Characterization of Intestinal Lactobacillus reuteri Strains as Potential Probiotics.

This study was conducted to evaluate the probiotic properties of Lactobacillus reuteri isolated from human infant feces (less than 3 months). Out of thirty-two representative L. reuteri strains isolated from the infant human feces, nine isolates (i.e. LR5, LR6, LR9, LR11, LR19, LR20, LR25, LR26 and LR34) showed survival in acid, bile and simulated stomach-duodenum passage conditions, indicating their high tolerance to gastric juice, duodenal juice and bile environments. The nine isolates did not show strong hydrophobic properties because the percentages of adhesion to the apolar solvent, n-hexadecane, did not exceed 40%, showing that their surfaces were rather hydrophilic. Functionality of these nine probiotic isolates was supported by their antagonistic activity and their ability to deconjugate bile salts. The safety of the nine indigenous L. reuteri isolates was supported by the absence of transferable antibiotic resistance determinants, DNase activity, gelatinase activity and hemolysis. The results obtained so far suggest that the nine strains are resistant to low pH, bile salts and duodenum juice, so they could survive when passing through the upper part of the gastrointestinal tract and fulfill their potential probiotic action in the host organism. According to these results, the L. reuteri strains isolated from human infant feces possess interesting probiotic properties that make them potentially good candidates for probiotics.

Diversity of bacillus species isolated from okpehe, a traditional fermented soup condiment from Nigeria.

The diversity of Bacillus species isolated from the fermented soup condiment okpehe in Nigeria was studied using a combination of phenotypic and genotypic methods. Fifty strains presumptively characterized as Bacillus spp. using the API 50 CHB test were further identified by PCR of randomly amplified polymorphic DNA (RAPD) and by amplified ribosomal DNA restriction analysis (ARDRA) genotyping methods. ARDRA fingerprinting with HhaI, HinfI, and Sau3AI restriction enzymes did not allow successful differentiation between the Bacillus species, except for distinguishing B. cereus from other Bacillus species. This problem was overcome with the combination of RAPD PCR and ARDRA genotypic fingerprinting techniques. Sequencing of 16S rRNA genes of selected strains representative of the major clusters revealed that the Bacillus strains associated with this fermentation were B. subtilis, B. amyloliquefaciens, B. cereus, and B. licheniformis (in decreasing order of incidence). The presence of enterotoxin genes in all B. cereus strains was demonstrated by multiplex PCR. The high incidence of detection (20%) of possibly pathogenic B. cereus strains that contained enterotoxin genes indicated that these fermented foods may constitute a potential health risk.

Weissella beninensis sp. nov., a motile lactic acid bacterium from submerged cassava fermentations, and emended description of the genus Weissella.

Four Gram-positive, catalase-negative, short rod-shaped or coccoid, heterofermentative lactic acid bacterial strains (2L24P13(T), 1L48P15, 1L24P31 and 1L24P34) with unusual phenotypic and genotypic properties were isolated from submerged fermenting cassava on MRS agar. All strains were motile, grew at 15 degrees C, produced dl-lactic acid from glucose with gas formation and produced ammonia from arginine. Acid was produced from d-fructose, d-galactose, d-glucose, lactose, maltose, d-mannose, melibiose, d-raffinose, sucrose, N-acetylglucosamine and d-mannitol, but not from d-arabinose or xylose. 16S rRNA gene sequence analysis revealed that the strains belonged to the genus Weissella and were most closely related to Weissella ghanensis LMG 24286(T). Low DNA-DNA reassociation values were obtained between the isolates and W. ghanensis DSM 19935(T). Based on the genetic and phenotypic results, the strains are considered to represent a novel species, for which the name Weissella beninensis sp. nov. is proposed. The type strain is 2L24P13(T) (=DSM 22752(T)=LMG 25373(T)).

Functional properties of lactic acid bacteria isolated from ethnic fermented vegetables of the Himalayas.

