Feasibility between Bifidobacteria Targeting and Changes in the Acoustic Environment of tumor Tissue for Synergistic HIFU.

High intensity focused ultrasound (HIFU) has been recently shown as a rapidly developing new technique for non-invasive ablation of local tumors whose therapeutic efficiency can be significantly improved by changing the tissue acoustic environment (AET). Currently, the method of changing AET is mainly to introduce a medium with high acoustic impedance, but there are some disadvantages such as low retention of the introduced medium in the target area and a short residence time during the process. In our strategy, anaerobic bacterium Bifidobacterium longum (B. longum) which can colonize selectively in hypoxic regions of the animal body was successfully localized and shown to proliferate in the hypoxic zone of tumor tissue, overcoming the above disadvantages. This study aimed to explore the effects of Bifidobacteria on AET (including the structure and acoustic properties of tumor tissues) and HIFU ablation at different time. The results show that the injection of Bifidobacteria increased the collagen fibre number, elastic modulus and sound velocity and decreased neovascularization in tumor tissues. The number of collagen fibres and neovascularization decreased significantly over time. Under the same HIFU irradiation intensity, the B. longum injection increased the coagulative necrosis volume and decreased the energy efficiency factor (EEF). This study confirmed that Bifidobacteria can change the AET and increase the deposition of ultrasonic energy and thereby the efficiency of HIFU. In addition, the time that Bifidobacteria stay in the tumor area after injection is an important factor. This research provides a novel approach for synergistic biologically targeted HIFU therapy.

Gut microbiota of preterm infants supplemented with probiotics: sub-study of the ProPrems trial.

BACKGROUND: The ProPrems trial, a multi-center, double-blind, placebo-controlled randomized trial, previously reported a 54% reduction in necrotizing enterocolitis (NEC) of Bell stage 2 or more from 4.4 to 2.0% in 1099 infants born before 32 completed weeks' gestation and weighing < 1500 g, receiving probiotic supplementation (with Bifidobacterium longum subsp. infantis BB-02, Streptococcus thermophilus TH-4 and Bifidobacterium animalis subsp. lactis BB-12). This sub-study investigated the effect of probiotic supplementation on the gut microbiota in a cohort of very preterm infants in ProPrems. RESULTS: Bifidobacterium was found in higher abundance in infants who received the probiotics (AOR 17.22; 95% CI, 3.49-84.99, p < 0.001) as compared to the placebo group, and Enterococcus was reduced in infants receiving the probiotic during the supplementation period (AOR 0.27; 95% CI, 0.09-0.82, p = 0.02). CONCLUSION: Probiotic supplementation with BB-02, TH-4 and BB-12 from soon after birth increased the abundance of Bifidobacterium in the gut microbiota of very preterm infants. Increased abundance of Bifidobacterium soon after birth may be associated with reducing the risk of NEC in very preterm infants.

Bifidogenic effects of Cordyceps sinensis fungal exopolysaccharide and konjac glucomannan after ultrasound and acid degradation.

The bifidogenic effects of exopolysaccharide (EPS) of a medicinal fungus (Cordyceps sinensis) and a well-known food polysaccharide konjac glucomannan (KGM) with different molecular weight (MW) ranges were evaluated through in vitro experiments in liquid cultures of Bifidobacteria. Native EPS and KGM were partially degraded with power ultrasound (US) to improve the water solubility, and further hydrolysed with trifluoroacetic acid to much lower MW. The acid-hydrolysed fractions (EPS-AH and KGM-AH) supported the growth of all five tested bifidobacterial species, while the US-degraded high MW fractions, EPS-US and KGM-US, could only slightly support the growth of some species. All EPS fractions increased the acetic acid production of most bifidobacterial species. Most remarkably, the high MW EPS-US, EPS-AH and KGM-US fractions significantly enhanced the cell viability with much higher colony forming unit (CFU) counts, suggesting a protective effect of these high MW polysaccharides for the bacterial survival. The results have shown that MW was a significant factor on the bifidogenic properties of partially degraded EPS and KGM.

Overexpression of Small Heat Shock Protein Enhances Heat- and Salt-Stress Tolerance of Bifidobacterium longum NCC2705.

