Carbohydrate Fermentation Patterns Research Articles

원유에서 분리한 Lactobacillus zeae RMK354의 생리적 특성 및 ACE 억제능

본 연구는 국내 각 지역의 목장에서 수거한 원유에서 분리된 젖산균을 대상으로 10% 환원탈지유에 <TEX>$37^{\circ}C$</TEX>에서 pH 4.4에 도달할 때까지 배양한 다음 각각에서 얻어진 유청으로 ACE 저해율을 측정한 결과 88.6%인 우수한 균주를 선발하였다. 선정된 균은 Gram 양성, rod형태의 homo균이며, 당 발효실험과 16S rRNA 분석결과 Lactobacillus zeae로 판명되었고, Lactobacillus zeae RMK354로 명명하였다. 발효유에 적합한 starter인지 확인하기 위해 생리적 특성을 조사하였다. L. zeae RMK354는 배양온도 <TEX>$40^{\circ}C$</TEX>에서 빠른 생장을 보였고, pH4.3에 도달하는데 10시간이 소요되었다. 16종의 항생제 중 polymyxin B와 vancomycin에 대해 내성이 높았으며, 효소활성실험에서 esterase와 leucine arylamidase의 활성도가 높았다. 담즙에 대한 내성이 있는 것으로 나타났으며, pH 내성 실험결과 pH 2에서 큰 변화가 없음에 따라 내신성이 있었다. 항균력 시험에서는 Escherichia coli와 Staphylococcus aureus에 대해 억제력이 없었으나 Salmonella typhimurium에 대해 60.0%의 항균력을 보였다. 이러한 결과를 토대로 ACE 억제활성능이 우수한 기능성 발효유 제품의 스타터로 L. zeae RMK354는 적합하다고 할 수 있다. In order to develop a new starter for fermented milk, 1037 bacterial strains were isolated from raw milk. The strain that showed excellent acid producing and angiotensin converting enzyme (ACE) inhibitory activity (88.6%) was selected and identified as a Lactobacillus zeae based on the result of API carbohydrate fermentation pattern and 16S rDNA sequence. Lactobacillus zeae RMK354 was investigated further to study its physiological characteristics. It showed strong ACE inhibitory activity compared with commercial LAB starters tested. The optimum growth temperature of L. zeae RMK354 was <TEX>$40^{\circ}C$</TEX> and it took 10 hr to reach pH 4.3 under this condition. L. zeae RMK354 showed more sensitive to penicillin-G, bacitracin, novobiocin, in a comparison of 14 different antibiotics, and showed most resistance to polymyxin B and vancomycin. It showed higher esterase and leucine arylamidase activities compared with 16 other enzymes. It was comparatively tolerant to bile juice and able to survive at pH 2 for 3 hr. It showed inhibitory activity against Salmonella Typhimurium with the rate of 60%. Based on these and previous results, L. zeae RMK354 could be an excellent starter culture for fermented milk with high level of ACE inhibitory activity.

Screening of Indigenous Strains of Lactic Acid Bacteria for Development of a Probiotic for Poultry

In an attempt to develop a probiotic formulation for poultry feed, a number of lactic acid bacteria (LAB) were isolated from chicken intestinal specimens and a series of in vitro experiments were performed to evaluate their efficacy as a potential probiotic candidate. A total of 650 LAB strains were isolated and screened for their antagonistic potential against each other. Among all the isolates only three isolates (TMU121, 094 and 457) demonstrated a wide spectrum of inhibition and were thus selected for detailed investigations. All three selected isolates were able to inhibit the growth of E. coli and Salmonella species, although to variable extent. The nature of the inhibitory substance produced by the isolates TMU121 and 094 appeared to be associated with bacteriocin, as their activity was completely lost after treatment with proteolytic enzymes, while pH neutralization and catalase enzyme had no effect on the residual activity. In contrast, isolate TMU457 was able to resist the effect of proteolytic enzymes while pH neutralization completely destroyed its activity. Attempts were made to study the acid, bile tolerance and cell surface hydrophobicity of these isolates. TMU121 showed high bile salt tolerance (0.3%) and high cell surface hydrophobicity compared to the other two strains studied, while TMU094 appeared the most pH resistant strain. Based on these results, the three selected LAB isolates were considered as potential ingredients for a chicken probiotic feed formulation and were identified to species level based on their carbohydrate fermentation pattern by using API 50CH test kits. The three strains were identified as Lactobacillus fermentum TMU121, Lactobacillus rhamnosus TMU094, and Pediococcus pentosaceous TMU457.

