Bifidobacteria Ecology of non-Human Primates: Characterization of Novel Species with Unexpected Functionalities for Probiotic Applications and a Co-Evolutionary Host-Microbe Analysis

Bifidobacterium spp. are known as probiotic strains and recently new features emphasize their importance for human health, as EPSs and folate production. The relationship between Bifidobacterium spp. and their hosts is unknown, but probably links to peculiarities in the bifidobacterial cell-wall structures or to bifidobacterial ability to metabolize substrates from the host diet. Recently, a richness and diversity of bifidobacteria was observed in Callithrix jacchus and Saguinus midas, introducing the existence of a storehouse in primate guts. Several techniques were developed to deepen the microbial diversity, mainly based on the PCR. The RFLP-PCR of 16S rRNA gene represents a fast tool to distinguish human or animal origin bifidobacteria, useful in “Microbial Source Tracking” and probiotic selection. The project aim was the exploration of the bifidobacterial occurrence and diversity in evolutionary primate hosts to improve the knowledge about bifidobacteria distribution in non-human primates, and to identify bifidobacteria with new probiotic features (EPSs and folate production). 17 subjects from Strepsirrhini, Eulemur macaco, Eulemur rubriventer, Hapalemur alaotrensis and Lemur catta, and from Simiiformes, the New World Monkeys Callithrix jaccus, Pithecia pithecia, Saguinus oedipus and Saguinus imperator, and the Old World Monkeys, Chlorocebo aethiops and Macaca Sylvanus, were studied. Strains tested for probiotics traits, acid and bile tolerance, revealed B. aesculapii, B. myosotis and B. spp. MRM_8.19 strains as the most resistance. The folate production on strains from ring-tailed lemur and common marmoset revealed autotrophy only in strains from common marmoset. The distribution of microbial communities in non-human primates from 8 babies of common marmosets, golden faced saki and Barbary macaques and 11 adults of ring-tail lemurs, black lemurs, red-bellied lemur, Alaotran bamboo lemur, Barbary macaques, grivet, cotton top-tamarin and emperor tamarin, was carried out using ARDRA and rep-PCR. Results revealed a richness in both abundance and diversity of Bifidobacterium in primates

In silico RFLP Analysis of 16S rRNA Genes: A Helpful Application for Distinguishing Bifidobacteria from Human and Animal Source

Bifidobacterial species are widespread in gastrointestinal tracts of mammalian and other animals; they can be found in extra body environment only after a fecal contamination or human intentional addition (as the case of probiotics). Interestingly their occurrence is strictly linked to their hosts with a clear demarcation between animal and human species. PCR-restriction fragment length polymorphism (PCR-RFLP) on the 16S rRNA gene, using Alul, and TaqI restriction enzymes, have been utilized to distinguish the animal or human source of 64 strains belonging to 13 Bifidobacterium species (Delcenserie et al. [15]). Our aim was to test this method updating an in silico restriction analysis on the available 16S rRNA gene sequences of all 55 currently described taxa of Bifidobacterium genus. Our results confirmed the reliability of this method, optimized with the use of three restriction enzymes: Alul, TaqI and MaeIII, as a fast and simple strategy to determine the origin (human or animal) of bifidobacteria. Interestingly, the bifidobacterial species recently isolated from non-human primates cluster in the group of animal source except the bifidobacterial species isolated from higher non-human primates closest to humans such as apes (chimpanzee, orangutan and gorilla) that clusters with human group. Moreover, B. minimum, B. subtile and B. mongoliense isolated only from extrabody environment of which the source is unknown clustered with animal species. The in silico RFLP-PCR confirmed its powerful ability to attribute the primary source of occurrence (human or animal) for bifidobacterial species to the human or animal habitat

A reverse metabolic approach to weaning: in silico identification of immune-beneficial infant gut bacteria, mining their metabolism for prebiotic feeds and sourcing these feeds in the natural product space

Weaning is a period of marked physiological change. The introduction of solid foods and the changes in milk consumption are accompanied by significant gastrointestinal, immune, developmental, and microbial adaptations. Defining a reduced number of infections as the desired health benefit for infants around weaning, we identified in silico (i.e., by advanced public domain mining) infant gut microbes as potential deliverers of this benefit. We then investigated the requirements of these bacteria for exogenous metabolites as potential prebiotic feeds that were subsequently searched for in the natural product space