Bifidobacteria are probiotics that are incorporated live into various dairy products. They confer health-promotive effects via gastrointestinal tract colonization. However, to provide their health-beneficial properties, they must battle the various abiotic stresses in that environment, such as bile salts, acids, oxygen, and heat. In this study, Bifidobacterium longum salt- and heat-stress tolerance was enhanced by homologous overexpression of a small heat shock protein (sHsp). A positive contribution of overproduced sHsp to abiotic stress tolerance was observed when the bacterium was exposed to heat and salt stresses. Significantly higher survival of B. l ongum NCC2705 overexpressing sHsp was observed at 30 and 60 min into heat (55 °C) and salt (5 M NaCl) treatment, respectively. Thermotolerance analysis at 47 °C with sampling every 2 h also revealed the great potential tolerance of the engineered strain. Cell density and acid production rate increased for the sHsp-overexpressing strain after 8 and 10 h of both heat and salt stresses. In addition, tolerance to bile salts, low pH (3.5) and low temperature (4 °C) was also increased by homologous overexpression of the sHsp hsp20 in B. l ongum. Results revealed that hsp20 overexpression in B longum NCC2705 plays a positive cross-protective role in upregulating abiotic responses, ensuring the organism's tolerance to various stress conditions; therefore, sHsp-overexpressing B. l ongum is advised for fermented dairy foods and other probiotic product applications.

Membrane fatty acid composition and fluidity are involved in the resistance to freezing of Lactobacillus buchneri R1102 and Bifidobacterium longum R0175.

Determinations of membrane fatty acid composition and fluidity were used together with acidification activity and viability measurements to characterize the physiological state after freezing of Lactobacillus buchneri R1102 and Bifidobacterium longum R0175 cells harvested in the exponential and stationary growth phases. For both strains, lower membrane fluidity was achieved in cells harvested in the stationary growth phase. This change was linked to a lower unsaturated-to-saturated fatty acid ratio for both strains and a higher cyclic-to-saturated fatty acid ratio for L. buchneri R1102 alone. These membrane properties were linked to survival and to maintenance of acidification activity of the cells after freezing, which differed according to the strain and the growth phase. Survival of B. longum R0175 was increased by 10% in cells with low membrane fluidity and high relative saturated fatty acid contents, without any change in acidification activity. Acidification activity was more degraded (70 min) in L. buchneri R1102 cells displaying low membrane fluidity and high saturated and cyclic fatty acid levels. Finally, this study showed that membrane modifications induced by the growth phase differed among bacterial strains in terms of composition. By lowering membrane fluidity, these modifications could be beneficial for survival of B. longum R0175 during the freezing process but detrimental for maintenance of acidification activity of L. buchneri R1102.

Supercritical CO2 interpolymer complex encapsulation improves heat stability of probiotic bifidobacteria.

The probiotic industry faces the challenge of retention of probiotic culture viability as numbers of these cells within their products inevitably decrease over time. In order to retain probiotic viability levels above the therapeutic minimum over the duration of the product's shelf life, various methods have been employed, among which encapsulation has received much interest. In line with exploitation of encapsulation for protection of probiotics against adverse conditions, we have previously encapsulated bifidobacteria in poly-(vinylpyrrolidone)-poly-(vinylacetate-co-crotonic acid) (PVP:PVAc-CA) interpolymer complex microparticles under supercritical conditions. The microparticles produced had suitable characteristics for food applications and also protected the bacteria in simulated gastrointestinal fluids. The current study reports on accelerated shelf life studies of PVP:PVAc-CA encapsulated Bifidobacterium lactis Bb12 and Bifidobacterium longum Bb46. Samples were stored as free powders in glass vials at 30 °C for 12 weeks and then analysed for viable counts and water activity levels weekly or fortnightly. Water activities of the samples were within the range of 0.25-0.43, with an average a(w) = 0.34, throughout the storage period. PVP:PVAc-CA interpolymer complex encapsulation retained viable levels above the recommended minimum for 10 and 12 weeks, for B. longum Bb46 and B. lactis Bb12, respectively, thereby extending their shelf lives under high storage temperature by between 4 and 7 weeks. These results reveal the possibility for manufacture of encapsulated probiotic powders with increased stability at ambient temperatures. This would potentially allow the supply of a stable probiotic formulation to impoverished communities without proper storage facilities recommended for most of the currently available commercial probiotic products.

Sunlight inactivation of human polymerase chain reaction markers and cultured fecal indicators in river and saline waters.

