The nutrient requirements of Lactobacillus acidophilus LA-5 and their application to fermented milk.

Lactobacillus acidophilus LA-5 is a suitable probiotic for food application, but because of its slow growth in milk, an increase in its efficiency is desired. To shorten the time required for fermentation, the nutrient requirements of L. acidophilus LA-5 were analyzed, including the patterns of consumption of amino acids, purines, pyrimidines, vitamins, and metal ions. The nutrients required by L. acidophilus LA-5 were Asn, Asp, Cys, Leu, Met, riboflavin, guanine, uracil, and Mn2+, and when they were added to milk, the fermentation time of fermented milk prepared by L. acidophilus LA-5 alone was shortened by 9 h, with high viable cell counts that were maintained during storage of nutrient-supplemented fermented milk compared with the control. For fermented milk prepared by fermentation with Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, and L. acidophilus LA-5, viable cell counts of L. acidophilus LA-5 increased 1.3-fold and were maintained during storage of nutrient-supplemented fermented milk compared with the control. Adding nutrients had no negative effect on the quality of the fermented milk. The results indicated that suitable nutrients enhanced the growth of L. acidophilus LA-5 and increased its viable cell counts in fermented milk prepared by L. acidophilus LA-5 alone and mixed starter culture, respectively.

Impact of Phyllantus niruri and Lactobacillus amylovorus SGL 14 in a mouse model of dietary hyperoxaluria.

Hyperoxaluria is a pathological condition which affects long-term health of kidneys. The present study evaluates the impact of the combination of Lactobacillus amylovorus SGL 14 and the plant extract Phyllantus niruri (namely Phyllantin 14™) on dietary hyperoxaluria. Safety and efficacy of Phyllantin 14 have been evaluated in vivo. Mice C57BL6 fed a high-oxalate diet were compared to mice fed the same diet administered with Phyllantin 14 by gavage for 6 weeks. Control mice were fed a standard diet without oxalate. No adverse effects were associated to Phyllantin 14 supplementation, supporting its safety. Mice fed a high-oxalate diet developed significant hyperoxaluria and those administered with Phyllantin 14 showed a reduced level of urinary oxalate and a lower oxalate-to-creatinine ratio. Soluble and insoluble caecal oxalate were significantly lower in treated group, a finding in agreement with the colonisation study, i.e. mice were colonised with SGL 14 after 3 weeks. Microbiota analysis demonstrated that both oxalate diet and Phyllantin 14 can differently modulate the microbiota. In conclusion, our findings suggest that Phyllantin 14 supplementation represents a potential supportive approach for reducing urinary oxalate and/or for enhancing the efficacy of existing treatments.

Physicochemical and microstructural properties and probiotic survivability of symbiotic almond yogurt alternative using polymerized whey protein as a gelation agent.

A plain symbiotic almond yogurt-like product was formulated and developed using a plant-based starter YF-L02 (Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus supplemented with Lactobacillus acidophilus, Lactobacillus paracasei, and Bifidobacterium animalis) and inulin; 0.6% polymerized whey protein (PWP), 0.3% pectin, and 0.05% xanthan gum were optimized for the formula of the almond yogurt alternative. Two groups with/without calcium citrate and vitamin D2 were prepared and analyzed for chemical composition, changes in pH, viscosity, and probiotic survivability during storage at 4 °C for 10 weeks. The results showed that (1) over 10 weeks storage, the differences in the pH, viscosity, and probiotic survivability between the control and the fortified samples were not significant (P > 0.05); (2) the pH of both yogurt samples decreased 0.2 units while their viscosity slightly increased during storage; (3) the populations of L. paracasei and B. animalis remained above 106 cfu/g during the storage, whereas the population of L. acidophilus decreased dramatically during the first 4 weeks, especially the control group; (4) the microstructure was examined by scanning electron microscopy, revealing a compact and denser gel structure formed by 0.6% PWP with the presence of 0.3% pectin and 0.05% xanthan gum. In conclusion, PWP might be a proper gelation agent for the formulation of symbiotic almond yogurt alternative. PRACTICAL APPLICATION: In this study, polymerized whey protein was used as a gelation agent to formulate symbiotic almond yogurt alternatives with comparable physical texture and probiotic survivability to dairy yogurt during storage. This technology may be used for the development of plant-based fermented foods.

