Culture-Independent Analysis of Probiotic Products by Denaturing Gradient Gel Electrophoresis

In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products is crucial. Currently, analysis of most probiotics is still based on culture-dependent methods involving the use of specific isolation media and identification of a limited number of isolates, which makes this approach relatively insensitive, laborious, and time-consuming. In this study, a collection of 10 probiotic products, including four dairy products, one fruit drink, and five freeze-dried products, were subjected to microbial analysis by using a culture-independent approach, and the results were compared with the results of a conventional culture-dependent analysis. The culture-independent approach involved extraction of total bacterial DNA directly from the product, PCR amplification of the V3 region of the 16S ribosomal DNA, and separation of the amplicons on a denaturing gradient gel. Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level. This whole culture-independent approach can be performed in less than 30 h. Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner. Unfortunately, as reported in previous studies in which the culture-dependent approach was used, a rather high percentage of probiotic products suffered from incorrect labeling and yielded low bacterial counts, which may decrease their probiotic potential

Polyphasic taxonomic characterization of lactic acid bacteria isolated from spontaneous sorghum fermentations used to produce ting, a traditional South African food

Ting, an indigenous cooked fermented food made from sorghum flour, is consumed extensively in South Africa. Due to the spontaneous nature of the sorghum fermentation considerable variations in the sensory and microbial quality of the end-product may occur, thus hampering large-scale production of this food. The use of starter cultures purified from the fermented sorghum may be an alternative approach to obtain ting of consistent quality. The aim of this study was therefore to identify the lactic acid bacteria (LAB) associated with ting fermentation using a polyphasic approach. Phenotypic characterization and sequence analysis of the genes encoding the 16S subunit of the ribosomal RNA(rrs) and phenylalanyl tRNA synthase (pheS) were used. The results of these analyses showed that ting fermentation involved at least three different species of LAB, i.e. Lactobacillus fermentum, L. plantarumand L. rhamnosus. To our knowledge, this is the first report of polyphasic taxonomic characterization of LAB from this food. This research forms an essential first step towards the development of relevant starter cultures to produce ting of consistent quality

Molecular source tracking of predominant lactic acid bacteria in traditional Belgian sourdoughs and their production environments

To investigate the circulation of predominant sourdough lactic acid bacteria (LAB) species in the production environment of two Belgian artisan sourdough bakeries. Isolates were collected from sourdoughs, flour, hands of the baker and air in the bakery setting and taxonomically characterized using repetitive element sequence-based PCR fingerprinting, pheS and/or 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis. In parallel, PCR-DGGE (denaturing gradient gel electrophoresis) analysis of V3-16S rDNA amplicons was applied to visualize the predominant bacterial population in the sourdoughs and the corresponding bakery environment (flour, hands of the baker, air and bakery equipment). Both approaches revealed that sourdoughs produced at D01 and D10 were mainly dominated by Lactobacillus spicheri and L. plantarum and by L. sanfranciscensis, respectively, and that these LAB species also circulated in the corresponding bakery environment. Furthermore, AFLP fingerprinting demonstrated that sourdough and bakery environment isolates of these species were genetically indistinguishable. For more sensitive source-tracking, SYBR Green-based real-time PCR assays were developed using species-specific primers targeting the pheS gene of L. plantarum and L. sanfranciscensis, detected in air samples from D01 and D10, respectively. The results obtained in this study indicate that specific strains of LAB persist in artisan doughs over years and circulate in the bakery environment. Furthermore, the importance of air as a potential carrier of LAB in artisan bakery environments was demonstrated. PheS-based real-time PCR can be used to detect, quantify and/or monitor specific LAB species (e.g. starter cultures) in sourdough and bakery environment samples

EFFECT OF CONCHING ON PROCYANIDINS AND FUNCTIONAL PROPERTIES OF CHOCOLATE

Chocolate is a highly processed cocoa product known to be rich in flavonoids, compounds which have been long associated to both healthy and sensory properties. A multi-step process involving cocoa beans fermentation, drying, roasting, nib-grinding and refining, conching and tempering is implied in the production of chocolate.To our knowledge, scarce attention has been paid to the effect of conching on the procyanidins profile, on their polymerisation from monomers up to oligomers and polymers, as well as on the in vitro functional properties such as antiradical activity and reducing power. Two conching processes, characterized by different time/temperature combinations, a Long Time Conching (LTC) and a Short Time Conching (STC), were thus taken studied and the effect on flavan-3-ols monomers, on the degree of polymerization and on the in vitro functional properties of dark chocolates was studied. Procyanidin content was determined by HPLC analysis, while the total polyphenol index (TPI), the antiradical activity as well as the reducing power were evaluated by the reaction with the Folin–Ciocalteu reagent, the decolorization assays of the ABTS radical (TEAC) and the Ferric Reducing Antioxidant Power (FRAP) methods, respectively.The procyanidins content and profile were deeply affected by the different processing conditions: at the end of conching the STC-samples were characterized by a higher amount of monomers, compared to the LTC-ones which, in turn, resulted more polymerised as confirmed by the presence of P10 polymers. Both STC- and LTC conched products presented comparable TPI and FRAP values but products collected at different steps during the conching process, and in particular during LTC, showed a significant improvement of the radical scavenging properties. This result suggest the possibility to modulate the conching process in terms of time and temperature combination in order to improve the antioxidant activity of the final products.[...

Polyphasic taxonomic characterization of Lactobacillus rossiae isolates from Belgian and Italian sourdoughs reveals intraspecific heterogeneity

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Use of microbial communities for human and animal health

The present invention relates to a mixture of bacteria belonging to at least 6 or 7 different and specific bacterial species preferably for use to prevent or treat gastro-intestinal disorders. Preferably, said mixture of bacteria are grown together in a fermenter prior to administering said mixture to a subject in order to prevent or treat said disorder

Isolation and characterization of lactic acid bacteria and yeasts from the Brazilian grape sourdough

Sourdough is a mixture of flour and water fermented by lactic acid bacteria and yeast, with a large use in bakery products. This study was developed with Brazilian grape (Niagara rosada) sourdough obtained from spontaneous fermentation. The aim of this work was to characterize genotypic and phenotypically lactic acid bacteria and yeasts isolated from sourdough. The phenotypic identification for bacteria and yeasts was performed by using the kit API50CHL and 20CAUX and the genotypic characterization was performed by sequencing method. A total of four isolated strains were analyzed in this study. Two of these strains were phenotypically and genotypic identified as Lactobacillus paracasei and one as Saccharomyces cerevisiae. Another sample phenotypically identified as Candida pelliculosa did not show the same identity by sequencing. It shows the need to use phenotypic and genotypic characterization associated for the correct microorganism identification