In the present study, 124 enterococcal strains, isolated from traditional Italian cow, goat and buffalo cheeses, were characterized using phenotypic features and randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). The RAPD-PCR profiles obtained with four primers and five different amplification conditions were compared by numerical analysis and allowed an inter- and intraspecific differentiation of the isolates. Whole cell protein analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used as a reference method for species identification. The strains were identified as Enterococcus faecalis (82 strains), E. faecium (27 strains), E. durans (nine strains), E. gallinarum (four strains) and E. hirae (two strains). Species recognition by means of RAPD-PCR was in agreement with the SDS-PAGE results except for eight strains of E. faecium that clustered in separated groups. On the other hand, phenotypic identification based on carbohydrate fermentation profiles, using the rapid ID 32 STREP galleries, gave different results from SDS-PAGE in 12.1% of the cases. The majority of the strains had weak acidifying and proteolytic activities in milk. One E. faecium strain showed vanA (vancomycin resistance) genotype while four strains showed a beta-haemolytic reaction on human blood. Several strains showed antagonistic activity towards indicator strains of Listeria innocua, Clostridium tyrobutyricam and Propionibacterium freudenreichii subsp. shermanii.
"RAPD-PCR was carried out using the primer M13 (5 -GAG GGT GGC GGT TCT-3 ) (Huey and Hall, 1989). PCR reactions and amplification conditions were performed as described by Andrighetto et al. (2001) and Suzzi et al. (2000). Rep-PCR analysis was carried out using the primer GTG 5 (5 -GTG GTG GTG GTG GTG-3 ) and amplification conditions as previously described by Gevers et al. (2001). "
[Show abstract][Hide abstract] ABSTRACT: A total of 104 lactic acid bacteria were isolated from ogi, wara, fermenting cassava mash for local food product gari and raw milk for nono. They were characterized phenotypically and divided into six main groups, which are facultative heterofermentative rods, obligate heterofermentative rods, tetrad-forming homofermentative cocci, homofermentative cocci, heterofermentative cocci, and an unidentified group. A total of 40 strains with good acidification, hydrogen peroxide production, and fermentation of indigestible sugar such as raffinose were selected. They were further characterized employing genotypic fingerprinting techniques, such as RAPD-PCR with primer M13, rep-PCR with primer (GTG)5 and sequencing of the 16S rDNA genes for starter culture development. RAPD-PCR did not give accurate delineation of the strains, but with the combination of rep-PCR fingerprinting and 16S rDNA sequencing, representative strains from rep-PCR clusters at 80% Pearson's correlation coefficient (r) grouped the organisms to nine (9) clusters. These were identified as Lactobacillus fermentum (7), Lactobacillus plantarum (6), Lactobacillus pentosus (2), Pediococcus pentosaceus (10), Pediococcus acidilactici (4), Enterococcus faecium (7), and the unidentified Lactobacillus species (4) were assigned into Lactobacillus fermentum group. A polyphasic taxonomic approach for characterization proved useful in the accurate typing of the LAB strains.
"Thus, a comparison of RAPD-PCR data obtained with a combination of several primers designed for either conserved or variable regions of bacterial genome makes it possible to get an idea on the interspecific diversity of S. thermophilus. This modified PCR program used for RAPD PCR was found to be more efficient than that described by Botina et al. (2007) and Andrighetto et al. (2001, 2002). On the basis of RAPD, no clear correlation can be deduced between RAPD clusters and folate production by isolates. "
[Show abstract][Hide abstract] ABSTRACT: This study deals with the bio-prospecting of folate producing strains of Streptococcus thermophilus isolated from milk and different fermented milk products of Indian origin. From a total of 500 randomly selected colonies
isolated from 209 different samples, 117 isolates were identified as S. thermophilus by classical biochemical and molecular characterization. Frequency of incidence of S. thermophilus in the different samples of milk and milk products was variable with the highest in the dahi followed by yogurt and lassi and a very low incidence in case of milk and cheese.On screening for folate using a microbiological assay with a tri-enzyme
extraction, about 15% of strains was found to produce folate in the range of 40–50μg · L−1, 35% in the range of 20–30μg · L−1, and the remainder in the range of 4–16μg · L−1. Comparative analysis of the random amplification of polymorphic DNA PCR fingerprint profiles was used to characterize interspecific
diversity of the ten highest folate producers. The LacZ gene of two of the highest folate producing isolates were sequenced and submitted to GenBank under following accession numbers
of FJ161697 and FJ161698.
摘要 本文利用生物勘探方法研究了印度牛乳及发酵乳制品中产叶酸的嗜热链球菌株。从 209 个样本中随机选取了 500 个菌落, 通过经典的生物化学和分子生物学方法确定 117 株为嗜热链球菌。在 Dali 样品中, 嗜热链球菌含量较高,其次是酸乳和
lassi, 由此可见, 在这些牛乳和发酵乳制品中的菌株生物多样性为嗜热链球菌占主要部分。采用三重酶提取微生物学测定法筛选产叶酸菌株, 发现约 15% 的菌株产叶酸的范围为 40-50 μg·L-1, 35% 的菌株为 20-30 μg·L-1, 其余的则在 4-16 μg·L-1 之间。采用 RAPD - PCR 指纹图谱对比分析了 10 株产叶酸最高菌株的种间多样性。对两株产叶酸含量最高菌株的 LacZ 基因进行测序,将序列提交到 GenBank 中得到序列号为 FJ161697 和 FJ161698。
–Folate–Microbiological assay–Bioprospecting–Interspecific diversity
关键词嗜热链球菌 (Streptococcus thermophilus)–叶酸–微生物学测定法–生物勘探–种间多样性
"). DNA was amplified using methods and amplification conditions described by Andrighetto et al. (2001) for primer M13 and Ben Omar et al. (2000) for primer Lb2. Rep-PCR was performed using the primer GTG5 (5 0 -GTG GTG GTG GTG GTG-3 0 ) and methods as previously described by Gevers et al. (2001). "
[Show abstract][Hide abstract] ABSTRACT: The diversity of lactic acid bacteria associated with Hussuwa fermentation, a Sudanese fermented sorghum food, was studied using a polyphasic taxonomical approach. Predominant strains could be well characterised based on a combination of phenotypic tests and genotypic methods such as ARDRA, rep-PCR and RAPD-PCR, as well as 16S rRNA gene sequencing of representative strains. Thus, the majority (128 of 220, 58.3%) of strains exhibited phenotypic properties typical of heterofermentative lactobacilli and of these, 100 strains were characterised more closely using the genotyping methods. The majority (97/100) strains could be characterised as Lactobacillus fermentum strains. Seventy-two of 220 strains (32.7%) showed phenotypic properties that are characteristic of pediococci. Of 41 selected strains investigated by genotyping techniques, 38 (92.7%) could be characterised as Pediococcus acidilactici strains, while three (7.3%) could be characterised as Pediococcus pentosaceus strains. The Hussuwa fermentation thus appears to be dominated by L. fermentum strains and P. acidilactici strains. For this reason, we selected representative and predominant strains as potential starter cultures for Hussuwa fermentation. These strains, L. fermentum strains BFE 2442 and BFE 2282 and P. acidilactici strain BFE 2300, were shown on the basis of RAPD-PCR fingerprinting to predominate in a model fermentation when used as starter cultures inoculated at 1 x 10(6) CFU/g and to lower the pH of the fermentation to below pH 4.0 within 48 h. These cultures should be studied for further development as starter preparations in pilot scale studies in actual field fermentations.
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