Analysis of the Genome Sequence ofLactobacillus gasseriATCC 33323 Reveals the Molecular Basis of an Autochthonous Intestinal Organism

This study presents the complete genome sequence of Lactobacillus gasseri ATCC 33323, a neotype strain of human origin and a native species found commonly in the gastrointestinal tracts of neonates and adults. The plasmid-free genome was 1,894,360 bp in size and predicted to encode 1,810 genes. The GC content was 35.3%, similar to the GC content of its closest relatives, L. johnsonii NCC 533 (34%) and L. acidophilus NCFM (34%). Two identical copies of the prophage LgaI (40,086 bp), of the Sfi11-like Siphoviridae phage family, were integrated tandomly in the chromosome. A number of unique features were identified in the genome of L. gasseri that were likely acquired by horizontal gene transfer and may contribute to the survival of this bacterium in its ecological niche. L. gasseri encodes two restriction and modification systems, which may limit bacteriophage infection. L. gasseri also encodes an operon for production of heteropolysaccharides of high complexity. A unique alternative sigma factor was present similar to that of B. caccae ATCC 43185, a bacterial species isolated from human feces. In addition, L. gasseri encoded the highest number of putative mucus-binding proteins (14) among lactobacilli sequenced to date. Selected phenotypic characteristics that were compared between ATCC 33323 and other human L. gasseri strains included carbohydrate fermentation patterns, growth and survival in bile, oxalate degradation, and adhesion to intestinal epithelial cells, in vitro. The results from this study indicated high intraspecies variability from a genome encoding traits important for survival and retention in the gastrointestinal tract.

Extracellular proteolytic activities expressed by Bacillus pumilus isolated from endodontic and periodontal lesions

The purpose of the present study was to identify 12 Bacillus isolates that had been obtained from root canals of teeth requiring endodontic therapy and from periodontal pockets in severe marginal periodontitis, and to determine whether these isolates exhibited extracellular proteolytic activity and, using in vitro assays, whether any such activity could degrade substrates that would be pathophysiologically relevant with regard to the production of endodontic and periodontal lesions. Biochemical and carbohydrate fermentation patterns were used in the identification of all strains, which was confirmed by determination of the16S rRNA gene sequence for strain BJ0055. Screening for production of extracellular proteolytic activity by all strains was done with a general proteinase substrate. All isolates were identified as representing Bacillus pumilus and all exhibited extracellular proteolytic activity. The putative pathophysiological relevance of extracellular proteinase production in strain BJ0055 was assessed using fluorophore-labelled elastin and collagen and several chromogenic peptides. Probable classes of proteinases acting on each substrate were investigated using class-specific inhibitors. Activity-pH profiles were determined in buffers at different pH values. Extracellular activities that were caseinolytic, elastinolytic, collagenolytic, glutamyl endopeptidase-like, and alanyl tripeptidyl peptidase-like were observed. No trypsin-like activities were detected. Serine- and chymotrypsin-like serine proteinase activities were detected, with activity observed at neutral and alkaline, but not acidic, pH. B. pumilus strains isolated from endodontic and periodontal lesions exhibited extracellular activities that degrade elastin, collagen and other substrates. These activities may be virulence factors that contribute to tissue damage in apical periodontitis and severe marginal periodontitis.

Proposal to reclassify the three biotypes of Bifidobacterium longum as three subspecies: Bifidobacterium longum subsp. longum subsp. nov., Bifidobacterium longum subsp. infantis comb. nov. and Bifidobacterium longum subsp. suis comb. nov.

