Hernandulcin Production in Cell Suspensions of Phyla Scaberrima: Exploring Hernandulcin Accumulation through Physical and Chemical Stimuli.

Hernandulcin (HE) is a non-caloric sweetener synthesized by the Mexican medicinal plant Phyla scaberrima. Herein we present the results of HE production through cell suspensions of P. scaberrima as well as the influence of pH, temperature, biosynthetic precursors and potential elicitors to enhance HE accumulation. The incorporation of mevalonolactone (30-400 mg L-1 ) farnesol (30-400 mg L-1 ), AgNO3 (0.025-0.175 M), cellulase (5-60 mg L-1 ; 0.3 units/mg), chitin (20-140 mg L-1 ) and (+)-epi-α-bisabolol (300-210 mg L-1 ) to the cell suspensions, resulted in a differential accumulation of HE and biomass. Among elicitors assayed, chitin, cellulase and farnesol increased HE production from 93.2 to ∼160 mg L-1 but, (+)-epi-α-bisabolol (obtained by a synthetic biology approach) increased HE accumulation up to 182.7 mg L-1 . HE produced by the cell suspensions was evaluated against nine strains from six species of gastrointestinal bacteria revealing moderate antibacterial activity (MIC, 214-465 µg mL-1 ) against Staphylococcus aureus, Escherichia coli and Helicobacter pylori. Similarly, HE showed weak toxicity against Lactobacillus sp. and Bifidobacterium bifidum (>1 mg mL-1 ), suggesting a selective antimicrobial activity on some species of gut microbiota. According to our results, chitin and (+)-epi-α-bisabolol were the most effective molecules to enhance HE accumulation in cell suspensions of P. scaberrima.

Promotive effects of sesamin on proliferation and adhesion of intestinal probiotics and its mechanism of action.

The effect of sesamin on intestinal flora was studied by in vitro animal fecal anaerobic fermentation system, and were analyzed by 16S rDNA sequencing. Results showed that sesamin modulated the composition of intestinal microorganisms and reshaped gut microbiome. The abundance of probiotics Lactobacillaceae and Bifidobacteriaceae increased, while the abundance of Enterobacteriaceae decreased. The properties of probiotics (Bifidobacterium bifidum and Lactophilus acidophilus) adhesion to epithelial colon cells (NCM460) were assessed by gram staining and plate counting methods. Results showed that sesamin increased the adhesive index of probiotics. Analysis of RT-qPCR, Western blot and immunofluorescence staining indicated that sesamin up-regulated the expression of the adhesive protein (ß-cadherin and E-cadherin) of NCM460 cells. In conclusion, sesamin could promote the proliferation and adhesion of intestinal probiotics leading to modulating gut microbiota, which provided basis for sesamin as a food-borne functional factor for improving intestinal health.

2'-Fucosyllactose promotes Bifidobacterium bifidum DNG6 adhesion to Caco-2 cells.

Adhesion to the intestinal mucosa is the prerequisite for bifidobacteria to colonize and exert biological functions, whereas the choice of carbon source affects the ability of bifidobacteria to adhere to and interact with intestinal epithelial cells. However, knowledge about the relationship between human milk oligosaccharide consumption by bifidobacteria and its adhesion is still limited. In this study, we aim to investigate the effect of 2'-fucosyllactose (2'-FL) as the carbon source on the growth and adhesion properties of Bifidobacterium bifidum DNG6, and make comparisons with galactooligosaccharides and glucose. We found that the growth and adhesion properties of B. bifidum DNG6 grown in different carbon sources were varied. The 2'-FL as a carbon source improves the adhesion ability of B. bifidum DNG6. The expression of adhesion-associated genes was significantly higher in B. bifidum DNG6 grown in 2'-FL after incubation with Caco-2 cells compared with that in galactooligosaccharides and glucose. Our results indicated that 2'-FL may promote B. bifidum DNG6 adhesion to Caco-2 cells through high expression of genes encoding adhesion proteins. The findings of this study contribute to a better understanding of the involvement of human milk oligosaccharides in the adhesion of bifidobacteria and further support the potential application of 2'-FL as a prebiotic in infant nutritional supplements.