Possible application in food industry of a recently isolated exopolysaccharydes producing bifidobacterial species

Health awareness among consumers have recently generated more demands for low-fat or fat-free dairy products. However, since milk fat contr\uecbutes to the flavor, body and texture development of the dairy products, removal leads to textural and functional defects in low fat fermented milk products. ln this perspective, the exopolysaccharides (EPS) produced by food grade lactic acid bacteria (LAB) have gained much importance as biothickeners and texturizers. EPS producing LAB as 'biothickeners' should offer natural, more acceptable and a preferred approach to many additives. EPS \uecmpart highly desirable rheological changes in the food matrix such as increased viscosity, improved texture and reduced syneresis. Further, EPS may \uecnduce positive physiological responses \uecnclud\uecng lower cholesterol levels, reduced formation of pathogenic biofilms, modulation of adhesion to epithelial cells and increased levels of bifidobacterio showing a prebiotic potential. Hence, the choice of EPS producing starter cultures seems to give several advantages over nonproducing ones. Bifidobocterium aesculapi, a novel species recently described, resulted able to ferment lactose as well as producing an exocellular gelling matrix, which was identified and quantified as EPS. Seven strains of B. aesculapii and the type strains of the two related species 8. stellenboschense and B. scardovii were tested for their abil\uecty to ferment who\uece milk. Texture parameters (Textureanalyzer), volatile aromatic compounds (detected by GC/MS-So|id Phase Micro Exraction) and pH were assayed. B. aesculapii strains fermented whole milk conferring to the obtained products the highest viscosity index, hardness, consistency and cohesiveness values. The volatile profiles showed quali-quantitative differences among the samples and, from a sensoriaI point of view, they represent a specific product fingerprinting. However, 2,3 butanedione, 3-hyd roxy-2-buta none, 2-butanone, propanone and acetic acid were the most representative detected molecules. The data obtained suggest that B. aesculapii, belonging to probiotic bacteria, should be tested for further application in food \uecndustry

USE OF EPS PRODUCING BIFIDOBACTERIUM AESCULAPII STRAINS TO IMPROVE TEXTURE AND QUALITY OF SOY MILK BASED FERMENTED MILKS

Functional foods represent one of the fastest growing areas in the food industry because considered a dietary strategy to reduce illness. The development of dairy functional products has become an important subject. Consumers have increased the demand for low/fat-free, but also smooth and creamy, functional dairy products. Since milk fat contributes to flavour and texture of the dairy products, its removal leads to textural defects. So several attempts have been made to increase the textural and quality properties of these products. Among the strategies, the use of strains able to produce exopolysaccharides (EPS) has been suggested as an alternative to additives such as xanthan, pectin and carrageenan. So, the use of EPS-producing Bifidobacterium aesculapii strains was proposed for to obatain functional fermented milks from soy milk. Seven strains of B. aesculapii, recently described, able to ferment lactose and producing an exocellular gelling matrix, were employed for the fermentation of soy-milk. The strains were inoculated (7.5 log cfu/ml) and incubated at 37°C. The resulting fermented milks were subjected to EPS and volatile molecule profiles (GC/MS-SPME) analysis and Texture Analyzer. Also the survival of B. aesculapii in the products was checked during the refrigerated storage. The results showed all the strains were able to grow in soymilk and to reach a final concentration of 9.0 log cfu/ml. The strains produced large amount of EPS in products. The fermented milks were characterized by specific volatile molecule profiles. Generally, the strains able to produce the major amount of EPS resulted in more viscous products. The results obtained have highlighted the chance to exploit EPS producing Bifidobacterium aesculapii strains to produce fermented milks characterized by specific “aromagramma and texture properties. These results could be exploited also to expand the market of fermented soy-milk products by using a biotechnological approach enhancing also the overall product quality

Identification of Bifidobacterium spp. using hsp60 PCR-RFLP analysis: An update

A PCR-RFLP technique has been applied on 13 species of Bifidobacterium in order to update a previous study carried out by Baffoni et al. [1]. This method is based on the restriction endonuclease activity of HaeIII on the PCR-amplified hsp60 partial gene sequence, and allows a rapid and efficient identification of Bifidobacterium spp. strains at species and subspecies level

