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Bacteriocin is a kind of natural substance that can effectively inhibit bacteria, but its production usually limited by environment. Co-culture is a strategy to stimulate bacteriocin production. Bifidocin A produced by Bifidobacterium animalis BB04, is a novel bacteriocin with a broad-spectrum antimicrobial active of foodborne bacteria. In order to enhance bifidocin A production, bacteriocin-inducing strains were screened firstly in co-cultivation. Then, the molecular mechanism of co-cultural induction was investigated by transcriptomic and proteomic analysis. Finally, the key inducing metabolites were identified by using targeted metabolomic technology. The results showed that Wickerhamomyces anomalus Y-5 in co-cultivation could significantly enhance bifidocin A production, with a 3.00-fold increase compared to mono-culture. The induction may not depend on direct contact with cells and may instead be attributed to be continuous exposure to inducing substances at specific concentration. In co-cultivation, W. anomalus Y-5 up-regulated Hxk2 and Tap42 to activate Glucose-cAMP and Tor and HOG-MAPK pathway, stimulated the expression of the retrograde gene, produced glutamine and glycerol to maintain activity. During this process, glutamine, inosine, guanosine, adenine, uracil, fumaric acid and pyruvic acid produced by W. anomalus Y-5 could induce the synthesis of bifidocin A. In conclusion, W. anomalus Y-5 in co-cultivation induced the synthesis of bifidocin A by regulating various signaling pathways to produce inducing substances. These findings establish a foundation for high-efficient synthesis of bifidocin A and provide a new perspective into the industrial production of bacteriocin., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Bifidobacteria are recognized as health-promoting bacteria that reside in the human gut, helping in the digestion of fiber, preventing infections, and producing essential compounds like vitamins. To date, Bifidobacterium animalis subsp. lactis , together with Bifidobacterium adolescentis , Bifidobacterium bifidum , Bifidobacterium breve, and Bifidobacterium longum , represents one of the species that are used as probiotic bacteria. Despite the extensive and detailed scientific research conducted on this microbial taxon, the molecular mechanisms by which B. animalis subsp. lactis exerts health benefits to its host are still largely unknown. Thus, we dissected the genetic repertoire and phylogenetic relationship of 162 strains of B. animalis subsp. lactis to select a representative reference strain of this taxon suitable for investigating its interaction with the host. The B. animalis subsp. lactis PRL2013 strain, which was isolated by a mucosal sample of a healthy adult, was chosen as the reference of the monophyletic cluster of human origin and revealed a greater adhesion index than that observed for another B. animalis subsp. lactis strain used in the industry as a probiotic supplement. Transcriptomics analyses of PRL2013 strain, when exposed to human cell monolayers, revealed 291 significantly upregulated genes, among which were found genes predicted to encode extracellular structures that may directly interact with human cells, such as extracellular polymeric substances, wall teichoic acids, and pili., Importance: To date, many Bifidobacterium animalis subsp. lactis strains have been isolated from human fecal samples. However, their presence in these samples does not necessarily suggest an ability to colonize the human gut. Furthermore, probiotics of non-human origin may not effectively interact with the gut epithelium, resulting in transient bacteria of the gut microbiota. In vitro experiments with human cells revealed that B. animalis subsp. lactis PRL2013, an autochthonous member of the human gut, shows colonization capability, leading to future applications in functional foods., Competing Interests: The authors declare no conflict of interest.

Bifidobacterium animalis subsp. lactis BLa80 is a new probiotic strain with extensive applications in food products both domestically and internationally. Given the rising consumption of this probiotic, its safety assessment is increasingly crucial in the food industry. This study evaluates the safety of strain BLa80 using a combination of in vitro and in vivo assays along with genomic analysis. Methods included exposing the strain to artificial gastric and intestinal fluids, as well as a medium containing bile salts, to stimulate human digestive conditions. The strain showed high tolerance to gastric fluid at pH of 2.5 and to 0.3 % bile salts. It maintained a 99.92 % survival rate in intestinal fluid. Additional tests assessed hemolytic activity, antibiotic susceptibility (revealing sensitivity to 7 antibiotics), and biogenic amine production using HPLC-ELSD, confirming the absence of histamine, and other harmful amines. Bile salt hydrolase activity was demonstrated qualitatively, and metabolic byproducts were quantitatively analyzed using a D-/l-lactic acid assay kit, showing that BLa80 produces 1.48 mg/mL of l-lactic acid and no harmful d-lactic acid. Genomic analysis confirmed the absence of virulence or pathogenicity genes, and a 90-day oral toxicity study in rats confirmed no toxic effects at various doses. Overall, these findings support the safety classification of the strain BLa80., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest in relation to the topics or materials covered in this manuscript., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In this study, the utilization mechanism of oligosaccharides by Bifidobacterium was investigated through the transcriptome sequencing and non-targeted metabolomics technology of Bifidobacterium animalis cultured with fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS). The results showed that FOS affected the synthesis of adenosine triphosphate binding transporters (ABC transporters) by increasing the expression levels of msmE, msmG, and gluA. Similarly, GOS improved aminoacyl-tRNA synthases by upregulating the expression of tRNA-Ala, tRNA-Pro, and tRNA-Met. Bifidobacterium animalis cultured with FOS and GOS produced different metabolites, such as histamine, tartaric acid, and norepinephrine, with the functions of inhibiting inflammation, alleviating depression and diseases related to brain and nervous system and maintaining body health. Furthermore, the transcriptome and metabolome analysis results revealed that FOS and GOS promoted the growth and metabolism of Bifidobacterium animalis by regulating the related pathways of carbohydrate, energy, and amino acid metabolism. Overall, the experimental results provided significant insights into the prebiotic effects of FOS and GOS., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Functional constipation (FC) can seriously affect the physical and mental health of children. The goal of this study is to assess the efficacy and safety of Bifidobacterium animalis subsp. lactis XLTG11 in treating FC in children through a randomized, double-blinded, placebo-controlled approach. Eligible children were randomized into either the intervention group (IG, n = 65, receiving conventional treatment with probiotics) or the control group (CG, n = 66, receiving conventional treatment without probiotics). The primary outcome measure was fecal frequency. Fecal gut microbiota analysis and PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) were used to predict gene family abundances based on 16S information. Over the course of treatment, the weekly frequency of feces within each group increased significantly (F = 41.97, p < 0.001). The frequency of feces (times/week (t/w)) in the IG was significantly higher than that in the CG (3.69 ± 2.62 t/w vs.3.18 ± 1.43 t/w, 4.03 ± 2.54 t/w vs. 2.89 ± 1.39 t/w and 3.74 ± 2.36 t/w vs. 2.94 ± 1.18 t/w and 3.45 ± 1.98 vs. 3.17 ± 1.41 t/w for the 1st, 2nd, 3rd, and 4th week after intervention, respectively) (F = 7.60, p = 0.0067). After the intervention, dominate species shifted to Bifidobacterium longum, Bifidobacterium breve, and Escherichia coli in the IG. Additionally, genes related to short-chain fatty acid (SCF) metabolism were upregulated, while methane metabolism was downregulated. Administration of XLTG11 at a dose of 1 × 10 10  CFU/day to children increased fecal frequency, induced beneficial changes in gut microbiota, and regulated SCFs and methane metabolism-related genes., (© 2024. The Author(s).)