A total of 94 strains of Lactic acid bacteria (LAB), previously isolated from ethnic fermented vegetables and tender bamboo shoots of the Himalayas, were screened for functional properties such as acidification capacity, enzymatic activities, degradation of antinutritive factors and oligosaccharides, production of biogenic amines, hydrophobicity and adherence to mucus secreting HT29 MTX cells. Strong acidification and coagulation activities of LAB strains were recorded. Most of the LAB strains showed antimicrobial activities against the used indicator strains; however, only Lb. plantarum IB2 (BFE 948) isolated from inziangsang, a fermented leafy vegetable product, produced a bacteriocin against Staphylococcus aureus S1. LAB strains showed enzymatic activities and also degraded oligosaccharides. Almost all the strains of LAB were non-producers of biogenic amines except few strains. Some strains of Lb. plantarum showed more than 70% hydrophobicity. Adherence to the mucus secreting HT29 MTX cells was also shown by seven strains indicating their probiotic nature.

Pediococcus argentinicus sp. nov. from Argentinean fermented wheat flour and identification of Pediococcus species by pheS, rpoA and atpA sequence analysis.

A Gram-positive, small coccus-shaped lactic acid bacterium, strain LMG 23999(T), was isolated from Argentinean wheat flour. 16S rRNA gene sequence analysis revealed that the phylogenetic position of the novel strain was within the genus Pediococcus, with Pediococcus stilesii, Pediococcus pentosaceus and Pediococcus acidilactici as its closest relatives (97.7, 97.3 and 96.9 % gene sequence similarity, respectively). Fluorescent amplified fragment length polymorphism fingerprinting of whole genomes and whole-cell protein electrophoresis confirmed the unique taxonomic status of the novel strain. DNA-DNA hybridizations, DNA G+C content determination, comparative sequence analysis of the pheS, rpoA and atpA genes and physiological and biochemical characterization demonstrated that strain LMG 23999(T) (=CCUG 54535(T)=CRL 776(T)) represents a novel species for which the name Pediococcus argentinicus sp. nov. is proposed. Multi-locus sequence analysis based on pheS, rpoA and atpA genes was found to be a suitable method for the identification of species of the genus Pediococcus.

A genus-specific PCR method for differentiation between Leuconostoc and Weissella and its application in identification of heterofermentative lactic acid bacteria from coffee fermentation.

A genus-specific PCR analysis method was developed for a rapid and reliable differentiation between the two heterofermentative lactic acid bacteria genera Leuconostoc and Weissella. Primer sets specific for target regions of the 16S rRNA genes were designed and the specificity of the PCR was evaluated using the type strains of 13 species of Leuconostoc and 11 species of Weissella. In addition, the newly developed genus-specific PCR analysis was applied to characterize 72 lactic acid bacteria (LAB) strains isolated from coffee fermentation and which were presumptively classified as Leuconostoc or Weissella species. Additionally, a total of 34 LAB isolates from various other fermented foods were included. The investigations of these strains were conducted to test the effectiveness of correct characterization of field isolates using the genus-specific PCR approach. The correct assignment to one of these two genera by the application of the genus-specific primers was confirmed by further identifying the strains using repetitive extragenic palindromic-PCR and 16S rRNA gene sequencing.

Functional characteristics of Lactobacillus spp. from traditional Maasai fermented milk products in Kenya.

In this study functional characteristics of 23 representative Lactobacillus strains isolated from the Maasai traditional fermented milk 'Kule naoto' were determined. The Lb. acidophilus group strains showed resistance to gastric juice and bile. In addition, some Lb. acidophilus strains expressed bile salt hydrolase activity, and had ability to assimilate cholesterol in vitro. In-vitro adhesion to HT29 MTX cells of up to 70% was recorded. Lb. fermentum strains showed almost 100% survival under simulated stomach acidic conditions and physiological salt concentrations of bile salts, hydrophobicity values were over 80%. Most strains of the Lb. casei and Lb. acidophilus groups showed aggregation abilities of above 50%. Many strains expressed a protective effect against N-methyl-N'-nitro-N-nitrosoguanidine induced DNA damage according to the 'comet assay' and none was virulent. The antibiotic minimum inhibitory concentration of selected strains was established. According to these results, the Lactobacillus spp associated with 'Kule naoto', contain potentially probiotic (functional) strains.