Evaluation of pyroligneous acid as a therapeutic agent against Salmonella in a simulated gastrointestinal tract of poultry.

Pyroligneous acid (PA) was evaluated as a potential alternative to therapeutic antibiotics in poultry. Antimicrobial activity of PA was studied at acidic pH (2.0) and neutral pH (7.0) of the liquid against Salmonella enterica and Lactobacillus acidophilus. Acidic PA gave a MIC value of 0.8% (v/v) and 1.6% (v/v), and neutralized PA gave a MIC value of 1.6% (v/v) and 3.2% (v/v) against S. enterica and L. acidophilus respectively. Acidic PA was evaluated at different concentrations in a simulated poultry digestive tract and cecal fermentation to study its effect on the cecal microflora and fermentation profile. PA at a concentration of 1.6% (v/v) completely inhibited S. enterica and was also found to have a similar effect on lactobacilli count as compared with the control (p = 0.17). Additionally, PA at this concentration was found not to have a significant effect on acetic acid production after 24 h of cecal fermentation (p = 0.20). Graphical abstract.

Survival of Lactobacillus acidophilus LA-5 and Escherichia coli O157:H7 in Minas Frescal cheese made with oregano and rosemary essential oils.

The effects of the incorporation of the essential oils from Origanum vulgare L. (OVEO; 0.07 µL/g) and Rosmarinus officinalis L. (ROEO; 2.65 µL/g) in combination in Minas Frescal cheese on the counts of the probiotic Lactobacillus acidophilus LA-5 and Escherichia coli O157:H7 were evaluated during refrigerated storage (7 ±â€¯0.5 °C). The terpenes of OVEO and ROEO, survival of the probiotic strain during in vitro digestion, as well as the physicochemical and sensory aspects were also monitored in Minas Frescal cheese. All terpenes decreased in cheese when the storage time increased. The incorporation of OVEO and ROEO delayed the increase in L. acidophilus LA-5 counts in cheese, but did not affect its ability to survive in cheese under simulated gastrointestinal conditions. The decreases in counts of E. coli O157:H7 observed in the first 15 days of refrigerated storage were strongly correlated (r ≥ 0.82) with the terpenes detected in cheese. Scores attributed for aroma, flavor, overall impression and purchase intention of cheese with OVEO and ROEO increased with the increase of the storage time. The incorporation of OVEO and ROEO in combination could be a strategy to control E. coli O157:H7 in probiotic Minas cheese during storage; however, the amounts of these substances should be cautiously selected considering possible negative sensory impacts in this product.

Characteristics of probiotic yoghurts supplemented with Pu-erh tea infusion.

BACKGROUND: Recently, interest in the development of functional foods enriched with bioactive components has increased. Dairy products supplemented with tea extracts known for their health-promoting properties are good examples of such products. However, most of the scientific studies and applications focus on green tea. The present study was established to estimate the effect of Pu-erh tea supplementation on the viability of starter microorganisms and selected physico-chemical and sensory properties of probiotic ABT-yoghurt. METHODS: ABT-yoghurts (Lactobacillus acidophilus La-5, Bifidobacterium animalis ssp. lactis BB-12, Streptococcus thermophilus) were produced from cow’s milk with 0%, 5%, 10% or 15% (v/v) of Pu-erh tea infusion added before the fermentation stage. The products obtained were subjected to the following analyses one day after production (colour profile) and after 7, 14 and 21 days of cold storage: ferric reducing antioxidant power (FRAP) and anti-radical power (ARP) measured against DPPH radical, titratable acidity, pH, texture parameters (back extrusion test), viability of starter cultures and sensory quality (hedonic scale experiment). RESULTS: Pu-erh tea supplementation significantly enhanced the antioxidant potential of probiotic yoghurts as a 3–6.5-fold increase in FRAP and a 10–24-fold increase in ARP values were observed in comparison to plain ABT-yoghurt. Pu-erh tea slightly enhanced the viability of L. acidophilus and reduced the pH of probiotic yoghurts. Higher concentrations of Pu-erh tea caused decreased firmness and consistency while cohesiveness and index of viscosity remained unaffected upon supplementation. The addition of Pu-erh tea infusion modified the colour and sensory properties of the probiotic yoghurts but the sensory quality of the tea yoghurts was rated lower when compared to the plain one. Among all tea yoghurts, the one with 15% Pu-erh tea additive received the highest scores in sensory assessment. CONCLUSIONS: Pu-erh tea may be successfully applied as a functional additive to probiotic yoghurts, signifi- cantly enhancing the antioxidant properties of fermented milk and ensuring a high rate of starter bacteria viability during storage. However, the level of fortification must be carefully chosen as some doses negatively influence texture parameters and sensory quality.