Background: Preterm children with their aberrant gut microbiota and susceptibility to infections and inflammation constitute a considerable target group for probiotic therapy to generate the age-appropriate healthy microbiota., Methods: 68 preterm neonates were randomized into five intervention groups: Beginning from the median age of 3 days, 13 children received Lactobacillus rhamnosus GG (LGG) directly orally, and 17 via the lactating mother. 14 children received LGG with Bifidobacterium lactis Bb-12 (Bb12) orally, and 10 via the lactating mother. 14 children received placebo. The children's faecal microbiota was assessed at the age of 7 days by 16S rRNA gene sequencing., Results: The gut microbiota compositions of the children directly receiving the probiotic combination (LGG + Bb12) were significantly different from those of the children receiving the other intervention modes or placebo (p = 0.0012; PERMANOVA), the distinction being due to an increase in the relative abundance of Bifidobacterium animalis (P < 0.00010; ANCOM-BC), and the order Lactobacillales (P = 0.020; ANCOM-BC)., Conclusion: The connection between aberrant primary gut microbiota and a heightened risk of infectious and non-communicable diseases invites effective microbiota modulation. We show that the direct, early, and brief probiotic intervention of LGG + Bb12 10 9 CFU each, is sufficient to modulate the gut microbiota of the preterm neonate., Impact: Preterm children have a higher risk of several health problems partly due to their aberrant gut microbiota. More research is needed to find a safe probiotic intervention to modify the gut microbiota of preterm children. The maternal administration route via breast milk might be safer for the newborn. In our study, the early and direct administration of the probiotic combination Lactobacillus rhamnosus GG with Bifidobacterium lactis Bb-12 increased the proportion of bifidobacteria in the preterm children's gut at the age of 7 days, but the maternal administration route was not as effective., (© 2023. The Author(s).)

BackgroundAntimicrobial resistance (AMR) is caused by AMR determinants, mainly genes (ARGs) in the bacterial genome. Bacteriophages, integrative mobile genetic elements (iMGEs) or plasmids can allow ARGs to be exchanged among bacteria by horizontal gene transfer (HGT). Bacteria, including bacteria with ARGs, can be found in food. Thus, it is conceivable that in the gastrointestinal tract, bacteria from the gut flora could take up ARGs from food.AimThe study objective was to gain insight into the ARG set carried by commonly used probiotic bacteria that may enter the human body with non-fermented foods, fermented foods, or probiotic dietary supplements (FFPs) and to assess ARG mobility.MethodsNext generation sequencing whole genome data from 579 isolates of 12 commonly employed probiotic bacterial species were collected from a public repository. Using bioinformatical tools, ARGs were analysed and linkage with mobile genetic elements assessed.ResultsResistance genes were found in eight bacterial species. The ratios of ARG positive/negative samples per species were: Bifidobacterium animalis (65/0), Lactiplantibacillus plantarum (18/194), Lactobacillus delbrueckii (1/40), Lactobacillus helveticus (2/64), Lactococcus lactis (74/5), Leucoconstoc mesenteroides (4/8), Levilactobacillus brevis (1/46), Streptococcus thermophilu s (4/19). In 66% (112/169) of the ARG-positive samples, at least one ARG could be linked to plasmids or iMGEs. No bacteriophage-linked ARGs were found.ConclusionThe finding of potentially mobile ARGs in probiotic strains for human consumption raises awareness of a possibility of ARG HGT in the gastrointestinal tract. In addition to existing recommendations, screening FFP bacterial strains for ARG content and mobility characteristics might be considered.

Some probiotic bifidobacteria are highly robust and shelf-stable, whereas others are difficult to produce, due to their sensitivity to stressors. This limits their potential use as probiotics. Here, we investigate the molecular mechanisms underlying the variability in stress physiologies of Bifidobacterium animalis subsp. lactis BB-12 and Bifidobacterium longum subsp. longum BB-46, by applying a combination of classical physiological characterization and transcriptome profiling. The growth behavior, metabolite production, and global gene expression profiles differed considerably between the strains. BB-12 consistently showed higher expression levels of multiple stress-associated genes, compared to BB-46. This difference, besides higher cell surface hydrophobicity and a lower ratio of unsaturated to saturated fatty acids in the cell membrane of BB-12, should contribute to its higher robustness and stability. In BB-46, the expression of genes related to DNA repair and fatty acid biosynthesis was higher in the stationary than in the exponential phase, which was associated with enhanced stability of BB-46 cells harvested in the stationary phase. The results presented herein highlight important genomic and physiological features contributing to the stability and robustness of the studied Bifidobacterium strains. IMPORTANCE Probiotics are industrially and clinically important microorganisms. To exert their health-promoting effects, probiotic microorganisms must be administered at high counts, while maintaining their viability at the time of consumption. In addition, intestinal survival and bioactivity are important criteria for probiotics. Although bifidobacteria are among the most well-documented probiotics, the industrial-scale production and commercialization of some Bifidobacterium strains is challenged by their high sensitivity to environmental stressors encountered during manufacturing and storage. Through a comprehensive comparison of the metabolic and physiological characteristics of 2 Bifidobacterium strains, we identify key biological markers that can serve as indicators for robustness and stability in bifidobacteria.