Decay rates for sunlight inactivation of polymerase chain reaction (PCR) markers for total Bacteroidales, human-specific Bacteroidales, Escherichia coli, and Bifidobacterium adolescentis relative to cultured E. coli were investigated. The experiment used 100-L chambers of fresh water and seawater (paired with dark controls) seeded with human sewage and exposed to natural sunlight over three summer days. Culturable E. coli levels in sunlight-exposed chambers decreased by at least 3 logs on day 1, and by day 3 a total reduction of 4.5 to 5.5 logs was achieved in fresh water and seawater, respectively. In contrast, PCR detection of the four gene targets in sunlight-exposed chambers reduced by no more than 2 logs over the duration of the study (k(t) < 0.071 log(e) units h(-1)). Under these experimental conditions, PCR markers are considerably more conservative than culturable E. coli and can persist for extended periods of time following inactivation of E. coli.

Using a simplex centroid to study the effects of pH, temperature and lactulose on the viability of Bifidobacterium animalis subsp. lactis in a model system.

This paper reports on the effects of lactulose (0-10 g/l) on Bifidobacterium animalis subsp. lactis, along with the influence of pH (4.5-8.5) and temperature (15-45 °C); the three factors were combined through a simplex centroid. The experiments were performed in a laboratory medium and the data of cells counts were modeled through the Weibull equation for the evaluation of the first reduction time, the shape parameter and the death time. These fitting parameters were used as input values to build a desirability profile and a second-order model through the DoE approach (Design of Experiments). The medium containing glucose was used as control. The prebiotic enhanced the viability of the microbial target, by prolonging the first reduction time and inducing a shoulder phase in the death kinetic; moreover, in some combinations the statistical analysis highlighted a kind of interaction with the pH.

Cell viability of microencapsulated Bifidobacterium animalis subsp. lactis under freeze-drying, storage and gastrointestinal tract simulation conditions.

The purpose of this study was to improve the survival of Bifidobacterium animalis subsp. lactis 10140 during freeze-drying process by microencapsulation, using a special pediatric prebiotics mixture (galactooligosaccharides and fructooligosaccharides). Probiotic microorganisms were encapsulated with a coat combination of prebiotics-calcium-alginate prior to freeze-drying. Both encapsulated and free cells were then freeze-dried in their optimized combinations of skim milk and prebiotics. Response surface methodology (RSM) was used to produce a coating combination as well as drying medium with the highest cell viability during freeze-drying. The optimum encapsulation composition was found to be 2.1 % Na-alginate, 2.9 % prebiotic, and 21.7 % glycerol. Maximum survival predicted by the model was 81.2 %. No significant (p > 0.05) difference between the predicted and experimental values verified the adequacy of final reduced models. The protection ability of encapsulation was then examined over 120 days of storage at 4 and 25 °C and exposure to a sequential model of infantile GIT conditions including both gastric conditions (pH 3.0 and 4.0, 90 min, 37 °C) and intestinal conditions (pH 7.5, 5 h, 37 °C). Significantly improved cell viability showed that microencapsulation of B. lactis 10140 with the prebiotics was successful in producing a stable symbiotic powdery nutraceutical.

Role of calcium alginate and mannitol in protecting Bifidobacterium.

Fourier transform infrared (FTIR) spectroscopy was carried out to ascertain the mechanism of Ca-alginate and mannitol protection of cell envelope components and secondary proteins of Bifidobacterium animalis subsp. lactis Bb12 after freeze-drying and after 10 weeks of storage at room temperature (25°C) at low water activities (a(w)) of 0.07, 0.1, and 0.2. Preparation of Ca-alginate and Ca-alginate-mannitol as microencapsulants was carried out by dropping an alginate or alginate-mannitol emulsion containing bacteria using a burette into CaCl(2) solution to obtain Ca-alginate beads and Ca-alginate-mannitol beads, respectively. The wet beads were then freeze-dried. The a(w) of freeze-dried beads was then adjusted to 0.07, 0.1, and 0.2 using saturated salt solutions; controls were prepared by keeping Ca-alginate and Ca-alginate-mannitol in aluminum foil without a(w) adjustment. Mannitol in the Ca-alginate system interacted with cell envelopes during freeze-drying and during storage at low a(w)s. In contrast, Ca-alginate protected cell envelopes after freeze-drying but not during 10-week storage. Unlike Ca-alginate, Ca-alginate-mannitol was effective in retarding the changes in secondary proteins during freeze-drying and during 10 weeks of storage at low a(w)s. It appears that Ca-alginate-mannitol is more effective than Ca-alginate in preserving cell envelopes and proteins after freeze-drying and after 10 weeks of storage at room temperature (25°C).

