obligately and facultatively heterofermentative sourdough lactic acid bacteria were screened for their capacity to grow optimally in the presence of arabinose, ribose and xylose as carbon sources. Lactobacillus alimentarius 15F, Lact. brevis 10A, Lact. fermentum 1F and Lact. plantarum 20B showed higher growth rate, cell yield, acidification rate and production of acetic acid when some pentoses instead of maltose were added to the SDB medium. Lactobacillus plantarum 20B used arabinose also in a synthetic medium where complex growth factors such as yeast extract were omitted. Other Lact. plantarum strains did not show the same property. Pentosan extract was treated with alpha-L-arabinofuranosidase from Aspergillus niger or endo-xylanase from Bacillus subtilis to produce hydrolysates containing mainly arabinose and xylose, respectively. In particular, the hydrolysate containing arabinose substantiated the growth and the production of lactic acid and, especially, of acetic acid by Lact. plantarum 20B. Sourdough fermentation by Lact. plantarum 20B with addition of pentosan extract and alpha-L-arabinofuranosidase increased the acidification rate, titratable acidity and acetic acid content compared with traditional sourdough. A facultatively heterofermentative strain, Lact. plantarum 20B, also produced a sourdough with an optimal fermentation quotient.     

Fermentation of fructans by epiphytic lactic acid bacteria

A total of 712 strains of lactic acid bacteria isolated from forage grasses were studied for their ability to ferment fructans of phlein- as well as inulin-type. Only 16 strains utilized phlein and eight of these also fermented inulin. They were identified as Lactobacillus paracasei subsp. paracasei, Lact. plantarum, Lact. brevis and Pediococcus pentosaceus . In the species Lact. paracasei subsp. paracasei , all strains gave positive results, whereas the other positive strains possessed unique properties within their own species. In all but two cases (strains of the species Lact. plantarum ), the phlein was more intensively fermented than the inulin, as indicated by a lower pH and a higher lactic acid concentration. On the basis of the outcome of this study it seems worthwhile to inoculate grasses of low sugar content before ensiling with an active strain that can ferment fructans.