Objective: To investigate the effect of Bifidobacterium animalis B94 on the prevention and treatment of liver injury in rats and to elucidate the underlying mechanism of this relationship., Methods: Specific pathogen-free (SPF) rats were selected as the healthy control group, liver injury group and B94 treatment group, with 6 rats in each group. After the model was established, the experimental animals were tested for serum liver function indicators, gut microbiota composition, metabolite composition, and histopathology., Results: The albumin/globulin ratio and serum TBA, alanine aminotransferase, aspartate aminotransferase, and indirect bilirubin levels in the B94 treatment group were significantly lower than those in the liver injury group. 16S rRNA analysis showed that the gut microbiota of the three groups of rats were significantly different. Metabolic profile analysis showed that there were significant differences in the gut metabolomes of the three groups. Haematoxylin-eosin staining of the intestinal mucosa and liver tissues showed that the degree of liver and intestinal tissue damage in the B94 treatment group was significantly lower than that in the liver injury group., Conclusion: Bifidobacterium animalis B94 can affect the process of liver injury in rats by improving liver function, reducing intestinal damage, and regulating gut microbiota and metabolite production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Wang, Yao, Ni, Zhao, Wei and Chen.)

Bifidobacterium animalis subsp. lactis Bb12 is a widely used probiotic that provides numerous health benefits to its host, many due to its immunomodulatory properties. Although the precise mechanism of modulation is still under investigation, several reports associate the interaction of TLR2 with components of the bacterial cell wall inducing a signaling cascade that culminates with the production of cytokines and co-stimulatory molecules. MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of immune responses, including those toward probiotics. In this study, we analyzed the miRNA expression profile in swine monocytes exposed to Bb12 by using an anti-TLR2 blocking strategy and Bb12 involvement in the regulation of the TLR2 pathway. As a result, the expression of 40 miRNAs was influenced by the treatments (p < 0.01), and 15 differentially expressed miRNAs with validated miRNA-mRNA interactions with around 26 proteins related to the TLR2 pathway were identified. The miRNAs upregulated in response to Bb12 included miR-15a-5p, miR-16-5p, miR-26a-5p, miR-29b-3p, and miR-30d-5p, and the following showed downregulation: miR-181a-5p, miR-19b-3p, miR-21-5p, miR-23a-5p, and miR-221-3p. The expression of let-7c-5p, let-7f-5p, miR-146b-5p, miR-150-5p, and miR-155-5p was increased by Bb12 only when TLR2 was blocked. The identified miRNA common targets were downstream proteins from bacterial recognition via TLR2, such as MyD88, TRAF6, and MAPK members; transcription factors such as NF-κB and AP-1; and cytokines such as IL-6, IL-10, and TNF-α. TLR2 participation was abrogated by anti-TLR2 antibody and suggests that bacterial recognition is complemented by other receptors since there were still changes in the microtranscriptome., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Bifidobacterium animalis CP-9 was a commensal strain isolated from human breast milk. In this study, genetic and 90-day oral toxicity were assessed in rodents for its safety. Ames test as well as in vivo bone marrow micronucleus and spermatocyte chromosomal aberration were surveyed in mice. B. animalis CP-9 exhibited no mutagenic activity in the Ames test at the highest tested dosage (5000 µg/plate) with or without metabolic activation. No evidence of in vivo genetic toxicity was observed at the maximum tested dosage of 10 g/kg body weight (BW). Furthermore, there was no statistically significant difference of the biochemical and histological parameters in the rats administrated with B. animalis CP-9 at dosages of 0, 0.25, 0.5, or 1.5 g/kg BW/day. No indication of concern for pathogenicity was exhibited during evaluation of Bifidobacterium ssp. generally, or B. animalis specifically. It was noted that B. animalis CP-9 was able to survive in gastric acid-like and high bile salt environment, and showed strong adhesion to the intestinal epithelial cells, Caco-2. Intriguingly, B. animalis CP-9 decreased olic acid-induced triglyceral (TG) accumulation in the Caco-2 cells, and viable B. animalis CP-9 had a better bacteriostatic activity compared to another well-documented B. animalis ssp. lactis, BB-12. Based on the present study, B. animalis CP-9 can be a safe probiotic supplement and may improve the health of host. PRACTICAL APPLICATION: Although the health benefits of probiotics are well known, the safety of a probiotic product is acquired particularly for a long-term consumption. We conduct the safety of B. animalis CP-9 isolated from human breast milk, and demonstrate no toxicity concern in vitro and in vivo. Hence, B. animalis CP-9 powder can be used as a commercial and safe probiotic supplement with some health benefits., (© 2022 Institute of Food Technologists®.)

Background: The physiology and application characteristics of probiotics are closely associated with the growth phase. Bifidobacterium animalis subsp. lactis A6 is a promising probiotic strain isolated from the feces of a healthy centenarian in China. In this study, RNA-seq was carried out to investigate the metabolic mechanism between the exponential and the stationary phase in B. lactis A6., Results: Differential expression analysis showed that a total of 815 genes were significantly changed in the stationary phase compared to the exponential phase, which consisted of 399 up-regulated and 416 down-regulated genes. The results showed that the transport and metabolism of cellobiose, xylooligosaccharides and raffinose were enhanced at the stationary phase, which expanded carbon source utilizing profile to confront with glucose consumption. Meanwhile, genes involved in cysteine-cystathionine-cycle (CCC) pathway, glutamate dehydrogenase, branched-chain amino acids (BCAAs) biosynthesis, and Clp protease were all up-regulated in the stationary phase, which may enhance the acid tolerance of B. lactis A6 during stationary phase. Acid tolerance assay indicated that the survival rate of stationary phase cells was 51.07% after treatment by pH 3.0 for 2h, which was 730-fold higher than that of 0.07% with log phase cells. In addition, peptidoglycan biosynthesis was significantly repressed, which is comparable with the decreased growth rate during the stationary phase. Remarkably, a putative gene cluster encoding Tad pili was up-regulated by 6.5 to 12.1-fold, which is consistent with the significantly increased adhesion rate to mucin from 2.38% to 4.90% during the transition from the exponential phase to the stationary phase., Conclusions: This study reported growth phase-associated changes of B. lactis A6 during fermentation, including expanded carbon source utilizing profile, enhanced acid tolerance, and up-regulated Tad pili gene cluster responsible for bacterial adhesion in the stationary phase. These findings provide a novel insight into the growth phase associated characteristics in B. lactis A6 and provide valuable information for further application in the food industry., (© 2022. The Author(s).)