Effect of different tea extracts on the physicochemical and sensory parameters of stirred probiotic yoghurts.

BACKGROUND: In yoghurt production, additives and flavours are added after the fermentation process, but in the case of yoghurts with the addition of tea extracts all studies concern set-type yoghurts. For this reason, the aim of this study was to analyse the effect of green, black, red (Pu-erh) and white tea extracts addition on sensory characteristics, pH, viscosity and colour of stirred probiotic yoghurts stored for two weeks. METHODS: Skim milk powder (10% w/w), sucrose (4% w/w) and typical yoghurt cultures with the addition of Lactobacillus acidophilus LA-5 were applied for yoghurt manufacture. Then yoghurts were stirred and 4, 8, 12% black, red, green and white tea extracts were added. The samples were analysed after 1, 3, 7 and 14 days of storage at 5ºC. Instrumental colour (L*a*b*) determination, rheological measurements and sensory evaluation were carried out. RESULTS: The pH analysis of the final yoghurt samples showed that the type and quantity of tea extract addition had little effect. Apparent viscosity was affected by 12% addition of tea extracts but not by storage time. The instrumental analysis of the colour confirmed the visual assessment. Sensory evaluation revealed that yoghurts with the addition of green tea extract were the most accepted. The addition of extracts made of red, black and white tea resulted in lower values of all the sensory characteristics tested. CONCLUSIONS: The stirring method can be applied to the production of probiotic yoghurts with various tea extracts. The acidity of the yoghurts was practically not affected by the addition of the infusion derived from the different types of tea. 4% addition of tea extract did not affect the viscosity of the yoghurts. The best sensory scores among tea yoghurts were obtained by yoghurt with 4% green tea addition.

Lactobacillus acidophilus DDS-1 Modulates Intestinal-Specific Microbiota, Short-Chain Fatty Acid and Immunological Profiles in Aging Mice.

Distribution of the microbiota varies according to the location in the gastrointestinal (GI) tract. Thus, dysbiosis during aging may not be limited to faecal microbiota and extend to the other parts of the GI tract, especially the cecum and colon. Lactobacillus acidophilus DDS-1, a probiotic strain, has been shown to modulate faecal microbiota and its associated metabolic phenotype in aging mice. In the present study, we investigated the effect of L. acidophilus DDS-1 supplementation on caecal- and mucosal-associated microbiota, short-chain fatty acids (SCFAs) and immunological profiles in young and aging C57BL/6J mice. Besides differences in the young and aging control groups, we observed microbial shifts in caecal and mucosal samples, leading to an alteration in SCFA levels and immune response. DDS-1 treatment increased the abundances of beneficial bacteria such as Akkermansia spp. and Lactobacillus spp. more effectively in caecal samples than in mucosal samples. DDS-1 also enhanced the levels of butyrate, while downregulating the production of inflammatory cytokines (IL-6, IL-1ß, IL-1α, MCP-1, MIP-1α, MIP-1ß, IL-12 and IFN-γ) in serum and colonic explants. Our findings suggest distinct patterns of intestinal microbiota, improvements in SCFA and immunological profiles with DDS-1 supplementation in aging mice.

Improved Viability of Microencapsulated Probiotics in a Freeze-Dried Banana Powder During Storage and Under Simulated Gastrointestinal Tract.