Selection of a Bifidobacterium animalis subsp. lactis strain with a decreased ability to produce acetic acid.

We have characterized a new strain, Bifidobacterium animalis subsp. lactis CECT 7953, obtained by random UV mutagenesis, which produces less acetic acid than the wild type (CECT 7954) in three different experimental settings: De Man-Rogosa-Sharpe broth without sodium acetate, resting cells, and skim milk. Genome sequencing revealed a single Phe-Ser substitution in the acetate kinase gene product that seems to be responsible for the strain's reduced acid production. Accordingly, acetate kinase specific activity was lower in the low acetate producer. Strain CECT 7953 produced less acetate, less ethanol, and more yoghourt-related volatile compounds in skim milk than the wild type did. Thus, CECT 7953 shows promising potential for the development of dairy products fermented exclusively by a bifidobacterial strain.

Enhanced growth and bioconversion of isoflavones in prebiotic-soymilk fermented by UV-treated lactobacilli and bifidobacteria.

The aim of this study was to evaluate the effects of ultraviolet (UV) radiation (ultraviolet A (UVA), ultraviolet B (UVB) and ultraviolet C (UVC) at 30-90 J/m²) on the membrane properties of lactobacilli and bifidobacteria, and their bioconversion of isoflavones in prebiotic-soymilk. UV treatment caused membrane permeabilization and alteration at the acyl chain, polar head and interface region of membrane bilayers via lipid peroxidation. Such alteration subsequently led to decreased (p < 0.05) viability of lactobacilli and bifidobacteria immediately after the treatment. However, the effect was transient where cells treated with UV, particularly UVA, grew better in prebiotic-soymilk than the control upon fermentation at 37°C for 24 h (p < 0.05). In addition, UV treatment also increased (p < 0.05) the intracellular and extracellular ß-glucosidase activity of lactobacilli and bifidobacteria. This was accompanied by an increased (p < 0.05) bioconversion of glucosides to bioactive aglycones in prebiotic-soymilk. Our present study illustrated that treatment of lactobacilli and bifidobacteria with UV could develop a fermented prebiotic-soymilk with enhanced bioactivity.

Exposure of intestinal epithelial cells to UV-killed Lactobacillus GG but not Bifidobacterium breve enhances the effector immune response in vitro.

BACKGROUND: Intestinal bacteria and intestinal epithelial cells (IEC) may modulate the mucosal immune response. In this study, immune modulation by Lactobacillus GG (LGG) and Bifidobacterium breve (Bb1, Bb2) in the presence or absence of IEC was addressed in an in vitro transwell co-culture model. METHODS: UV-killed LGG,Bb1, Bb2 or Toll-like receptor (TLR) 2 or nucleotide oligomerization domain (NOD) 2 ligands were added directly to unstimulated or anti-CD3/CD28-stimulated PBMC, or applied apically to human IEC (HT-29) co-cultured with PBMC. A mixture of live bacteria was used as reference. The effect on T helper 1 (IFN-gamma, IL-12), T helper 2 (IL-13), inflammatory (TNF-alpha) and regulatory (IL-10) cytokine secretion was determined. RESULTS: Both UV-killed LGG and Bb enhanced IL-12, IFN-gamma, TNF-alpha and IL-10, and reduced IL-13 secretion when added directly to stimulated PBMC, similar to live bacteria. IEC reduced IL-13, IFN-gamma and IL-10 secretion by stimulated PBMC. Apically added LGG, TLR2 and NOD2 ligands,but not Bb, enhanced IFN-gamma, IL-12 and/or TNF-alpha secretion. Bacteria did not induce cytokine secretion when added to HT-29/unstimulated PBMC co-cultures, whereas direct incubations with PBMC did. CONCLUSION: UV-killed LGG as well as Bb supported a T helper 1 and/or regulatory phenotype when added directly to activated PBMC, similar to live bacteria. In contrast, LGG, TLR2 or NOD2 ligands - but not Bb - enhanced T helper 1 type cytokine secretion when added to IEC, while IL-10 secretion remained suppressed. Co-cultures combining IEC and PBMC may reveal differences between bacterial strains relevant for the in vivo situation.