Antibiotic Susceptibility Profiles of Lactic Acid Bacteria from the Human Vagina and Genetic Basis of Acquired Resistances.

Lactic acid bacteria can act as reservoirs of antibiotic resistance genes that can be ultimately transferred to pathogens. The present work reports on the minimum inhibitory concentration (MIC) of 16 antibiotics to 25 LAB isolates of five Lactobacillus and one Bifidobacterium species from the human vagina. Acquired resistances were detected to kanamycin, streptomycin, chloramphenicol, gentamicin, and ampicillin. A PCR analysis of lactobacilli failed to identify genetic determinants involved in any of these resistances. Surprisingly, a tet(W) gene was detected by PCR in two Bifidobacterium bifidum strains, although they proved to be tetracycline-susceptible. In agreement with the PCR results, no acquired genes were identified in the genome of any of the Lactobacillus spp. strains sequenced. A genome analysis of B. bifidum VA07-1AN showed an insertion of two guanines in the middle of tet(W) interrupting the open reading frame. By growing the strain in the presence of tetracycline, stable tetracycline-resistant variants were obtained. An amino acid substitution in the ribosomal protein S12 (K43R) was further identified as the most likely cause of VA07-1AN being streptomycin resistance. The results of this work expand our knowledge of the resistance profiles of vaginal LAB and provide evidence for the genetic basis of some acquired resistances.

Melanins of Inonotus Obliquus: Bifidogenic and Antioxidant Properties.

Extracts and melanins from Inonotus obliquus are widely used in medicine due to their high antioxidant properties. This study is dedicated to define the influence of the physicochemical and antioxidant properties of Inonotus obliquus melanins and their bifidogenic effects on Bifidobacterium bifidum 1 and Bifidobacterium animalis subsp. lactis. For this purpose, melanins precipitated from Inonotus obliquus aqueous extracts, obtained by a few methods, and separated melanin fractions by organic solvents were used. For the melanin physicochemical properties analysis spectrophotometry, electron paramagnetic resonance (EPR) spectroscopy and dynamic light scattering methods were applied. Melanins and their fractions difference in particle size and charge, antioxidant properties, and redox potential were revealed. It was shown that the redox potential, the size of melanin particles and the z-potential had maximum influence on bifidobacteria growth. The greatest activating effect on bifidobacteria was established by using melanin isolated from aqueous microwave extracts in concentrations of 10-13, 10-10, 10-5 g/cm3. The use of this melanin with antioxidant activity 0.67 ± 0.06 mg/g (expressed as ascorbic acid equivalent), and with redox potential -5.51 ± 2.22 mV as a prebiotic allowed the growth of Bifidobacterium bifidum 1 s to increase by 1.4 times in comparison with ascorbic acid by 24 h of cultivation.

Optimization of composite cryoprotectant for freeze-drying Bifidobacterium bifidum BB01 by response surface methodology.

Freeze drying has been well applied in the preparation of high-efficiency probiotic powders. However, the process is generally accompanied by probiotic viability deficiency, which is the bottleneck for further application. To improve the viability of Bifidobacterium bifidum BB01 during freeze-drying, we optimized the cryoprotectant of B. bifidum BB01 by response surface methodology (RSM) with a Central Composite Design (CCD). In this study, two values of B. bifidum BB01 with different protectant factors were investigated, including freeze-drying survival rate and the viable counts of per unit weight of freeze-dried powder. The optimized cryoprotectants were obtained as follows: glycine of 5.5%, sodium bicarbonate of 0.8%, xylo-oligosaccharides of 7%, arginine of 4.5% and skim milk of 25%. The survival rate and the viable counts of per unit weight of powder were 90.37 ± 1.9% and (2.78 ± 0.13) × 1011cfu·g-1, respectively, both close to the predicted value (88.58% and 2.71 × 1011 cfu·g-1). Our research demonstrated that RSM was successful in optimizing composite cryoprotectant for freeze-dried powder of B. bifidum which can as well protect the probiotic cells.