In the year 2002, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium suis were unified into a single species, Bifidobacterium longum, preserving the former species names through the creation of the three biotypes 'longum', 'infantis' and 'suis'. Consequently, the use of the species names B. infantis and B. suis was to be discontinued. The above taxonomic rearrangement of B. longum was based on DNA-DNA hybridizations and 16S rRNA and HSP60 gene sequence analysis. However, a variety of other genotypic techniques including ribotyping, amplified rDNA restriction analysis (ARDRA), randomly amplified polymorphic DNA (RAPD)-PCR, BOX-PCR, PCR-denaturing gradient gel electrophoresis (DGGE), comparison of the recA, tuf and ldh gene sequences, plasmid profiling and considerable variation in carbohydrate fermentation patterns as well as results of starch and PAGE electrophoresis experiments clearly discriminate former B. longum, B. infantis and B. suis strains. In the present paper we compile this published information and propose the description of Bifidobacterium longum subsp. longum subsp. nov., Bifidobacterium longum subsp. infantis comb. nov. and Bifidobacterium longum subsp. suis comb. nov. The International Committee on Systematics of Prokaryotes Subcommittee on the taxonomy of Bifidobacterium, Lactobacillus and related organisms is in favour of this proposal. The type strains of Bifidobacterium longum subsp. longum subsp. nov., subsp. infantis comb. nov. and subsp. suis comb. nov. are E194b (variant a)T (ATCC 15707T=DSM 20219T), S12T (=ATCC 15697T=DSM 20088T) and Su859T (ATCC 27533T=DSM 20211T), respectively.

Metabolic and genetic profiling of clinical O157 and non-O157 Shiga-toxin-producing Escherichia coli

A collection of clinical Shiga-toxin-producing Escherichia coli (STEC) strains, mainly belonging to serotypes O26, O103, O111, O145 and O157, was characterised by a polyphasic approach including molecular serotyping, PCR-based detection of virulence factors ( stx1, stx2, eae, EHEC-hlyA, saa, katP, espP), carbohydrate fermentation profiles using API50 tests and random amplification of polymorphic DNA (RAPD) fingerprinting. An RAPD protocol based on the combination of 2 primers resulted in sufficiently complex patterns enabling discrimination to the serotype level. Moreover, carbohydrate fermentation profiles obtained after evaluating up to 50 different carbohydrates led to separation of different STEC serotypes. Virulence typing results confirm the association of Shiga toxins and intimin subtypes with specific serotypes and clinical diagnosis. Clinical diagnosis of strains did not correlate with either RAPD profiles or carbohydrate fermentation patterns.

The biodiversity of predominant lactic acid bacteria in dolo and pito wort for the production of sorghum beer

To quantify and identify the predominant lactic acid bacteria (LAB) in dolo and pito wort processing, and to examine their biodiversity at strain level. The processing of dolo and pito wort was studied at four production sites in Burkina Faso and Ghana. The succession of dominant micro-organisms, pH and titratable acidity were determined from sorghum malt through mashing and acidification to final wort. In the sorghum malt and during mashing, the LAB counts were 5.7-7.5 log CFU g(-1). Similar levels of yeasts and gram-negative, catalase-positive bacteria were observed. These levels decreased to 3.7-4.5 log CFU g(-1) and<or=2-3 log CFU g(-1), respectively, at the end of mashing, including a mild heat treatment. During acidification at ambient temperature (30-33 degrees C) lasting for 12-16 h, LAB counts increased to 8.8-9.9 log CFU g(-1), pH decreased from 5.55+/-0.12 to 3.72+/-0.24, and the titratable acidity calculated as lactic acid, increased from 0.13% to 0.61%. The gram-negative, catalase-positive bacteria and yeasts observed in the malt and during mashing were no longer detected. A total of 556 strains of LAB were isolated and purified. The LAB isolates were characterized and identified by a polyphasic approach based on phenotypic and genotypic methods, such as carbohydrate fermentation patterns using API 50 CHL, intergenic transcribed spacers-polymerase chain reaction/restriction fragment length polymorphism (ITS-PCR/RFLP), pulsed-field gel electrophoresis (PFGE) and 16S rRNA gene sequencing. Lactobacillus fermentum was identified as the dominant LAB species in the malt during mashing and during acidification. The other species observed during acidification were Lactobacillus delbrueckii ssp. delbrueckii, Lact. delbrueckii ssp. bulgaricus and Pediococcus acidilactici. These bacteria comprised a minor fraction of the bacterial population and no distinct microbial succession was observed for the LAB. At species level, the LAB profiles were similar for the four production sites; however, a pronounced diversity was observed at strain level. For one site, which had implemented a cleaning procedure between batches only, Lact. fermentum was found. Lact. fermentum was found to be the dominant LAB species throughout the entire process to final dolo and pito wort, including the acidification. Lact. delbrueckii ssp. delbrueckii, Lact. delbrueckii ssp. bulgaricus and P. acidilactici occurred in low numbers. At strain level, a high diversity based on PFGE-RFLP was observed for Lact. fermentum within and between sites. This study for the first time gives details of the involvement of LAB in the production of dolo and pito wort, for West African traditional sorghum beer production. One species, Lact. fermentum was predominant throughout the process, and seems to harbour potential starter cultures to be selected according to technological characteristics determined at strain level.