Bifidobacterium catulorum sp. nov., a novel taxon from the faeces of the baby common marmoset (Callithrix jacchus)

In our previous study based on hsp60 PCR-restriction fragment length polymorphism and 16S rRNA gene sequencing, we stated that the bifidobacterial strains isolated from the individual faecal samples of five baby common marmosets constituted different phylogenetically isolated groups of the genus Bifidobacterium. In that study, we also proposed that these isolated groups potentially represented novel species of the genus Bifidobacterium. Out of them, Bifidobacterium aesculapii, Bifidobacterium myosotis, Bifidobacterium tissieri and Bifidobacterium hapali, have been described recently. Another strain, designated MRM 8.19T, has been classified as member of the genus Bifidobacterium on the basis of positive results for fructose-6-phosphate phosphoketolase activity and analysis of partial 16S rRNA, hsp60, clpC, dnaJ, dnaG and rpoB gene sequences. Analysis of 16S rRNA and hsp60 gene sequences revealed that strain MRM 8.19T was related to B. tissieri DSM 100201T (95.8\u200a%) and to Bifidobacterium bifidum ATCC 29521T (93.7\u200a%), respectively. The DNA G+C composition was 63.7\u2009mol% and the peptidoglycan structure was l-Orn(Lys)-l-Ser. Based on the phylogenetic, genotypic and phenotypic data reported, strain MRM 8.19T represents a novel taxon within the genus Bifidobacterium for which the name Bifidobacterium catulorum sp. nov. (type strain MRM 8.19T=DSM 103154T=JCM 31794T) is proposed

Exopolysaccharide (EPS)-producing Bifidobacterium aesculapii: screening for the presence of rfb_P gene and EPS production

Nowadays, health awareness among consumers has increased the demand for low/fat-free, but at the same time, smooth and creamy dairy products. Thus, since milk fat contributes to flavour, body and texture development of the dairy products, with its removal leading to textural and functional defects and EPSs production by food grade LAB have gained importance as biothickeners and texturizers. During the technological food process/storage, EPSs guarantee the microorganism survival/viability and better rheological properties. Furthermore, EPSs have beneficial effects on health, are involved in adhesion mechanisms, control of pathogens and induction of positive physiological response. Searching for EPSs-producing bacteria is of interest in screening of new strains as probiotic in low-fat and free-dairy foods. Several Bifidobacterium strains are able to produce EPSs, but mechanism of biosynthesis is only hypothesized; however, the priming-glycosyltransferase rfb_P seems the key enzyme in the EPS-units biosynthesis. In our work, a degenerated primer pair was designed on available rfb_P partial gene sequences of Bifidobacterium spp., which were were aligned with ClustalW. Gemi program was used for primers design. After a manual editing, PCR primers specificity was checked against the bacterial genome database using MFE-primer 2.0. Selected primers were tested in vivo: 7 Bifidobacterium aesculapii strains, MRM_3.1T, MRM_4.2, MRM_4.6, MRM_4.7, MRM_4.8, MRM_5.13, MRM_8.7, which produce a gelling exocellular matrix, were tested; Bifidobacterium longum subsp. infantis ATCC15697T and Bifidobacterium saguini DSM23967T were used as positive and negative controls, respectively. The expected amplicon products were electroforetically verified. To correlate the presence of amplicon with the metabolic production, EPSs were extracted and quantified from each strain with the method by Dubois et al. (1956). In silico and in vivo amplifications were performed to test the specificity of our designed degenerated primer pair and results, supported the reliability of our set for a rapid screening of EPS-producing Bifidobacterium spp. Furthermore, quantification analysis\u2026 have also highlighted.

Bifidobacterium myosotis sp. Nov., Bifidobacterium tissieri sp. nov. and Bifidobacterium hapali sp. nov., isolated from faeces of baby common marmosets (Callithrix jacchus L.)