Functional properties of Lactobacillus plantarum strains isolated from Maasai traditional fermented milk products in Kenya.

Lactobacillus plantarum was the major species among the lactic acid bacterial strains isolated from traditional fermented milk of the Maasai in Kenya. Selected strains were characterized for their functional properties using in vitro standard procedures. All strains expressed acid tolerance at pH 2.0 after 2-h exposure of values that ranged from 1% to 100%, while bile tolerance of acid-stressed cells at 0.3% oxgal varied from 30% to 80%. In vitro adhesion to the mucus-secreting cell line HT 29 MTX and binding capacity to extracellular protein matrices was demonstrated for several strains. The four strains tested in a simulated stomach duodenum passage survived with recovery rates ranging from 17% to 100%. Strains were intrinsically resistant to several antibiotics tested. From these in vitro studies, a number of Lb. plantarum strains isolated from the Maasai traditional fermented milk showed probiotic potential. The strains are good candidates for multifunctional starter culture development.

Microbiological and biochemical profile of cv. Conservolea naturally black olives during controlled fermentation with selected strains of lactic acid bacteria.

The effect of controlled fermentation processes on the microbial association and biochemical profile of cv. Conservolea naturally black olives processed by the traditional anaerobic method was studied. The different treatments included (a) inoculation with a commercial starter culture of Lactobacillus pentosus, (b) inoculation with a strain of Lactobacillus plantarum isolated from a fermented cassava product and (c) uninoculated spontaneous process. Microbial growth, pH, titratable acidity, organic acids and volatile compounds were monitored throughout the fermentation. The initial microbiota consisted of Gram-negative bacteria, lactic acid bacteria and yeasts. Inhibition of Gram-negative bacteria was evident in all processes. Both starter cultures were effective in establishing an accelerated fermentation process and reduced the survival period of Gram-negative bacteria by 5 days compared with the spontaneous process, minimizing thus the likelihood of spoilage. Higher acidification of the brines was observed in inoculated processes without any significant difference between the two selected starter cultures (113.5 and 117.6mM for L. plantarum and L. pentosus, respectively). L. pentosus was also determined as the major species present during the whole process of spontaneous olive fermentation. It is characteristic that lactic acid fermentation was also initiated rapidly in the spontaneous process, as the conditions of fermentation, mainly the low salt level (6%, w/v) favored the dominance of lactic acid bacteria over yeasts. Lactic, acetic and propionic were the organic acids detected by HPLC in considerable amounts, whereas citric and malic acids were also present at low levels and degraded completely during the processes. Ethanol, methanol, acetaldehyde, ethyl acetate were the major volatile compounds identified by GC. Their concentrations varied among the different treatments, reflecting varying degrees of microbial activity in the brines. The results obtained from this study could help the Greek table olive industry to improve the existing processing schemes in order to increase product consistency and quality expanding the international market for naturally black olives.

Diversity of predominant lactic acid bacteria associated with cocoa fermentation in Nigeria.