Probiotic strains from the Bifidobacterium or Lactobacillus genera improve health outcomes in models of metabolic and cardiovascular disease. Yet, underlying mechanisms governing these improved health outcomes are rooted in the interaction of gut microbiota, intestinal interface, and probiotic strain. Central to defining the underlying mechanisms governing these improved health outcomes is the development of adaptable and non-invasive tools to study probiotic localization and colonization within the host gut microbiome. The objective of this study was to test labeling and tracking efficacy of Bifidobacterium animalis subspecies lactis 420 (B420) using a common clinical imaging agent, indocyanine green (ICG). ICG was an effective in situ labeling agent visualized in either intact mouse or excised gastrointestinal (GI) tract at different time intervals. Quantitative PCR was used to validate ICG visualization of B420, which also demonstrated that B420 transit time matched normal murine GI motility (~8 hours). Contrary to previous thoughts, B420 did not colonize any region of the GI tract whether following a single bolus or daily administration for up to 10 days. We conclude that ICG may provide a useful tool to visualize and track probiotic species such as B420 without implementing complex molecular and genetic tools. Proof-of-concept studies indicate that B420 did not colonize and establish residency align the murine GI tract., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Probiotics are consumed in fermented dairy products or as capsules for their putative health benefits. However, little research has been done to evaluate the effects of the delivery matrix on the health benefits of probiotics in humans. To examine the effects of delivering Bifidobacterium animalis subsp. lactis BB-12 (BB-12) (log 10 10 ± 0.5 CFU/day) via a yogurt smoothie versus a capsule, we monitored the fecal microbiota, gut transit times (GTTs), and fecal excretion of short-chain fatty acids (SCFAs) in healthy adults. In a randomized, four-period, crossover study performed in a partially blind manner, 36 adults were recruited and randomly assigned to four treatments: control yogurt smoothie (YS), yogurt smoothie with BB-12 added prefermentation (PRE), yogurt smoothie with BB-12 added postfermentation (POST), and capsule containing BB-12 (CAP). Participants' fecal microbiota was assessed using 16S rRNA sequencing, GTTs via SmartPill, and fecal SCFAs by gas chromatography (GC) before (baseline) and after each intervention. Participants had significantly higher percentage of Streptococcus after consuming YS versus CAP ( P = 0.01). Bifidobacterium -specific terminal restriction fragment length polymorphism analysis revealed a significantly higher percentage of B. animalis after consuming PRE and POST compared to baseline, YS, CAP, and final washout ( P < 0.0001). The predominant SCFAs were negatively correlated with GTTs. Consumption of BB-12 delivered in a yogurt smoothie or capsule did not significantly alter the composition of the gut microbiota, GTTs, or fecal SCFA concentration of the study cohort. However, daily consumption of BB-12 in yogurt smoothie may result in higher relative abundance of B. animalis in healthy adults. (This trial has been registered at ClinicalTrials.gov under identifier NCT01399996.) IMPORTANCE Bifidobacterium animalis subsp. lactis BB-12 is a probiotic strain that has been used worldwide since 1985. It has commonly been delivered in fermented dairy products for perceived benefits associated with gut health and enhanced immune function. In addition to fermented dairy products, many new probiotic-containing alternatives such as probiotic-containing juice, probiotic-containing chocolate, and capsules have been developed. While these products provide more options for people to access probiotics, little research has been done on the effect of delivery matrix (dairy versus nondairy) on their efficacy in humans. In addition, it was unclear how yogurt fermentation may influence the survival of BB-12 in the product or on its performance in vivo. The significance of our study is in simultaneously assessing the effect of BB-12, alone and in different delivery vehicles, on the gut transit time, fecal short-chain fatty acids, and the composition of the gut microbiota of the study cohort.

Probiotics are known to protect against liver damage induced by the alcohol and acetaldehyde accumulation associated with alcohol intake. However, there have been few studies of the direct effect of probiotics on alcohol metabolism, and the types of probiotics that were previously analyzed were few in number. Here, we investigated the effects of 19 probiotic species on alcohol and acetaldehyde metabolism. Four probiotic species that had a relatively high tolerance to alcohol and metabolized alcohol and acetaldehyde effectively were identified: Lactobacillus gasseri CBT LGA1, Lactobacillus casei CBT LC5, Bifidobacterium lactis CBT BL3, and Bifidobacterium breve CBT BR3. These species also demonstrated high mRNA expression of alcohol and acetaldehyde dehydrogenases. ProAP4, a mixture of these four probiotics species and excipient, was then administered to rats for 2 weeks in advance of acute alcohol administration. The serum alcohol and acetaldehyde concentrations were significantly lower in the ProAP4-administered group than in the control and excipient groups. Thus, the administration of ProAP4, containing four probiotic species, quickly lowers blood alcohol and acetaldehyde concentrations in an alcohol and acetaldehyde dehydrogenasedependent manner. Furthermore, the serum alanine aminotransferase activity, which is indicative of liver damage, was significantly lower in the ProAP4 group than in the control group. The present findings suggest that ProAP4 may be an effective means of limiting alcohol-induced liver damage.