Freeze-dried banana powder represents an ideal source of nutrients and has not yet been used for probiotic incorporation. In this study, microencapsulation by freeze drying of probiotics Lactobacillus acidophilus and Lactobacillus casei was made using whey protein isolate (WPI), fructooligosaccharides (FOS), and their combination (WPI + FOS) at ratio (1:1). Higher encapsulation yield was found for (WPI + FOS) microspheres (98%). Further, microcapsules of (WPI + FOS) were used to produce a freeze-dried banana powder which was analyzed for bacterial viability under simulated gastrointestinal fluid (SGIF), stability during storage at 4 °C and 25 °C, and chemical and sensory properties. Results revealed that (WPI + FOS) microcapsules significantly increased bacteria stability in the product over 30 days of storage at 4 °C averaging (≥ 8.57 log CFU/g) for L. acidophilus and (≥ 7.61 log CFU/g) for L. Casei as compared to free cells. Bacteria encapsulated in microspheres (WPI + FOS) were not significantly affected by the SGIF, remaining stable up to 7.05 ± 0.1 log CFU/g for L.acidophilus and 5.48 ± 0.1 log CFU/g for L.casei after 90 min of incubation at pH 2 compared to free cells which showed minimal survival. Overall, encapsulated probiotics enriched freeze-dried banana powders received good sensory scores; they can therefore serve as safe probiotics food carriers.

Quality of yogurt fortified with magnesium lactate.

BACKGROUND: Magnesium is a micronutrient which plays an important role in a wide range of fundamental cellular reactions. Deficiency of magnesium leads to serious biochemical and symptomatic changes. The present study was carried out to establish the influence of magnesium lactate fortification on the physico- chemical, microbiological and rheological properties of fat-free yogurt manufactured using different starters. METHODS: In this study, yogurts were produced from fat-free milk, standardized with skimmed milk powder to 6% protein content, and then divided into two parts. One part was left without supplementa- tion as a control and in the second part, magnesium L-lactate hydrate was added in the amount of 317.30 mg 100 g–1, which was equal to 35 mg of Mg2+ 100 g–1 of milk. Both mixtures were blended, pasteurized at 85°C for 30 minutes, cooled to 45°C and then divided into three parts, inoculated with: (1) YC-X11 yogurt cul- ture, (2) YF-L811 yogurt culture and (3) VITAL yogurt culture supplemented with probiotics (Lactobacillus acidophilus, Bifidobacterium lactis) respectively. Fermentation was performed at 43°C and the final yogurts were cooled to 5°C. After 24 hours of cold storage, the pH values, titratable acidity, syneresis, color, texture profile, viscosity, sensory analysis and microbiology of the yogurts were analyzed. RESULTS: The results showed that addition of magnesium lactate significantly reduced syneresis and increased the hardness of fat-free yogurts. There was no impact on the viability of starter bacteria in the yogurts after 24 hours of refrigerated storage. CONCLUSIONS: Magnesium lactate showed good potential for the fortification of dairy foods, according to physicochemical data. Further research is needed regarding the influence of storage time and to establish whether the observed effects are largely due to the magnesium cation or lactate anion.

Phyto-Fabrication of ZnO Nanoparticles Using Piper betel Aqueous Extract and Evaluation of its Applicability in Dentistry.