The fermentation of cocoa relies on a complex succession of bacteria and filamentous fungi, all of which can have an impact on cocoa flavor. So far, few investigations have focused on the diversity of lactic acid bacteria involved in cocoa fermentation, and many earlier investigations did not rely on polyphasic taxonomical approaches, which take both phenotypic and genotypic characterization techniques into account. In our study, we characterized predominant lactic acid bacteria from cocoa fermentations in Nigeria, using a combination of phenotypic tests, repetitive extragenic palindromic PCR, and sequencing of the 16S rRNA gene of representative strains for accurate species identification. Thus, of a total of 193 lactic acid bacteria (LAB) strains isolated from common media used to cultivate LAB, 40 (20.7%) were heterofermentative and consisted of either L. brevis or L. fermentum strains. The majority of the isolates were homofermentative rods (110 strains; 57% of isolates) which were characterized as L. plantarum strains. The homofermentative cocci consisted predominantly of 35 (18.1% of isolates) Pediococcus acidilactici strains. Thus, the LAB populations derived from these media in this study were accurately described. This can contribute to the further assessment of the effect of common LAB strains on the flavor characteristics of fermenting cocoa in further studies.

Phenotypic and genotypic identification of lactic acid bacteria isolated from ethnic fermented bamboo tender shoots of North East India.

Mesu, soidon, soibum and soijim are ethnic fermented bamboo tender shoot products prepared by the people in North East India. Microbiological analysis of mesu, soidon, soibum and soijim showed the population dominated by lactic acid bacteria (LAB) ranging up to 10(8) cfu g(-1). The phenotypic characterisation of predominant LAB isolated from the fermented bamboo shoot products was based on general morphology, physiological tests, API and Biolog systems. The genotypic characterisation of LAB was based on RAPD-PCR, rep PCR, species-specific PCR techniques, 16S rRNA gene sequencing and DNA-DNA hybridisation. Predominant functional LAB strains associated with the fermented bamboo shoot products were identified as Lactobacillus brevis, Lb. plantarum, Lb. curvatus, Pediococcus pentosaceus, Leuconostoc mesenteroides subsp. mesenteroides, Leuc. fallax, Leuc. lactis, Leuc. citreum and Enterococcus durans.

Leuconostoc holzapfelii sp. nov., isolated from Ethiopian coffee fermentation and assessment of sequence analysis of housekeeping genes for delineation of Leuconostoc species.

A Gram-positive, ovoid lactic acid bacterium, strain LMG 23990(T), was isolated from Ethiopian coffee fermentation. 16S rRNA gene sequence analysis indicated that the novel strain belongs to the genus Leuconostoc, with Leuconostoc citreum and Leuconostoc lactis as the closest neighbours (99.6 and 99.0 % 16S rRNA gene sequence similarity, respectively). Genotypic fingerprinting by fluorescent amplified fragment length polymorphism, whole-cell protein electrophoresis, DNA-DNA hybridizations, comparative sequence analysis of pheS, rpoA, atpA, and physiological and biochemical tests allowed us to differentiate strain LMG 23990(T) from all established Leuconostoc species. Strain LMG 23990(T) (=CCUG 54536(T)) therefore represents a novel species, for which the name Leuconostoc holzapfelii sp. nov. is proposed.

Lactobacillus ghanensis sp. nov., a motile lactic acid bacterium isolated from Ghanaian cocoa fermentations.

Three Gram-positive, catalase-negative, motile, rod-shaped strains, designated L486, L489(T) and L499, were isolated from fermenting cocoa. These organisms produced DL-lactic acid from glucose without gas formation. Ammonia was not produced from arginine. Acid was produced from amygdalin, D-cellobiose, aesculin, D-fructose, D-glucose, D-galactose, D-mannitol, D-mannose, N-acetylglucosamine, L-rhamnose, sucrose, salicin and D-trehalose. The cell walls contained peptidoglycan of the d-meso-diaminopimelic acid type. A 16S rRNA gene sequence analysis revealed that the isolates belong phylogenetically to the genus Lactobacillus and are closely related to Lactobacillus nagelii, Lactobacillus vini and Lactobacillus satsumensis. Low DNA-DNA reassociation values were obtained between the isolates and the phylogenetically closest neighbours. On the basis of the genetic and phenotypic results, the isolates are considered to represent a novel species, for which the name Lactobacillus ghanensis is proposed. The type strain is L489(T) (=DSM 18630(T)=CCUG 53453(T)).