Spontaneous phage-resistant mutants of Streptococcus thermophilus: Isolation and technological characteristics

Spontaneous phage-resistant mutants were isolated from 12 commercial Streptococcus thermophilus strains by secondary-culture method. They were characterized by cell and colony morphology, carbohydrate fermentation patterns, phage-resistance stability, efficiency of plaquing (EOP), acidifying and proteolytic powers and milk acidification kinetics. Only 22.6% ( n = 100 ) of isolates proved to be true phage-resistant mutants. Phage resistance stability was a variable parameter among the mutants isolated, while EOP values were mostly very low (<10 −7). Acidifying and proteolytic powers did not differ ( p > 0.05 ) between phage-sensitive strains and their respective phage-resistant derivatives. Groups of mutants were found showing a milk acidifying activity which was slower or faster than that observed for the parent strain. Some mutants exhibiting high phage resistance and good technological capacities could be used as improved strains for industrial processes.

Detection of Antifungal Properties in Lactobacillus paracasei subsp. paracasei SM20, SM29, and SM63 and Molecular Typing of the Strains

Lactobacilli isolated from different food and feed samples such as raw milk, cheese, yoghurt, olives, sour dough, as well as corn and grass silage, were screened for their antifungal activities. Out of 1,424 isolates tested, 82 were shown to be inhibitory to different yeasts (Candida spp. and Zygosaccharomyces bailii) and a Penicillium sp., which were previously isolated from spoiled yoghurt and fruits. Carbohydrate fermentation patterns suggested that a substantial portion, 25%, belonged to the Lactobacillus casei group, including L. casei, L. paracasei, and L. rhamnosus. The isolates SM20 (DSM14514), SM29 (DSM14515), and SM63 (DSM14516) were classified by PCR using species-specific primers to target the corresponding type strains (L. casei, L. paracasei, and L. rhamnosus) as controls. Further molecular typing methods such as randomly amplified polymorphic DNA, pulsed-field gel electrophoresis, and sequencing analysis of the 16S rRNA gene allowed classifying strains SM20, SM29, and SM63 as L. paracasei subsp. paracasei in accordance with the new reclassification of the L. casei group proposed by Collins et al. (Int. J. Syst. Bacteriol. 39:105–108).

In vitro inhibition of Escherichia coli O157:H7 by bifidobacterial strains of human origin

The ability of bifidobacteria isolated from infant feces to inhibit enterohemorrhagic Escherichia coli serotype O157:H7 in vitro and reduce its adhesion to human enterocyte-like Caco-2 cells was evaluated in comparison to American Type Culture Collection bifidobacterial reference strains. Five Bifidobacterium isolates from infant feces were identified and characterized by morphology, fructose-6-phosphate phosphoketolase (F6PPK) assay, polymerase chain reaction using bifidobacterial 16S rDNA specific primers, carbohydrate fermentation patterns, resistance to lysozyme, acid, bile and hydrogen peroxide as well as their ability to inhibit E. coli O157:H7 using the agar spot technique. Infant isolates showed greater resistance to bile, acid, lysozyme and more antimicrobial activity against E. coli O157:H7 than ATCC strains. Two infant isolates identified as B. bifidum RBL 71 and B. bifidum RBL 460 showed good adhesion and significant potential for reducing adhesion of E. coli O157:H7 to Caco-2 cells. This effect was dependent on bifidobacterial cell concentration. These results show that bifidobacteria isolated from infants may be useful for improving probiotic formulae with respect to protection against E. coli O157:H7 infection.