BACKGROUND: Facile, environmental friendly synthesis of metal oxide nanoparticles is of paramount importance when its biological applications are considered. OBJECTIVE: Current study reports phyto-fabrication of Zinc oxide nanoparticles using aqueous extract of Piper betel leaves as stabilizing and capping agent using co-precipitation method. RESULTS: P betel synthesized ZnO nanoparticles (PZnO) was characterized using Powder X-ray diffraction, UV-Visible spectroscopy, scanning electron microscopy and dynamic light scattering. PXRD pattern demonstrated hexagonal wurtzite structure with preferred orientation (100) plane confirmed by JCPDS file 36-1451. UV- Vis spectroscopy showed peak at 370 nm due to band edge of semiconductor, the PZnO. The average particle size determined by DLS measurement was 69 nm and morphologically the particles were short rod shaped and agglomerated as demonstrated by SEM images. Antibacterial activity of PZnO against dental pathogens namely Streptococcus mutans and Lactobacillus acidphillus was performed using well-diffusion method and antioxidant activity against 2, 2 diphenyl 1 picrylhydrazyl (DPPH) free radicals and were compared with ZnO used in clinical dentistry (DZnO). PZnO showed higher antioxidant activity of ~70% at 200 µg/mL with consistent activity at lower aliquots. PZnO showed higher antimicrobial activity than DZnO against both tested microbes and also exhibited inhibitory effects in concentration as low as 3.25 µg/mL. Cytotoxicity study using Balb 3T3 mouse fibroblast cell lines showed <40% cellular growth inhibition by both PZnO and DZnO, indicating their benignity towards selected cell lines. CONCLUSION: Phyto-fabricated facile PZnO nanoparticles having demonstrable antibacterial and antioxidant activity can be considered for use in clinical dentistry after further clinical trials.

Effect of ultrasound on lactic acid production by Lactobacillus strains in date (Phoenix dactylifera var. Kabkab) syrup.

Date syrup is rich in fermentable sugars and may be used as a substrate for different microbial fermentations, including lactic acid fermentation processes. The beneficial effects of ultrasounds (US) on bioprocesses have been reported for several microorganisms, due to the enhancement of cell growth, as well as improvements in yields and productivities. Therefore, US treatments (30 kHz, 100 W, 10-30 min) were applied to two lactobacilli (Lactobacillus helveticus PTCC 1332 and Lactobacillus acidophilus PTCC 1643), during fermentation using date syrup as substrate. The effects on lactic acid fermentation were evaluated by analyzing cell growth (dry cell weight and viable cell count), substrate consumption (quantification of glucose and fructose), and product formation (quantification of lactic acid) over time. The effects of US were also evaluated on cell membrane permeability. Both lactobacilli were able to grow well on date syrup without the need for addition of further ingredients. The US effects were highly dependent on treatment duration: treatments of 10- and 20-min stimulated lactobacilli growth, while the treatment extension to 30 min negatively affected cell growth. Similarly, the 10- and 20-min treatments increased sugar consumption and lactic acid production, contrarily to the 30-min treatment. All US treatments increased cell membrane permeability, with a more pronounced effect at more extended treatments. The results of this work showed that application of appropriate US treatments could be a useful tool for stimulation of lactic acid production from date syrup, as well as for other fermentative processes that use date syrup as substrate.

Mucin- and carbohydrate-stimulated adhesion and subproteome changes of the probiotic bacterium Lactobacillus acidophilus NCFM.

Adhesion to intestinal mucosa is a crucial property for probiotic bacteria. Adhesion is thought to increase host-bacterial interactions, thus potentially enabling health benefits to the host. Molecular events connected with adhesion and surface proteome changes were investigated for the probiotic Lactobacillus acidophilus NCFM cultured with established or emerging prebiotic carbohydrates as carbon source and in the presence of mucin, the glycoprotein of the epithelial mucus layer. Variation in adhesion to HT29-cells and mucin was associated with carbon source and mucin-induced subproteome abundancy differences. Specifically, while growth on fructooligosaccharides (FOS) only stimulated adhesion to intestinal HT-29 cells, cellobiose and polydextrose in addition increased adhesion to mucin. Adhesion to HT-29 cells increased by about 2-fold for bacteria grown on mucin-supplemented glucose. Comparative 2DE-MS surface proteome analysis showed different proteins in energy metabolism appearing on the surface, suggesting they exert moonlighting functions. Mucin-supplemented bacteria had relative abundance of pyruvate kinase and fructose-bisphosphate aldolase increased by about 2-fold while six spots with 3.2-2.1 fold reduced relative abundance comprised elongation factor G, phosphoglycerate kinase, BipAEFTU family GTP-binding protein, ribonucleoside triphosphate reductase, adenylosuccinate synthetase, 30S ribosomal protein S1, and manganese-dependent inorganic pyrophosphatase. Surface proteome of cellobiose- compared to glucose-grown L. acidophilus NCFM had phosphate starvation inducible protein stress-related, thermostable pullulanase, and elongation factor G increasing 4.4-2.4 fold, while GAPDH, elongation factor Ts, and pyruvate kinase were reduced by 2.0-1.5 fold in relative abundance. Addition of recombinant L. acidophilus NCFM elongation factor G and pyruvate kinase to a coated mucin layer significantly suppressed subsequent adhesion of the bacterium. BIOLOGICAL SIGNIFICANCE: Human diet is important for intestinal health and food components, especially non-digestible carbohydrates can beneficially modify the microbiota. In the present study, effects of emerging and established prebiotic carbohydrates on the probiotic potential of Lactobacillus acidophilus NCFM were investigated by testing adhesion to a mucin layer and intestinal cells, and comparing this with changes in abundancy of surface proteins thought to be important for host interactions. Increased adhesion was observed following culturing of the bacterium with fructooligosaccharides, cellobiose or polydextrose, as well as mucin-supplemented glucose as carbon source. Enhanced adhesion ability can prolong bacterial residence in GIT yielding positive health effects. Higher relative abundance of certain surface proteins under various conditions (i.e. grown on cellobiose or mucin-supplemented glucose) suggested involvement of these proteins in adhesion, as confirmed by competition in case of two recombinantly produced moonlighting proteins. Combination of Lactobacillus acidophilus NCFM with different carbohydrates revealed potential bacterial determinants of synbiotic interactions, including stimulation of adhesion.