The current study was conducted to develop a quantitative polymerase chain reaction (qPCR) assay for Bifidobacterium animalis ssp. lactis BB-12 quantification in microcapsules matrix with full-fat goat milk and inulin-type fructans. DNA was isolated from milk, feed solutions (before spray drying) and microcapsules (after spray drying) using DNAzol. Two primer pairs targeting Bal-23S or Tuf sequences were evaluated by qPCR. The qPCR efficiency was higher (89.5%) using the Tuf primers than Bal-23S primers (84.8%). Tuf primer pair was able to selectively detect B. animalis ssp. lactis BB-12. After, quantification of bifidobacteria in the microcapsules matrix by Tuf qPCR assay was compared to conventional enumeration by plate counting. The analysis of probiotic feed solutions and microcapsules showed higher (P < 0.05) bacterial enumeration determined by Tuf qPCR assay compared to those obtained by plate counting. This qPCR assay was considered a rapid and sensitive alternative for the quantification of B. animalis ssp. lactis BB-12 in probiotic microcapsules compared to plate counting., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Human gut bifidobacteria rely on ATP-binding cassette (ABC) transporters for oligosaccharide uptake. Multiple oligosaccharide-specific solute-binding protein (SBP) genes are occasionally associated with a single ABC transporter, but the significance of this multiplicity remains unclear. Here, we characterize BlMnBP1 and BlMnBP2, the two SBPs associated to the β-manno-oligosaccharide (MnOS) ABC transporter in Bifidobacterium animalis subsp. lactis. Despite similar overall specificity and preference to mannotriose (K d ≈80 nM), affinity of BlMnBP1 is up to 2570-fold higher for disaccharides than BlMnBP2. Structural analysis revealed a substitution of an asparagine that recognizes the mannosyl at position 2 in BlMnBP1, by a glycine in BlMnBP2, which affects substrate affinity. Both substitution types occur in bifidobacterial SBPs, but BlMnBP1-like variants prevail in human gut isolates. B. animalis subsp. lactis ATCC27673 showed growth on gluco and galactomannans and was able to outcompete a mannan-degrading Bacteroides ovatus strain in co-cultures, attesting the efficiency of this ABC uptake system. By contrast, a strain that lacks this transporter failed to grow on mannan. This study highlights SBP diversification as a possible strategy to modulate oligosaccharide uptake preferences of bifidobacterial ABC-transporters during adaptation to specific ecological niches. Efficient metabolism of galactomannan by distinct bifidobacteria, merits evaluating this plant glycan as a potential prebiotic., (© 2019 John Wiley & Sons Ltd.)

Health promoting or probiotic bacteria are commonly incorporated into a variety of functional foods and drug formulations, due to their purported ability to confer benefit to host health. Despite the extensive commercial exploitation of probiotic formulations there are still major knowledge gaps regarding the precise molecular mechanism of action and corresponding genetic/genomic properties of probiotic bacteria. In the current study, we describe a metagenomic approach which allows determination of the composition of probiotic supplements through next-generation sequencing analyses based on rRNA-associated sequences to assess bacterial composition of the product combined with a shotgun metagenomics approach directed to decode the genome sequences of the probiotic strains for each product assayed. The here developed approach has been tested for 10 probiotic supplements, revealing inconsistencies between the identified probiotic strains and the declared strains as indicated by the producers. Furthermore, the decoded bacterial genome sequence of Bifidobacterium animalis subsp. lactis BB-12 from a 1995 frozen dried stock revealed genetic evidence for genome evolution and stability of this microorganism when compared with the re-constructed genome of the identical strain from a probiotic supplement of 2017., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bifidobacteria are members of the gut microbiota of animals, including mammals, birds, and social insects. In this study, we analyzed and determined the pangenome of Bifidobacterium animalis species, encompassing B. animalis subsp. animalis and the B. animalis subsp. lactis taxon, which is one of the most intensely exploited probiotic bifidobacterial species. In order to reveal differences within the B. animalis species, detailed comparative genomics and phylogenomics analyses were performed, indicating that these two subspecies recently arose through divergent evolutionary events. A subspecies-specific core genome was identified for both B. animalis subspecies, revealing the existence of subspecies-defining genes involved in carbohydrate metabolism. Notably, these in silico analyses coupled with carbohydrate profiling assays suggest genetic adaptations toward a distinct glycan milieu for each member of the B. animalis subspecies, resulting in a divergent evolutionary development of the two subspecies. IMPORTANCE The majority of characterized B. animalis strains have been isolated from human fecal samples. In order to explore genome variability within this species, we isolated 15 novel strains from the gastrointestinal tracts of different animals, including mammals and birds. The present study allowed us to reconstruct the pangenome of this taxon, including the genome contents of 56 B. animalis strains. Through careful assessment of subspecies-specific core genes of the B. animalis subsp. animalis / lactis taxon, we identified genes encoding enzymes involved in carbohydrate transport and metabolism, while unveiling specific gene acquisition and loss events that caused the evolutionary emergence of these two subspecies., (Copyright © 2019 American Society for Microbiology.)

Background: Bifidobacterium animalis subsp. lactis HN019 is widely used as a probiotic in various dairy preparations. To ensure optimal health benefits, an adequate number of probiotics should be viable in products, but the culture-based methods require longer periods to account for the strain. Objective: Develop a standard curve to account viable load of B. animalis subsp. lactis HN019 by real-time PCR. Methods: The growth curve was developed according to plate count method, and cycle threshold (C T ) curve was prepared using C T values. These two curves were then combined to construct the standard curve. To validate the method, the strain was proliferated in whole milk, and viable loads were enumerated by plate counting and relative PCR counting followed by determining the SEM between the two counts. Results: The growth curve and C T curve, respectively, showed the highest viable load (2.13 × 10 8 CFU/mL) and lowest C T value (18.29) after 18 h, and during the entire growth phase (0-18 h), viable loads are inversely proportional to the C T values. The standard curve revealed the model y = 2E + 18e -1.233 x ( y = cells/mL, x = C T value; R ² = 0.992). In validation, the highest SEM (± 0.70 × 10 8 ) was found between the plate count (1.15 × 10 8 cells/mL) and relative PCR count (1.29 × 10 8 cells/mL) after cultivating for 14 h. Conclusions: The method could be readily used in dairy industries to quantify viable B. animalis subsp. lactis HN019 by a shorter period. Highlights: Culture-independent enumeration of viable B. animalis subsp. lactis HN019 by real-time PCR.

β -Galactosidase (E.C.3.2.1.23) catalyzes the hydrolysis of lactose into glucose and galactose and the synthesis of galacto-oligosaccharides as well. The β -galactosidases from bacteria, especially lactobacilli, and yeast have neutral pH and are much more likely to be developed as food additives. However, the challenges of cumbersome purification, product toxicity, and low yield in protein production have limited the commercialization of many excellent candidates. In this study, we identified a β -galactosidase gene ( bg42-106 ) in Bifidobacterium animalis ACCC05790 and expressed the gene product in Escherichia coli BL21(DE3) and Pichia pastoris GS115, respectively. The recombinant bG42-106 purified from E. coli cells was found to be optimally active at pH 6.0 and 60°C and had excellent stability over a wide pH range (5.0-8.0) and at high temperature (60°C). The specific activity of bG42-106 reached up to 2351 U/mg under optimal conditions. The galacto-oligosaccharide yield was 24.45 g/L after incubation with bG42-106 at 60°C for 2 h. When recombinant bG42-106 was expressed in Pichia pastoris GS115, it was found in the culture medium but only at a concentration of 1.73 U/ml. To increase its production, three strategies were employed, including codon optimization, disulfide formation, and fusion with a Cherry tag, with Cherry-tag fusion being most effective. The culture medium of P. pastoris that expressed Cherry-tagged bG42-106 contained 24.4 U/mL of β -galactosidase activity, which is 14-fold greater than that produced by culture of P. pastoris harboring wild-type bG42-106.