Safety Evaluations of Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI.

Over the past decade, a variety of lactic acid bacteria have been commercially available to and steadily used by consumers. However, recent studies have shown that some lactic acid bacteria produce toxic substances and display properties of virulence. To establish safety guidelines for lactic acid bacteria, the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) has suggested that lactic acid bacteria be characterized and proven safe for consumers’ health via multiple experiments (e.g., antibiotic resistance, metabolic activity, toxin production, hemolytic activity, infectivity in immune-compromised animal species, human side effects, and adverse-outcome analyses). Among the lactic acid bacteria, Bifidobacterium and Lactobacillus species are probiotic strains that are most commonly commercially produced and actively studied. Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI have been used in global functional food markets (e.g., China, Germany, Jordan, Korea, Lithuania, New Zealand, Poland, Singapore, Thailand, Turkey, and Vietnam) as nutraceutical ingredients for decades, without any adverse events. However, given that the safety of some newly screened probiotic species has recently been debated, it is crucial that the consumer safety of each commercially utilized strain be confirmed. Accordingly, this paper details a safety assessment of B. bifidum BGN4 and B. longum BORI via the assessment of ammonia production, hemolysis of blood cells, biogenic amine production, antimicrobial susceptibility pattern, antibiotic resistance gene transferability, PCR data on antibiotic resistance genes, mucin degradation, genome stability, and possession of virulence factors. These probiotic strains showed neither hemolytic activity nor mucin degradation activity, and they did not produce ammonia or biogenic amines (i.e., cadaverine, histamine or tyramine). B. bifidum BGN4 and B. longum BORI produced a small amount of putrescine, commonly found in living cells, at levels similar to or lower than that found in other foods (e.g., spinach, ketchup, green pea, sauerkraut, and sausage). B. bifidum BGN4 showed higher resistance to gentamicin than the European Food Safety Authority (EFSA) cut-off. However, this paper shows the gentamicin resistance of B. bifidum BGN4 was not transferred via conjugation with L. acidophilus ATCC 4356, the latter of which is highly susceptible to gentamicin. The entire genomic sequence of B. bifidum BGN4 has been published in GenBank (accession no.: CP001361.1), documenting the lack of retention of plasmids capable of transferring an antibiotic-resistant gene. Moreover, there was little genetic mutation between the first and 25th generations of B. bifidum BGN4. Tetracycline-resistant genes are prevalent among B. longum strains; B. longum BORI has a tet(W) gene on its chromosome DNA and has also shown resistance to tetracycline. However, this research shows that its tetracycline resistance was not transferred via conjugation with L. fermentum AGBG1, the latter of which is highly sensitive to tetracycline. These findings support the continuous use of B. bifidum BGN4 and B. longum BORI as probiotics, both of which have been reported as safe by several clinical studies, and have been used in food supplements for many years.

Bifidobacterium bifidum Extracellular Sialidase Enhances Adhesion to the Mucosal Surface and Supports Carbohydrate Assimilation.