Phenotypic and genotypic characteristics of shiga toxin-producing Escherichia coli strains isolated from children in São Paulo, Brazil

The biochemical and serological characteristics, virulence properties, and genetic relatedness of Shiga toxin-producing Escherichia coli (STEC) strains isolated in S o Paulo, from April 1989 through March 1990, were determined. This is also the first report on clinic findings of human STEC infections in Brazil. The only three STEC strains identified in that period were lysine decarboxylase negative, belonged to serotype O111ac: non-motile, were Stx1 producers, carried the eae and astA genes, and 2 of them also presented the EHEC-hly sequence. The children carrying STEC were all boys, with less than two years old, and had no previous history of hospitalization. None of them presented blood in stools. Vomiting, cough and coryza were the most common clinical manifestations observed. Although the STEC strains were isolated during summer months, and presented similar phenotypic and genotypic characteristics, carbohydrate fermentation patterns and PFGE analysis suggested that these diarrheal episodes were not caused by a single clone.

Phenotypic and genotypic properties of the genus Hafnia.

The present study characterised 73 Hafnia alvei isolates and five Escherichia isolates (originally identified as H. alvei) isolated from cases of diarrhoeal disease by the International Centre for Diarrhoeal Disease Research Branch (ICDDRB) in Bangladesh. Based upon the hydrolysis of arbutin and aesculin and the fermentation of salicin and D-arabinose, four distinct biotypes could be recognised among the 73 H. alvei isolates tested; biotype 1 (D-(-)-arabinose-positive only) accounted for 75% of all isolates analysed. Hydrolysis of aglycone compounds such as arbutin, salicin and aesculin appeared to be associated with expression of beta-glucosidase activity. ICDDRB isolates, when compared with type or reference strains of H. alvei, were shown not to belong to the genus Hafnia based upon resistance to Hafnia-specific bacteriophage 1672, possession of the phoE gene, expression of glutamate decarboxylase activity and significant 16S rDNA sequence divergence (approximately 8%) from the type strain, ATCC 13337T. True H. alvei strains, implicated in outbreaks of diarrhoeal disease in Canada, lacked the eaeA gene in contrast to ICDDRB isolates. Twenty-two H. alvei isolates were selected for further study. Based upon partial 16S rDNA sequencing, these 22 isolates fell into two genomic groups (genomospecies), identical to DNA groups previously established by DNA hybridisation studies. Markers such as motility, biotype, or enzymic or carbohydrate fermentation patterns did not correlate totally with DNA grouping, although malonate utilisation appeared to be the single best discriminatory phenotype. The results indicate that the genus Hafnia is heterogeneous and there do not appear to be any laboratory data available specifically linking these organisms to gastro-enteritis.

Identification of lactic acid bacteria constituting the predominating microflora in an acid-fermented condiment (tempoyak) popular in Malaysia

Tempoyak is a traditional Malaysian fermented condiment made from the pulp of the durian fruit ( Durio zibethinus). Salt is sometime added to proceed fermentation at ambient temperature. In various samples obtained from night markets, lactic acid bacteria (LAB) were the predominant microorganisms, ranging from log 8.4 to log 9.2 cfu g −1. No other microorganisms were present to such a level. These samples contained reduced amount of saccharose, glucose and fructose but increased amount of d- and l-lactic acid and acetic acid compared with samples of non-fermented durian fruit. Sixty-four isolates of LAB were divided into five groups by use of a few phenotypic tests. A total of 38 strains of LAB were selected for comparison by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of their whole cell protein patterns with a SDS–PAGE database of LAB. These strains were also examined for their carbohydrate fermentation patterns by use of API 50 CH. Isolates belonging to the Lactobacillus plantarum group were shown to be the predominant members of the LAB flora. In addition, isolates belonging to the Lactobacillus brevis group, Leuconostoc mesenteroides, Lactobacillus mali, Lactobacillus fermentum and an unidentified Lactobacillus sp. were also observed. A high degree of diversity among isolates belonging to the Lb. plantarum group was demonstrated by analysis of their plasmid profiles.