Amhezole, A Novel Fungal Secondary Metabolite from Aspergillus terreus for Treatment of Microbial Mouth Infection.

Bio-guided fractionation of Aspergillus terreus extract leads to isolation of a novel terpenoidal secondary metabolite. The isolated compound and the total alcoholic extract of Aspergillus terreus showed a remarkable activity against microbial mouth infections; namely, Candida albicans, Lactobacillus acidophilus, Streptococcus gordonii, and S. mutan. Moreover, the Minimum Inhibitory Concentration of the isolated compound was determined and showed low values. The combination of each of the alcoholic extract of A. terreus and the isolated compound Coe-Comfort tissue conditioner inhibited the growth of Candida albicans at concentrations of 500 and 7.81 µg/mL, respectively, Lactobacillus acidophilus at concentrations of 250 and 7.81 µg/mL, respectively, Streptococcus gordonii at concentrations of 1000 and 62.50 µg/mL, respectively, and S. mutans at concentrations of 1000 and 125 µg/mL, respectively. The oral dosing of the extract and the isolated compound did not show any significant effect on the activity of alanine aminotransferase, aspirate aminotransferase, and the levels of blood urea and serum creatinine. Copyright © 2017 John Wiley & Sons, Ltd.

Probiotic supplementation in diabetic hemodialysis patients has beneficial metabolic effects.

This study determined the effects of probiotic supplementation on glycemic control, lipid concentrations, biomarkers of inflammation and oxidative stress in 60 diabetic patients on hemodialysis in a parallel randomized double-blind placebo-controlled clinical trial. Participants were initially matched based on sex, duration of dialysis and diabetes, body mass index and age. Subsequently, they were randomly divided into two groups to take either a capsule containing the probiotics Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium bifidum or placebo for 12 weeks. Based on three-day dietary records throughout the trial, there was no significant change in dietary macro- and micro-nutrients or total dietary fiber to confound results. After the 12 weeks, analysis of patients who received probiotic supplements compared with the placebo showed they had significantly decreased fasting plasma glucose (-22.0 vs. +6.6 mg/dl), serum insulin (-6.4 vs. +2.3 µIU/ml), homeostasis model of assessment-estimated insulin resistance (-2.9 vs. +2.5), homeostasis model of assessment-estimated beta-cell function (-14.1 vs. +6.1) and HbA1c (-0.4 vs. -0.1%,), and improved quantitative insulin sensitivity check index (+0.03 vs. -0.02). Additionally, compared with the placebo, probiotic supplementation resulted in significant reductions in serum high-sensitivity C-reactive protein (-1933 vs. +252 ng/ml), plasma malondialdehyde (-0.3 vs. +1.0 µmol/l), subjective global assessment scores (-0.7 vs. +0.7) and total iron binding capacity (-230 vs. +33 µg/dl), and a significant increase in plasma total antioxidant capacity (+15 vs. -88 mmol/l). Thus, probiotic supplementation for 12 weeks among diabetic hemodialysis patients had beneficial effects on parameters of glucose homeostasis, and some biomarkers of inflammation and oxidative stress.