Bifidobacterium is a natural inhabitant of the human gastrointestinal (GI) tract. We studied the role of the extracellular sialidase (SiaBb2, 835 amino acids [aa]) from Bifidobacterium bifidum ATCC 15696 in mucosal surface adhesion and carbohydrate catabolism. Human milk oligosaccharides (HMOs) or porcine mucin oligosaccharides as the sole carbon source enhanced B. bifidum growth. This was impaired in a B. bifidum ATCC 15696 strain harboring a mutation in the siabb2 gene. Mutant cells in early to late exponential growth phase also showed decreased adhesion to human epithelial cells and porcine mucin relative to the wild-type strain. These results indicate that SiaBb2 removes sialic acid from HMOs and mucin for metabolic purposes and may promote bifidobacterial adhesion to the mucosal surface. To further characterize SiaBb2-mediated bacterial adhesion, we examined the binding of His-tagged recombinant SiaBb2 peptide to colonic mucins and found that His-SiaBb2 as well as a conserved sialidase domain peptide (aa 187 to 553, His-Sia) bound to porcine mucin and murine colonic sections. A glycoarray assay revealed that His-Sia bound to the α2,6-linked but not to the α2,3-linked sialic acid on sialyloligosaccharide and blood type A antigen [GalNAcα1-3(Fucα1-2)Galß] at the nonreducing termini of sugar chains. These results suggest that the sialidase domain of SiaBb2 is responsible for this interaction and that the protein recognizes two distinct carbohydrate structures. Thus, SiaBb2 may be involved in Bifidobacterium-mucosal surface interactions as well as in the assimilation of a variety of sialylated carbohydrates.IMPORTANCE Adhesion to the host mucosal surface and carbohydrate assimilation are important for bifidobacterium colonization and survival in the host gastrointestinal tract. In this study, we investigated the mechanistic basis for B. bifidum extracellular sialidase (SiaBb2)-mediated adhesion. SiaBb2 cleaved sialyl-human milk oligosaccharides and mucin glycans to produce oligosaccharides that supported B. bifidum growth. Moreover, SiaBb2 enhanced B. bifidum adhesion to mucosal surfaces via specific interactions with the α2,6 linkage of sialyloligosaccharide and blood type A antigen on mucin carbohydrates. These findings provide insight into the bifunctional role of SiaBb2 and the adhesion properties of B. bifidum strains.

Cellulase-Assisted Extraction of Polysaccharides from White Hyacinth Bean: Characterization of Antioxidant Activity and Promotion for Probiotics Proliferation.

Food-derived polysaccharides have advantages over synthetical compounds and have attracted interest globally for decades. In this study, we optimized the cellulase-assisted extraction of polysaccharides from white hyacinth bean (PWBs) with the aid of response surface methodology (RSM). The optimum extraction parameters were a pH of 7.79, a cellulase of 2.73%, and a ratio of water to material of 61.39, producing a high polysaccharide yield (3.32 ± 0.03)%. The scavenging ability of PWBs varied on three radicals (hydroxyl > 2,2-diphenyl-1-picrylhydrazyl (DPPH) > superoxide). Furthermore, PWBs contributed to the proliferation of three probiotic bacteria (Lactobacillus acidophilus LA5, Bifidobacterium bifidum BB01, and Lactobacillus bulgaricus LB6). These investigations of PWBs provide a novel bioresource for the exploitation of antioxidant and probiotic bacterial proliferation.

An In Vitro Approach to Study Effects of Prebiotics and Probiotics on the Faecal Microbiota and Selected Immune Parameters Relevant to the Elderly.

The aging process leads to alterations of gut microbiota and modifications to the immune response, such changes may be associated with increased disease risk. Prebiotics and probiotics can modulate microbiome changes induced by aging; however, their effects have not been directly compared. The aim of this study was to use anaerobic batch culture fermenters to assess the impact of various fermentable carbohydrates and microorganisms on the gut microbiota and selected immune markers. Elderly volunteers were used as donors for these experiments to enable relevance to an aging population. The impact of fermentation supernatants on immune markers relevant to the elderly were assessed in vitro. Levels of IL-1ß, IL-6, IL-8, IL-10 and TNF-α in peripheral blood mononuclear cell culture supernatants were measured using flow cytometry. Trans-galactooligosaccharides (B-GOS) and inulin both stimulated bifidobacteria compared to other treatments (p<0.05). Fermentation supernatants taken from faecal batch cultures supplemented with B-GOS, inulin, B. bifidum, L. acidophilus and Ba. coagulans inhibited LPS induced TNF-α (p<0.05). IL-10 production, induced by LPS, was enhanced by fermentation supernatants from faecal batch cultures supplemented with B-GOS, inulin, B. bifidum, L. acidophilus, Ba. coagulans and Bac. thetaiotaomicron (p<0.05). To conclude, prebiotics and probiotics could lead to potentially beneficial effects to host health by targeting specific bacterial groups, increasing saccharolytic fermentation and decreasing inflammation associated with aging. Compared to probiotics, prebiotics led to greater microbiota modulation at the genus level within the fermenters.