Genomic subpopulations within the species Pediococcus acidilactici detected by multilocus typing analysis: relationships between pediocin AcH/PA-1 producing and non-producing strains.

A high degree of genetic polymorphism among P. acidilactici strains was highlighted by a multilocus typing approach analysing several housekeeping genes and by sampling the whole genome using random amplified polymorphic DNA (RAPD) fingerprint analysis performed by using a single primer pedA gene targeted in low-stringency amplification conditions. Restriction fragment length polymorphism of the rpoC, ldhD/L and mle genes, and a modified RAPD analysis, permitted the grouping of Pediococcus acidilactici strains in seven genotypes (I-VII). Genotypic results obtained by analysing housekeeping genes involved in the transcription/translation machinery and in primary metabolism were supported by phylogenetic analysis based on the partial 16S rDNA sequencing of a reference strain of each of the seven clusters obtained. Three of the seven genotypes detected showed relationships with pediocin AcH/PA-1 production and carbohydrate fermentation patterns: all pediocin-producing and sucrose-positive strains were grouped in genotype VII, melibiose-, sucrose- and raffinose-positive strains in genotype VI, and arabinose-positive strains in genotype V.

Characterization of Lactobacillus carbohydrate fermentation activity using immobilized cell technique.

A microbial bioreactor based on calcium alginate immobilized Lactobacillus cells coupled to a pH electrode was developed for quantitative determination of carbohydrate fermentation activity. A high biomass (10(10) cfu mL(-)(1)) and particular pregrowth conditions were needed. Reduction of catabolite repression by monosaccharides was achieved by pregrowth in lactose. The evolution of acid production in a continuous flow-stopped flow bioreactor was monitored for different sugar solutions in contact with the immobilized bacteria. The resulting slopes (DeltamV/Deltat) were used to quantify the fermentation capability for a defined sugar related to that of glucose, which was taken as 100%. The procedure is simple, being based on pH variation that can give quantitative results compared to other reported techniques for carbohydrate fermentation pattern from which only qualitative results are obtained. In addition, it offers reduction in time and costs and is a suitable tool for the rapid analysis of isolated strains and in studies of modifications of sugar metabolism in mutants.

Classification and identification of strains of Lactobacillus brevis based on electrophoretic characterization of d-lactate dehydrogenase: Relationship between d-lactate dehydrogenase and beer-spoilage ability

The cell-free extracts of 60 strains which were identified phenotypically as being those of Lactobacillus brevis, including 48 isolates from the environment and 12 reference strains, were applied to polyacrylamide gel electrophoresis for extracting their NAD-dependent d- and l-lactate dehydrogenases (LDH). These strains were divided into 5 groups, i.e., Groups A, B, C, D, and E, on the basis of the electrophoretic mobilities of their d-LDH. The strains showed variations in their carbohydrate fermentation patterns. No relationship between the profile of d-LDH and the carbohydrate fermentation pattern was recognized. However, there appeared to be a relationship between the d-LDH profile and the beer-spoilage ability, because 40 out of 44 beer-spoilage strains identified as L. brevis were classified to Group B. We purified d-LDHs from the so-called complete beer-spoilage strain SBC 8002 of LDH Group B and from the non beer-spoilage strains JCM 1059 T of LDH Group A and AHU 1508 of LDH Group C. Although the purified d-LDHs had the same molecular weight (84 kDa), each possessed a different optimum pH, optimum temperature, and isoelectric point. The aforementioned parameter values for the enzyme from the so-called complete beer-spoilage strain SBC 8002 of LDH Group B were 10.0, 50°C, and 4.1, respectively; this strain was discriminated from the d-LDHs of the other two non beer-spoilage strains especially by its optimum temperature (50°C).