Cymbopogon citratus essential oil: effect on polymicrobial caries-related biofilm with low cytotoxicity

Abstract The objective of this study was to evaluate the effects of Cymbopogon citratus essential oil and its main compound (citral) against primary dental colonizers and caries-related species. Chemical characterization of the essential oil was performed by gas chromatography/mass spectroscopy (GC/MS), and the main compound was determined. Antimicrobial activity was tested against Actinomyces naeslundii, Lactobacillus acidophilus, S. gordonii, S. mitis, S. mutans, S. sanguinis and S. sobrinus. Minimum inhibitory and bactericide concentrations were determined by broth microdilution assay for streptococci and lactobacilli reference, and for clinical strains. The effect of the essential oil on bacterial adhesion and biofilm formation/disruption was investigated. Negative (without treatment) and positive controls (chlorhexidine) were used. The effect of citral on preformed biofilm was also tested using the same methodology. Monospecies and microcosm biofilms were tested. ANOVA or Kruskal-Wallis tests were used (α=0.05). Cytotoxicity of the essential oil to human keratinocytes was performed by MTT assay. GC/MS demonstrated one major component (citral). The essential oil showed an inhibitory effect on all tested bacterial species, including S. mutans and L. acidophilus. Essential oil of C. citratus (10X MIC) reduced the number of viable cells of lactobacilli and streptococci biofilms (p < 0.05). The essential oil inhibited adhesion of caries-related polymicrobial biofilm to dental enamel (p < 0.01). Citral significantly reduced the number of viable cells of streptococci biofilm (p < 0.001). The essential oil showed low cytotoxicity to human keratinocytes. Based on these findings, this study can contribute to the development of new formulations for products like mouthwash, against dental biofilms.

In Situ Electron Microscopy of Lactomicroselenium Particles in Probiotic Bacteria.

Electron microscopy was used to test whether or not (a) in statu nascendi synthesized, and in situ measured, nanoparticle size does not differ significantly from the size of nanoparticles after their purification; and (b) the generation of selenium is detrimental to the bacterial strains that produce them. Elemental nano-sized selenium produced by probiotic latic acid bacteria was used as a lactomicroselenium (lactomicroSel) inhibitor of cell growth in the presence of lactomicroSel, and was followed by time-lapse microscopy. The size of lactomicroSel produced by probiotic bacteria was measured in situ and after isolation and purification. For these measurements the TESLA BS 540 transmission electron microscope was converted from analog (aTEM) to digital processing (dTEM), and further to remote-access internet electron microscopy (iTEM). Lactobacillus acidophilus produced fewer, but larger, lactomicroSel nanoparticles (200-350 nm) than Lactobacillus casei (L. casei), which generated many, smaller lactomicroSel particles (85-200 nm) and grains as a cloudy, less electrodense material. Streptococcus thermophilus cells generated selenoparticles (60-280 nm) in a suicidic manner. The size determined in situ in lactic acid bacteria was significantly lower than those measured by scanning electron microscopy after the isolation of lactomicroSel particles obtained from lactobacilli (100-500 nm), but higher relative to those isolated from Streptococcus thermopilus (50-100 nm). These differences indicate that smaller lactomicroSel particles could be more toxic to the producing bacteria themselves and discrepancies in size could have implications with respect to the applications of selenium nanoparticles as prebiotics.

Effect of Aqueous Extract of the Seaweed Gracilaria domingensis on the Physicochemical, Microbiological, and Textural Features of Fermented Milks.