Lactobacillus spp. with in vitro probiotic properties from human faeces and traditional fermented products.

Lactobacillus strains from traditional African fermented milk products, as well as human intestinal isolates were identified and investigated in vitro for their technological and functional characteristics as potential new probiotic strains. To test survival under gastrointestinal conditions, first the protective effect of milk and the effects of medium composition, lysozyme, pepsin, and pH of the medium on bacterial viability were assessed in vitro using the Plackett-Burman statistical model and the commercially used L. johnsonii LA1 probiotic strain. The use of either an artificial gastric electrolyte solution or MRS did not play a significant role in the viability of the cultures, while lysozyme, acidic conditions (pH 2.5), pepsin and the presence of milk significantly influenced the survival of the strain. Therefore, these parameters were selected as important test variables in a model stomach passage survival trial. Five strains identified as L. plantarum and two identified as L. johnsonii showed good survival under simulated gastrointestinal conditions. These selected strains also showed antimicrobial activity, probably due to production of organic acids. All strains exhibited bile salt hydrolase activity, while only the L. plantarum strains showed beta-galactosidase activity.

Pediococcus stilesii sp. nov., isolated from maize grains.

A Gram-positive, coccus-shaped, lactic acid bacterium, strain LMG 23082T, was isolated from steeped maize grains. The organism is homofermentative and produces D- and L-lactic acid from glucose. 16S rRNA gene sequence analysis revealed that the organism belongs to the genus Pediococcus, with Pediococcus pentosaceus and Pediococcus acidilactici as nearest neighbours. Genotypic fingerprinting, whole-cell protein electrophoresis, DNA-DNA hybridizations and physiological and biochemical tests allowed differentiation of strain LMG 23082T from other established Pediococcus species. A remarkable feature was that, unlike other pediococci, this bacterium was capable of growth at pH 9.0. The strain studied represents a novel species for which the name Pediococcus stilesii sp. nov. is proposed with the type strain LMG 23082T (=BFE 1652T=FAIR-E 180T=CCUG 51290T), the only currently known isolate of the species.

Lactobacillus arizonensis is a later heterotypic synonym of Lactobacillus plantarum.

The 'Lactobacillus plantarum group' encompasses the taxa Lactobacillus plantarum subsp. plantarum, Lactobacillus plantarum subsp. argentoratensis, Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus arizonensis. In this study, the phylogenetic position of L. arizonensis was examined using 16S rRNA gene-specific methodologies (16S rRNA sequencing and ribotyping) and genomic DNA-based investigations [repetitive extragenic palindromic DNA (rep)-PCR and DNA-DNA hybridization]. Our results show that the L. arizonensis type strain could not be distinguished from the type strain of L. plantarum or from various L. plantarum reference strains. Therefore, it is proposed that the species L. arizonensis should be reclassified as a heterotypic synonym of L. plantarum.

Identification of predominant lactic acid bacteria isolated from traditionally fermented vegetable products of the Eastern Himalayas.

Gundruk, sinki and khalpi are lactic-fermented vegetable products of Sikkim in India, and inziangsang is a fermented leafy vegetable product of Nagaland and Manipur in India. A total of 65 samples of gundruk (25), sinki (12), khalpi (25) and inziangsang (3) were analysed for microbial counts. The population of lactic acid bacteria (LAB) as well as aerobic mesophilic counts were at the level of 10(7) cfu g(-1). Yeasts were detected only in few samples of sinki and khalpi. No moulds were detected. In order to identify the predominating organisms, a total of 269 strains of LAB were isolated from gundruk, sinki, khalpi and inziangsang samples. The phenotypic characteristics of these strains were determined followed by genotyping using RAPD-PCR, repetitive element PCR and species-specific PCR techniques. The major representatives of the LAB involved in these fermentations were identified as Lactobacillus brevis, Lactobacillus plantarum, Pediococcus pentosaceus, Pediococcus acidilactici and Leuconostoc fallax.