Characterization of lactococci isolated from minimally processed fresh fruit and vegetables

Lactic acid bacteria isolated from minimally processed fresh fruit and vegetable products were identified as Lactococcus lactis subsp. lactis on the basis of phenotypic tests, presence of lactococcal IS elements, and partial sequence analysis of the 16S rRNA gene. Isolated bacteria were differentiated using pulsed-field gel electrophoresis of SmaI digests of genomic DNA. Sprouted seeds were the best source of strains, and lactococci appear to be the dominant microflora on these products during the period they are intended to be eaten. Although these plant strains showed many similarities to strains of L. lactis used as dairy starter cultures, their carbohydrate fermentation patterns were unusual and probably reflect their environmental origin. Most strains fermented sucrose and xylose, and some also fermented raffinose and melibiose. Most of the bacteriocin-producing strains produced nisin, and nisin genes could also be detected in strains that showed no bacteriocin activity, or that produced a different bacteriocin with a narrow spectrum of activity. One strain produced nisin but was unable to ferment sucrose, properties that have been generally regarded as linked. These strains may have uses as biopreservatives for minimally processed plant products.

Detection of pediocin PA-1-producing pediococci by rapid molecular biology techniques

Five bacteriocinogenic lactic acid bacteria strains (347, X13, Z102, A172 and P20) independently isolated from fermented sausages were identified asPediococcus acidilacticiby carbohydrate fermentation patterns and other biochemical characteristics. This fact, together with their activity againstListeria monocytogenes, suggested that they could be pediocin PA-1 producers. Rapid molecular biology techniques were used to detect the pediocin PA-1 operon in these strains. PCR, dot-blot and Southern hybridization, and DNA sequencing results confirmed that all of them had the genetic determinants for pediocin PA-1 biosynthesis encoded in a 9.4 kb plasmid. The bacteriocin produced byP. acidilactici347 has been purified by a procedure that included ammonium sulphate precipitation and cation exchange, hydrophobic-interaction and reverse-phase chromatography. The amino acid sequence of this bacteriocin was identical to pediocin PA-1, confirming the expression of the pediocin PA-1 genes.

Isolation and Partial Physiological Characterization of Commercial Strains of Bifidobacteria

The genus Bifidobacterium has been suggested to be capable of performing gastrointestinal modifications, providing nutritional value when added to the diet as a probiotic and having an inducing effect on the immune system by enhancing cytokine production. As a consequence, the dairy food industry is introducing bifidobacteria to such products as yogurt, flavored milk, and cottage cheese. The objectives of this project were to characterize isolates of bifidobacteria from commercial suppliers, to isolate and characterize bifidobacteria from dairy products, and to establish and compare their physiological characteristics. Physiological characterization was performed based on phenotypic characteristics, carbohydrate fermentation patterns, enzyme profiles, fructose-6-phosphate phosphoketolase (F6PPK) assays, and antibiotic sensitivities. The results demonstrated that commercial bifidobacteria strains and those strains isolated from dairy products were physiologically different. This demonstrates that some bifidobacteria present in dairy products may be mischaracterized when identifying their presence based solely on phenotypic characteristics. The difficulty in growth, rapid assessment, and isolation of bifidobacteria from a mixed culture in dairy products was evaluated in this study. X-α-Gal medium was selected as the most suitable for isolation of bifidobacteria from mixed culture products and modified lactobacilli MRS medium for growing pure isolates of bifidobacteria.

Production of nisin-like bacteriocins by Lactococcus lactis strains isolated from vegetables.

Four bacteriocin producing lactic acid bacteria isolated from vegetables were identified as Lactococcus lactis strains on the basis of physiological and biochemical characteristics, carbohydrate fermentation patterns and analysis of total soluble protein pattern by SDS PAGE. The bacteriocins had a wide spectrum of activity as antagonism was detected not only towards a variety of lactic acid bacteria, but also to Staphylococcus aureus and Listeria monocytogenes. These bacteriocins were resistant to heating at 121 degree C for 15 minutes and showed highest activity at low pH (<5.0). They were inactivated by the proteolytic enzymes alpha-chymotrypsin and proteinase K, but not by lipase, alpha-amylase, catalase or lysozyme. These bacteriocinogenic Lactococcus strains were all immune to the bacteriocins produced as well as to commercial nisin. Bacteriocin producer culture supernatants showed a high degree (70 or 100%) of cross-reactivity in the nisin ELISA, suggesting similarity of the produced bacteriocins to nisin. The potential application of bacteriocin producing lactococci of vegetable origin for safety assurance of vegetable foods and controlling vegetable fermentations is discussed.