The effects of the Gracilaria domingensis seaweed aqueous extract in comparison with gelatin on the physicochemical, microbial, and textural characteristics of fermented milks processed with the mixed culture SAB 440 A, composed of Streptococcus thermophilus, Lactobacillus acidophilus, and Bifidobacterium animalis ssp. lactis, were investigated. The addition of G. domingensis aqueous extract did not affect pH, titratable acidity, and microbial viability of fermented milks when compared with the control (with no texture modifier) and the products with added gelatin. Fermented milk with added the seaweed aqueous extract showed firmness, consistency, cohesiveness, and viscosity index at least 10% higher than those observed for the control product (P < 0.05). At 4 h of fermentation, the fermented milks with only G. domingensis extract showed a texture comparable to that observed for products containing only gelatin. At 5 h of fermentation, firmness and consistency increased significantly (P < 0.05) in products with only seaweed extract added, a behavior not observed in products with the full amount of gelatin, probably due to the differences between the interactions of these ingredients with casein during the development of the gel network throughout the acidification of milk. The G. domingensis aqueous extract appears as a promising gelatin alternative to be used as texture modifier in fermented milks and related dairy products.

The influence of the dose of calcium bisglycinate on physicochemical properties, sensory analysis and texture profile of kefirs during 21 days of cold storage.

BACKGROUND: In the process of enrichment of dairy products a priority element is the proper selection of compounds that are a mineral carrier. Calcium bisglycinate is better absorbed by the body than inorganic forms of calcium. Moreover, the lactic acid which is produced in kefir fermentation and the presence of lactose have also a positive effect on the improvement of absorption of calcium. The aim of the present study was to determine the influence of the applied dose of calcium in the form of calcium bisglycinate on the physicochemical and sensory properties and texture of kefirs during 21-day period of cold storage. METHODS: Processed cow milk was enriched with 0, 5, 10, 15, 20, 25 and 30 mg of calcium (for 100 g of milk), repasteurized (72°C, 1 min), cooled down (26°C), inoculated with Commercial VITAL kefir culture (Danisco, Poland) and fermented for 16 hours (26°C). The assessment of the influence of addition   of calcium bisglycinate on acidity, syneresis, texture and sensory characteristics (1-9 points) of kefirs was conducted at four fixed dates (after 1 day, 7 days, 14 days and 21 days of storage). RESULTS: During successive weeks of cold storage in all experimental groups there was observed a tendency to decrease general acidity. On the 1st and 7th days of cold storage reduced whey leakage was observed in kefirs enriched with 25 mg and 30 mg Ca/100 g of milk. With increasing doses of enrichment with calcium both the hardness, adhesiveness and gumminess of kefirs decreased. The applied doses of calcium did not cause changes in the sensory characteristics such as colour and consistency of the fermented beverages. CONCLUSIONS: Calcium bisglycinate may be used to enrich kefirs with calcium even with 30 mg of calcium in 100 g of milk without the modification of the product's parameters.

Copper(II)-Bis(Thiosemicarbazonato) Complexes as Antibacterial Agents: Insights into Their Mode of Action and Potential as Therapeutics.

There is increasing interest in the use of lipophilic copper (Cu)-containing complexes to combat bacterial infections. In this work, we showed that Cu complexes with bis(thiosemicarbazone) ligands [Cu(btsc)] exert antibacterial activity against a range of medically significant pathogens. Previous work using Neisseria gonorrhoeae showed that Cu(btsc) complexes may act as inhibitors of respiratory dehydrogenases in the electron transport chain. We now show that these complexes are also toxic against pathogens that lack a respiratory chain. Respiration in Escherichia coli was slightly affected by Cu(btsc) complexes, but our results indicate that, in this model bacterium, the complexes act primarily as agents that deliver toxic Cu ions efficiently into the cytoplasm. Although the chemistry of Cu(btsc) complexes may dictate their mechanism of action, their efficacy depends heavily on bacterial physiology. This is linked to the ability of the target bacterium to tolerate Cu and, additionally, the susceptibility of the respiratory chain to direct inhibition by Cu(btsc) complexes. The physiology of N. gonorrhoeae, including multidrug-resistant strains, makes it highly susceptible to damage by Cu ions and Cu(btsc) complexes, highlighting the potential of Cu(btsc) complexes (and Cu-based therapeutics) as a promising treatment against this important bacterial pathogen.