Members of the Bifidobacterium genus are widely used as probiotics in fermented milk products. Bifidobacterium animalis subsp. animalis CNCM I-4602 grows and survives poorly in reconstituted skimmed milk (RSM). Availing of genome and transcriptome information, this poor growth and survival phenotype in milk was substantially improved by the addition of certain compounds, such as yeast extract, uric acid, glutathione, cysteine, ferrous sulfate, and a combination of magnesium sulfate and manganese sulfate. Carbohydrate utilization of CNCM I-4602 was also investigated, allowing the identification of several carbohydrate utilization gene clusters, and highlighting this strain's inability to utilize lactose, unlike the type strain of this subspecies, B. animalis subsp. animalis ATCC25527 and the B. animalis subsp. lactis subspecies. In addition, the ability of B. animalis subsp. animalis CNCM I-4602 to colonize a murine model was investigated, which showed that this strain persists in the murine gut for a period of at least 4 weeks. Associated in vivo transcriptome analysis revealed that, among other genes, a gene cluster encoding a predicted type IVb tight adherence (Tad) pilus was upregulated, indicating that this extracellular structure plays a role in the colonization/adaptation of the murine gastrointestinal tract by this strain.

Commercial probiotic bacteria must be tested for acquired antibiotic resistance elements to avoid potential transfer to pathogens. The European Food Safety Authority recommends testing resistance using microdilution culture techniques previously used to establish inhibitory thresholds for the Bifidobacterium genus. Many Bifidobacterium animalis subsp. lactis strains exhibit increased resistance to tetracycline, historically attributed to the ribosomal protection gene tet (W). However, some strains that harbor genetically identical tet (W) genes show various inhibition levels, suggesting that other genetic elements also contribute to observed differences. Here, we adapted several molecular assays to confirm the inhibition of B. animalis subsp. lactis strains Bl-04 and HN019 and employed RNA sequencing to assess the transcriptional differences related to genomic polymorphisms. We detected specific stress responses to the antibiotic by correlating ATP concentration to number of viable genome copies from droplet digital PCR and found that the bacteria were still metabolically active in high drug concentrations. Transcriptional analyses revealed that several polymorphic regions, particularly a novel multidrug efflux transporter, were differentially expressed between the strains in each experimental condition, likely having phenotypic effects. We also found that the tet (W) gene was upregulated only during subinhibitory tetracycline concentrations, while two novel tetracycline resistance genes were upregulated at high concentrations. Furthermore, many genes involved in amino acid metabolism and transporter function were upregulated, while genes for complex carbohydrate utilization, protein metabolism, and clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas systems were downregulated. These results provide high-throughput means for assessing antibiotic resistances of two highly related probiotic strains and determine the genetic network that contributes to the global tetracycline response. IMPORTANCE Bifidobacterium animalis subsp. lactis is widely used in human food and dietary supplements. Although well documented to be safe, B. animalis subsp. lactis strains must not contain transferable antibiotic resistance elements. Many B. animalis subsp. lactis strains have different resistance measurements despite being genetically similar, and the reasons for this are not well understood. In the current study, we sought to examine how genomic differences between two closely related industrial B. animalis subsp. lactis strains contribute to different resistance levels. This will lead to a better understanding of resistance, identify future targets for analysis of transferability, and expand our understanding of tetracycline resistance in bacteria., (Copyright © 2018 Morovic et al.)

Although probiotic lactobacilli and bifidobacteria are generally considered safe by various regulatory agencies, safety properties, such as absence of transferable antibiotic resistance, must still be determined for each strain prior to market introduction as a probiotic. Safety requirements for probiotics vary regionally and evaluation methods are not standardized, therefore methodologies are often adopted from food ingredients or chemicals to assess microbial safety. Four individual probiotic strains, Lactobacillus acidophilus NCFM ® , Lactobacillus paracasei Lpc-37 ® , Bifidobacterium animalis subsp. lactis strains Bl-04 ® , and Bi-07 ® , and their combination (HOWARU ® Restore) were examined for antibiotic resistance by broth microdilution culture, toxin genes by PCR and genome mining, and acute oral toxicity in rats. Only B. lactis Bl-04 exhibited antibiotic resistance above a regulated threshold due to a tetW gene previously demonstrated to be non-transferable. Genomic mining did not reveal any bacterial toxin genes known to harm mammalian hosts in any of the strains. The rodent studies did not indicate any evidence of acute toxicity following a dose of 1.7-4.1 × 10 12  CFU/kg body weight. Considering a 100-fold safety margin, this corresponds to 1.2-2.8 × 10 12  CFU for a 70 kg human. Our findings demonstrate a comprehensive approach of in vitro, in silico, and in vivo safety testing for probiotics., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In this study, Bifidobacterium animalis subsp. lactis BF052 was demonstrated the growth capability in soymilk and could be thus supplemented as a probiotic starter that employed soymilk as one of its food vehicles. The complete genome sequence of BF052 was therefore determined to understand the genetic basis of BF052 as a technological and functional probiotic starter. The whole genome sequence of BF052 consists of a circular genome of 1938 624 bp with a G+C content of 60.50%. This research highlights relevant genes involving in its adaptive responses to industrial and/or environmental stresses and utilization of α-galacto-oligosaccharides in BF052 strain compared with other representative bifidobacterial genomes., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Probiotics are increasingly applied to prevent and treat a range of infectious, immune related and gastrointestinal diseases. Despite this, the mechanisms behind the putative effects of probiotics are poorly understood. One of the suggested modes of probiotic action is modulation of the endogenous gut microbiota, however probiotic intervention studies in adults have failed to show significant effects on gut microbiota composition. The gut microbiota of young children is known to be unstable and more responsive to external factors than that of adults. Therefore, potential effects of probiotic intervention on gut microbiota may be easier detectable in early life. We thus investigated the effects of a 6 month placebo-controlled probiotic intervention with Bifidobacterium animalis subsp. lactis (BB-12®) and Lactobacillus rhamnosus (LGG®) on gut microbiota composition and diversity in more than 200 Danish infants (N = 290 enrolled; N = 201 all samples analyzed), as assessed by 16S rRNA amplicon sequencing. Further, we evaluated probiotic presence and proliferation by use of specific quantitative polymerase chain reaction (qPCR)., Results: Probiotic administration did not significantly alter gut microbiota community structure or diversity as compared to placebo. The probiotic strains were detected in 91.3% of the fecal samples from children receiving probiotics and in 1% of the placebo treated children. Baseline gut microbiota was not found to predict the ability of probiotics to establish in the gut after the 6 month intervention. Within the probiotics group, proliferation of the strains LGG® and BB-12® in the gut was detected in 44.7% and 83.5% of the participants, respectively. A sub-analysis of the gut microbiota including only individuals with detected growth of the probiotics LGG® or BB-12® and comparing these to placebo revealed no differences in community structure or diversity., Conclusion: Six months of probiotic administration during early life did not change gut microbiota community structure or diversity, despite active proliferation of the administered probiotic strains. Therefore, alteration of the healthy infant gut microbiota is not likely to be a prominent mechanism by which these specific probiotics works to exert beneficial effects on host health., Trial Registration: NCT02180581 . Registered 30 June 2014.