In a previous study on bifidobacterial distribution in New World monkeys, six strains belonging to the Bifidobacteriaceae were isolated from faecal samples of baby common marmosets (Callithrix jacchus L.). All the isolates were Gram-positive-staining, anaerobic, asporogenous and fructose-6-phosphate phosphoketolase-positive. Comparative analysis of 16S rRNA gene sequences revealed relatively low levels of similarity (maximum identity 96\u2009%) to members of the genus Bifidobacterium, and placed the isolates in three independent clusters: strains of cluster I (MRM_5.9T and MRM_5.10) and cluster III (MRM_5.18T and MRM_9.02) respectively showed 96.4 and 96.7\u2009% 16S rRNA gene sequence similarity to Bifidobacterium callitrichos DSM 23973T, while strains of cluster II (MRM_8.14T and MRM_9.14) showed 95.4\u2009% similarity to Bifidobacterium stellenboschense DSM 23968T. Phylogenetic analysis of partial hsp60 and clpC gene sequences supported an independent phylogenetic position of each cluster from each other and from the related type strains B. callitrichos DSM 23973T and B. stellenboschense DSM 23968T. Clusters I, II and III respectively showed DNA G+C contents of 64.9-65.1, 56.4-56.7 and 63.1-63.7\u200amol%. The major cellular fatty acids of MRM_5.9T were C14\u2009:\u20090, C16\u2009:\u20090 and C18\u2009:\u20091\u3c99c dimethylacetal, while C16\u2009:\u20090 was prominent in strains MRM_5.18T and MRM_8.14T, followed by C18\u2009:\u20091\u3c99c and C14\u2009:\u20090. Biochemical profiles and growth parameters were recorded for all the isolates. Based on the data provided, the clusters represent three novel species, for which the names Bifidobacterium myosotis sp. nov. (type strain MRM_5.9T\u2009=\u2009DSM 100196T\u2009=\u2009JCM 30796T), Bifidobacterium hapali sp. nov. (type strain MRM_8.14T\u2009=\u2009DSM 100202T\u2009=\u2009JCM 30799T) and Bifidobacterium tissieri sp. nov. (type strain MRM_5.18T\u2009=\u2009DSM 100201T\u2009=\u2009JCM 30798T) are proposed

Bifidobacterium aerophilum sp. nov., Bifidobacterium avesanii sp. nov. and Bifidobacterium ramosum sp. nov.: Three novel taxa from the faeces of cotton-top tamarin (Saguinus oedipus L.)

Forty-five microorganisms were isolated on bifidobacteria selective medium from one faecal sample of an adult subject of the cotton-top tamarin (Saguinus oedipus L.). All isolates were Gram-positive, catalase-negative, anaerobic, fructose-6-phosphate phosphoketolase positive, and asporogenous rod-shaped bacteria. In this study, only eight out of the forty-five strains were characterized more deeply, whereas the others are still currently under investigation. They were grouped by BOX-PCR into three clusters: Cluster I (TRE 17T, TRE 7, TRE 26, TRE 32, TRE 33, TRE I), Cluster II (TRE CT), and Cluster III (TRE MT). Comparative analysis of 16S rRNA gene sequences confirmed the results from the cluster analysis and revealed relatively low level similarities to each other (mean value 95%) and to members of the genus Bifidobacterium. All eight isolates showed the highest level of 16S rRNA gene sequence similarities with Bifidobacterium scardovii DSM 13734T (mean value 96.6%). Multilocus sequence analysis (MLSA) of five housekeeping genes (hsp60, rpoB, clpC, dnaJ and dnaG) supported their independent phylogenetic position to each other and to related species of Bifidobacterium. The G + C contents were 63.2%, 65.9% and 63.0% for Cluster I, Cluster II and Cluster III, respectively. Peptidoglycan types were A3\u3b1 l-Lys-l-Thr-l-Ala, A4\u3b2 l-Orn (Lys)-d-Ser-d-Glu and A3\u3b2 l-Orn-l-Ser-l-Ala in Clusters I, II and III, respectively. Based on the data provided, each cluster represented a novel taxon for which the names Bifidobacterium aerophilum sp. nov. (TRE 17T = DSM 100689 = JCM 30941; TRE 26 = DSM 100690 = JCM 30942), Bifidobacterium avesanii sp. nov. (TRE CT = DSM 100685 = JCM 30943) and Bifidobacterium ramosum sp. nov. (TRE M = DSM 100688 = JCM 30944) are proposed