Diversity and technological properties of predominant lactic acid bacteria from fermented cassava used for the preparation of Gari, a traditional African food.

Traditional fermentation of cassava is dominated by a lactic acid bacteria (LAB) population. Fermentation is important for improving product flavour and aroma as well as safety, especially by reduction of its toxic cyanogenic glucosides. The production of Gari from cassava in Benin typically occurs on a household or small industrial scale, and consequently suffers from inconsistent product quality and may not always be safe for consumption. Therefore, the diversity of LAB from a typical cassava fermentation for the preparation of Gari, and their technologically relevant characteristics were investigated with a view towards selection of appropriate starter cultures. A total of 139 predominant strains isolated from fermenting cassava were identified using phenotypic tests and genotypic methods such as rep-PCR and RAPD-PCR. DNA-DNA hybridisation and sequencing of the 16S rRNA genes were done for selected strains. Lactobacillus plantarum was the most abundantly isolated species (54.6% of isolates), followed by Leuconostoc fallax (22.3%) and Lactobacillus fermentum (18.0%). Lactobacillus brevis, Leuconostoc pseudomesenteroides and Weissella paramesenteroides were sporadically isolated. The L. plantarum strains were shown to be better acid producers and capable of faster acid production than the L. fallax or L. fermentum strains. The incidence of beta-glucosidase (linamarase) activity was also highest among strains of this species. Production of antagonistic substances such as H2O2 and bacteriocins, however, was more common among L. fallax and L. fermentum strains. Strains of all three species were capable of utilising the indigestible sugars raffinose and stachyose. Therefore, a starter culture containing a mixture of strains from all three species was recommended.

Lactobacillus curvatus subsp. melibiosus is a later synonym of Lactobacillus sakei subsp. carnosus.

On the basis of phenotypic and DNA-DNA reassociation studies, strain CCUG 34545T has been considered to represent a distinct Lactobacillus curvatus subspecies, Lactobacillus curvatus subsp. melibiosus. However, in several independent studies dealing with Lactobacillus sakei and L. curvatus strains, the subspecies division of L. curvatus has been found to be controversial. The original study distinguishing the two subspecies within both L. curvatus and L. sakei also lacked 16S rRNA gene sequence analyses. Therefore, the taxonomic position of L. curvatus subsp. melibiosus CCUG 34545T was re-evaluated in a polyphasic taxonomy study that included 16S rRNA gene sequence analysis, DNA-DNA reassociation, DNA G + C content determination, numerical analysis of ribotypes and whole-cell protein patterns and the examination of some fundamental phenotypic properties. The results obtained indicate that strain CCUG 34545T and its duplicate, CCUG 41580T, are Lactobacillus sakei subsp. carnosus strains and that L. curvatus subsp. melibiosus is a later synonym of L. sakei subsp. carnosus.

Isolation, identification and characterisation of the dominant microorganisms of kule naoto: the Maasai traditional fermented milk in Kenya.

From 22 samples of kule naoto, the traditional fermented milk products of the Maasai in Kenya, 300 lactic acid bacterial strains were isolated and phenotypically characterised by their ability to ferment different carbohydrates and by additional biochemical tests. Lactic acid bacteria (LAB), especially the genus Lactobacillus, followed by Enterococcus, Lactococcus and Leuconostoc, dominated the microflora of these samples. The major Lactobacillus species was Lactobacillus plantarum (60%), with a lower frequency of isolation for Lactobacillus fermentum, Lactobacillus paracasei and Lactobacillus acidophilus. Most strains produced enzymes such as beta-galactosidase and peptidases, which are of relevance to cultured dairy product processing, and exhibited similar patterns of enzymatic activity between species. Enterobacteriaceae could not be detected in 15 out of 22 samples (detection level 10(2)/ml). Conversely, yeasts (detection level 10(1)/ml) were detected in those samples in which Enterobacteriaceae were not found. The pH values of all these samples were < 4.5.