Peptidome similarity analysis enables researchers to gain insights into differential peptide profiles, providing a robust tool to discriminate strain-specific peptides, true intra-species differences among biological replicates or even microorganism-phenotype variations. However, no in silico peptide fingerprinting software existed to facilitate such phylogeny inference. Hence, we developed the Peptidomes for Phylogenies (P4P) web tool, which enables the survey of similarities between microbial proteomes and simplifies the process of obtaining new biological insights into their phylogeny. P4P can be used to analyze different peptide datasets, i.e. bacteria, viruses, eukaryotic species or even metaproteomes. Also, it is able to work with whole proteome datasets and experimental mass-to-charge lists originated from mass spectrometers. The ultimate aim is to generate a valid and manageable list of peptides that have phylogenetic signal and are potentially sample-specific. Sample-to-sample comparison is based on a consensus peak set matrix, which can be further submitted to phylogenetic analysis. P4P holds great potential for improving phylogenetic analyses in challenging taxonomic groups, biomarker identification or epidemiologic studies. Notably, P4P can be of interest for applications handling large proteomic datasets, which it is able to reduce to small matrices while maintaining high phylogenetic resolution. The web server is available at http://sing-group.org/p4p., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Aim: To develop a novel validated method for the isolation of Bifidobacterium animalis ssp. lactis BB-12 (BB-12) from faecal specimens and apply it to studies of BB-12 and Lactobacillus rhamnosus GG (LGG) recovered from the healthy human gastrointestinal (GI) tract., Methods and Results: A novel method for isolating and enumerating BB-12 was developed based on its morphologic features of growth on tetracycline-containing agar. The method identified BB-12 correctly from spiked stool close to 100% of the time as validated by PCR confirmation of identity, and resulted in 97-104% recovery of BB-12. The method was then applied in a study of the recovery of BB-12 and LGG from the GI tract of healthy humans consuming ProNutrients ® Probiotic powder sachet containing BB-12 and LGG. Viable BB-12 and LGG were recovered from stool after 21 days of probiotic ingestion compared to baseline. In contrast, no organisms were recovered 21 days after baseline in the nonsupplemented control group., Conclusions: We demonstrated recovery of viable BB-12, using a validated novel method specific for the isolation of BB-12, and LGG from the GI tract of healthy humans who consumed the probiotic supplement., Significance and Impact of the Study: This method will enable more detailed and specific studies of BB-12 in probiotic supplements, including when in combination with LGG., (© 2017 The Society for Applied Microbiology.)