Light sensitivity of Bifidobacterium longum in bioreactor cultivations.

Bifidobacteria are gaining commercial significance due to their probiotic properties. However, little is still known about the production of these bacteria and their behavior in bioreactors. Two Bifidobacterium longum strains were sensitive to light when grown in a transparent (glass) bioreactor under microaerophilic growth conditions (i.e. no gases added and slow mixing). The sensitivity was less clear the more anaerobic the initial conditions were. In a darkened bioreactor in microaerophilic conditions, the two strains grew with maximum specific growth rates of 0.36 h(-1) and 0.48 h(-1). In an illuminated bioreactor neither strain grew. In comparison, Lactobacillus reuteri was not sensitive to light under the same conditions.

Adhesion of inactivated probiotic strains to intestinal mucus.

It has been suggested that probiotics should be viable in order to elicit beneficial health effects. Inactivation of probiotics has been suggested to interfere with the binding to the mucosa and thereby with the immune modulating activity of probiotics. The effect of different inactivation methods on the mucus adhesion of nine probiotic strains was studied. Inactivation by heat or gamma-irradiation generally decreased the adhesive abilities. However, heat treatment increased the adhesion of Propionibacterium freudenreichii and gamma-irradiation enhanced the adhesion of Lactobacillus casei Shirota. Inactivation by u.v. was not observed to modulate the adhesion of the tested strains and it was concluded to be the most appropriate method for studying non-viable probiotics and preparing control products.

[The antagonistic properties of bacteria isolated from the digestive tract of female mink housed in the area of the Chernobyl Atomic Electric Power Station]. / Antagonisticheskie svoistva bakterii, vydelennykh iz pishchevaritel'nogo trakta samok norok, soderzhashchikhsia v zone ChAES.

Differences in species composition, number and level of antagonistic activity of bacteria isolated from the digestive tract of Chernobyl female minks of various age and with different immunological state have been established. Prevalence of anaerobes (bifidobacteria) and microaerophils (lactic acid bacteria) with the increase of microorganisms concentration along the channel: stomach, small and large intestine (10(7)-10(10)/g) was found in all the departments of digestive tract of minks. Among the identified lactic-acid bacteria Lactobacillus helveticus (10(7)-10(8)/g) prevailed in the stomach of the studied female minks, L. coryniformis (10(9)-10(10)/g) in the small intestine, L. casei (10(10)/g) in the large one. Antagonistic activity was most expressed in the strains of L. helveticus and L. casei, isolated from the younger (1.5 year-old) minks. Enterococcus faecalis isolated from the stomach of 1.5 year-old female minks was distinguished by the greatest antagonistic activity among identified enterococci. Strains of E. faecium isolated from the thin intestine of the young female minks (1.5 year-old) and from the large intestine of more nature animals (2.5 years) who received thymogen were characterized by the most expressed antibiosis among enterococci isolated bacteria a conclusion was made that the mechanisms of inhibitory effect of the studied microorganisms are underlied by not only their capacity to form organic acids but also by the capacity to produce antibiotic products.

[State of vaginal microbiocenosis and its correction in pregnant women living under conditions of constant exposure to low doses of radiation]. / Sostoianie vaginal'nogo mikrobiotsenoza i ego korrektsiia u beremennykh, prozhivaiushchikh v usloviiakh postoiannogo vozdeistviia malykh doz radiatsii.

Microflora of the vaginal mucosa was investigated bacteriologically in 44 pregnant women at the pregnancy term of 27-28 weeks. It was shown that in half of the women the vaginal microbiocenosis was within the normal and did not depend on urogynecological diseases in the case histories. In the women with impaired microbiocenosis the level of the impairment depended on gynecological diseases in the case histories. In the pregnant women without urogynecological diseases only slightly pronounced changes in the vaginal microbiocenosis were detected. The prophylactic use of eubiotics (bifidumbacterin and lactobacterin) was not efficient. In the women with urogynecological diseases in the case histories the levels of the microbiocenosis impairment were different. The prophylactic use of the eubiotics was efficient when the changes in the vaginal microbiocenosis were slightly pronounced or marked. When the changes were extreme the prophylactic use of the eubiotics failed.