Strains of Bifidobacterium animalis subsp. lactis are well-known health-promoting probiotics used commercially. B. animalis subsp. lactis has been isolated from different sources, and little is known about animal isolates of this taxon. The aim of this study was to examine the genotypic and phenotypic diversity between B. animalis subsp. lactis strains different animal hosts including Cameroon sheep, Barbary sheep, okapi, mouflon, German shepard and to compare to BB12, food isolates and the collection strain DSM 10140. Ten strains of B. animalis subsp. lactis from different sources were characterised by phenotyping, fingerprinting, and multilocus sequence typing (MLST). Regardless of origin, MLST and phylogenetic analyses revealed a close relationship between strains of B. animalis subsp. lactis with commercial and animal origin with the exception of isolates from ovine cheese, mouflon and German Shepard dog. Moreover, isolates from dog and mouflon showed significant differences in fermentation profiles and peptide mass fingerprints (MALDI-TOF). Results indicated phenotypic and genotypic diversity among strains of B. animalis subsp. lactis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Bifidobacteria are gut commensal microorganisms belonging to the Actinobacteria group. Some specific strains of Bifidobacterium animalis subsp. lactis are used in functional foods as they are able to exert health-promoting effects in the human host. Due to the limited genetic variability within this subspecies, it is sometimes difficult for a manufacturer to properly track its strain once included in dairy products or functional foods. In this paper, we present a peptidome-based analysis in which the proteomes of a set of B. animalis subsp. lactis strains were digested in silico with human gut endopeptidases. The molecular masses were compared along all the strains to detect strain-specific peptides. These peptides may be interesting towards the development of methodologies for strain identification in the final product., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The molecular details and impact of oligosaccharide uptake by distinct human gut microbiota (HGM) are currently not well understood. Non-digestible dietary galacto- and gluco-α-(1,6)-oligosaccharides from legumes and starch, respectively, are preferentially fermented by mainly bifidobacteria and lactobacilli in the human gut. Here we show that the solute binding protein (BlG16BP) associated with an ATP binding cassette (ABC) transporter from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 binds α-(1,6)-linked glucosides and galactosides of varying size, linkage, and monosaccharide composition with preference for the trisaccharides raffinose and panose. This preference is also reflected in the α-(1,6)-galactoside uptake profile of the bacterium. Structures of BlG16BP in complex with raffinose and panose revealed the basis for the remarkable ligand binding plasticity of BlG16BP, which recognizes the non-reducing α-(1,6)-diglycoside in its ligands. BlG16BP homologues occur predominantly in bifidobacteria and a few Firmicutes but lack in other HGMs. Among seven bifidobacterial taxa, only those possessing this transporter displayed growth on α-(1,6)-glycosides. Competition assays revealed that the dominant HGM commensal Bacteroides ovatus was out-competed by B. animalis subsp. lactis Bl-04 in mixed cultures growing on raffinose, the preferred ligand for the BlG16BP. By comparison, B. ovatus mono-cultures grew very efficiently on this trisaccharide. These findings suggest that the ABC-mediated uptake of raffinose provides an important competitive advantage, particularly against dominant Bacteroides that lack glycan-specific ABC-transporters. This novel insight highlights the role of glycan transport in defining the metabolic specialization of gut bacteria., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Bifidobacteria are commensal microorganisms of the human and animal intestinal tract, and their surface proteins can mediate bacterial communication and chemical sensing in the environment, as well as facilitate interactions between bacteria and the host. However, a systematic study of the outer surface-associated proteome of bifidobacteria has not been undertaken. In the present study, the proteins located on the surface of Bifidobacterium animalis ssp. lactis KLDS 2.0603 were systematically identified by a nongel proteomic approach, which consisted of the shaving of the bacterial surface with trypsin and an analysis of the released peptides by liquid chromatography-tandem mass spectrometry. A total of 105 surface-associated proteins were found, of which 15 proteins could potentially be involved in adhesion and interactions between bifidobacteria and the host. The proteins related to adhesion and interaction between bacteria and the host include pilus structure proteins (Fim A, Fim B), 10 moonlighting proteins, an NLP/P60 family protein, an immunogenic secreted protein, and a putative sugar-binding secreted protein. The results provide the basis for future studies on the molecular mechanisms of the interactions between bifidobacteria and the host., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Non-digestible milk oligosaccharides were proposed as receptor decoys for pathogens and as nutrients for beneficial gut commensals like bifidobacteria. Bovine milk contains oligosaccharides, some of which are structurally identical or similar to those found in human milk. In a controlled, randomized double-blinded clinical trial we tested the effect of feeding a formula supplemented with a mixture of bovine milk-derived oligosaccharides (BMOS) generated from whey permeate, containing galacto-oligosaccharides and 3'- and 6'-sialyllactose, and the probiotic Bifidobacterium animalis subsp. lactis (B. lactis) strain CNCM I-3446. Breastfed infants served as reference group. Compared with a non-supplemented control formula, the test formula showed a similar tolerability and supported a similar growth in healthy newborns followed for 12 weeks. The control, but not the test group, differed from the breast-fed reference group by a higher faecal pH and a significantly higher diversity of the faecal microbiota. In the test group the probiotic B. lactis increased by 100-fold in the stool and was detected in all supplemented infants. BMOS stimulated a marked shift to a bifidobacterium-dominated faecal microbiota via increases in endogenous bifidobacteria (B. longum, B. breve, B. bifidum, B. pseudocatenulatum)., (© 2015 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The survival of two probiotic strains -Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12 - incorporated into probiotic (PC) and into synbiotic (SC, with inulin + fructooligosaccharides, respectively, at 7.5 and at 2.5 g per 100 g) petit-suisse cheese was investigated in the beginning (day 1) and at the end (28 days) of storage at 4 °C when the food products were subjected to in vitro gastrointestinal simulated assays. Species-specific quantitative real time PCR (qPCR) combined with propidium monoazide (PMA-qPCR) was employed to quantify the strains. Initial La-5 and Bb-12 populations were always above 7 log CFU g(-1). The presence of the prebiotic ingredients in SC improved the Bb-12 and La-5 resistance after the 6 h assay, with higher populations in all the in vitro stages and throughout the storage period (p < 0.05), leading to equal or superior survival rates (SR) in SC of both probiotic strains, in the beginning as well as at the end of storage. The mean La-5 SR were 58% (PC) and 67% (SC), whereas the mean Bb-12 SR were 60% (PC) and 79% (SC). Our findings suggest that the addition of a prebiotic mixture in petit-suisse cheese was advantageous, since it improved both the Bb-12 and La-5 viability and tolerance under in vitro gastrointestinal simulated conditions, both in the fresh product and in the product refrigerated for 28 days.

To ensure quality management during the production processes of probiotics and for efficacy testing in vivo, accurate tools are needed for the identification and quantification of probiotic strains. In this study, a strain-specific qPCR assay based on Suppression Subtractive Hybridisation (SSH) for identifying unique sequences, was developed to quantify the strain Bifidobacterium animalis BAN in broiler feed. Seventy potential BAN specific sequences were obtained after SSH of the BAN genome, with a pool of closely related strain genomes and subsequent differential screening by dot blot hybridisation. Primers were designed for 30 sequences which showed no match with any sequence database entry, using BLAST and FASTA. Primer specificity was assessed by qPCR using 45 non-target strains and species in a stepwise approach. Primer T39&#95;S2 was the only primer pair without any unspecific binding properties and it showed a PCR efficiency of 80% with a Cq value of 17.32 for 20 ng BAN DNA. Optimised feed-matrix dependent calibration curve for the quantification of BAN was generated, ranging from 6.28 × 10(3)cfu g(-1) to 1.61 × 10(6)cfu g(-1). Limit of detection of the qPCR assay was 2 × 10(1)cfu g(-1) BAN. Applicability of the strain-specific qPCR assay was confirmed in a spiking experiment which added BAN to the feed in two concentrations, 2 × 10(6)cfu g(-1) and 2 × 10(4)cfu g(-1). Results showed BAN mean recovery rates in feed of 1.44 × 10(6) ± 4.39 × 10(5)cfu g(-1) and 1.59 × 10(4) ± 1.69 × 10(4)cfu g(-1), respectively. The presented BAN-specific qPCR assay can be applied in animal feeding trials, in order to control the correct inclusion rates of the probiotic to the feed, and it could further be adapted, to monitor the uptake of the probiotic into the gastrointestinal tract of broiler chickens., (Copyright © 2016 Elsevier B.V. All rights reserved.)