Multidrug resistance profile in Lactobacillus delbrueckii: a food industry species with probiotic properties.

Lactobacillus delbrueckii, a widely used lactic acid bacterium in the food industry, has been studied for its probiotic properties and reservoir of antibiotic-resistant genes, raising safety concerns for probiotic formulations and fermented products. This review consolidates findings from 60 articles published between 2012 and 2023, focusing on the global antibiotic resistance profile and associated genetic factors in L. delbrueckii strains. Resistance to aminoglycosides, particularly streptomycin, kanamycin, and gentamicin, as well as resistance to glycopeptides (vancomycin), fluoroquinolones (ciprofloxacin), and tetracyclines was predominant. Notably, although resistance genes have been identified, they have not been linked to mobile genetic elements, reducing the risk of dissemination. However, a significant limitation is the insufficient exploration of responsible genes or mobile elements in 80% of studies, hindering safety assessments. Additionally, most articles originated from Asian and Middle Eastern countries, with strains often isolated from fermented dairy foods. Therefore, these findings underscore the necessity for comprehensive analyses of new strains of L. delbrueckii for potential industrial and biotherapeutic applications and in combating the rise of antibiotic-resistant pathogens.

Amelioration of regulatory T cells by Lactobacillus delbrueckii and Lactobacillus rhamnosus in pristane-induced lupus mice model.

Regulatory T cells (Tregs) play an indispensable role in the control of immune responses and induction of peripheral tolerance. Dysregulation of Tregs is involved in the pathogenesis of systemic lupus erythematosus (SLE). Tolerogenic probiotics have shown beneficial effects in the control of autoimmune diseases. We evaluated the prophylactic and therapeutic effects of Lactobacillus delbrueckii and Lactobacillus rhamnosus on Tregs and their related molecules in pristane-induced lupus mice model. Fifty-four female BALB/c mice (3-5 weeks) were randomly divided into nine groups. Lupus was induced in all groups using pristane. Prophylactic groups were treated from Day 0 (at the time of pristane injection) and treatment groups were treated 2 months later with L. rhamnosus, L. delbrueckii, mix of both probiotics, and prednisolone. One group was considered as SLE-induced control group without any treatment. Presence of antinuclear antibodies (ANA), antidouble-stranded DNA (anti-dsDNA), antiribonucleoprotein (anti-RNP), proteinuria, and serum level of creatinine, urea, the expression of forkhead box P3 (Foxp3), interleukin 6 (IL-6), IL-10, transforming growth factor ß, and the number of Tregs were determined. SLE induction by pristane led to the formation of lipogranuloma, presence of ANA, anti-dsDNA, and anti-RNP. Probiotics consumption decreased the level of lipogranuloma, ANA, and anti-dsDNA. In addition, in probiotics receiving groups, Tregs and the expression level of Foxp3 increased, while IL-6 decreased. The effect of probiotics in the prophylactic group was more prominent. The results may indicate the effectiveness of L. delbrueckii and L. rhamnosus in the enhancement of Tregs and the decrease of inflammatory cytokines and disease severity in SLE-induced mice.

Short communication: Influence of an aqueous myrrh suspension on yogurt culture bacteria over yogurt shelf life.

Myrrh is an essential oil and natural flavoring approved by the US Food and Drug Administration, and it has antibacterial and antifungal activity against pathogens. Our objective was to determine the effect of an aqueous myrrh suspension on Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus counts in peptone solution and yogurt, as well as pH and titratable acidity of yogurt during 5 wk of storage at 1 to 4°C. The myrrh suspension (10% wt/vol) was prepared and incorporated into a pure culture dilution in peptone and into yogurt mix at a 1% (vol/vol) level. A control with no myrrh was also prepared, and 3 replications were conducted. Streptococcus thermophilus were enumerated using Streptococcus thermophilus agar with aerobic incubation at 37°C for 24 h, and Lactobacillus delbrueckii ssp. bulgaricus were enumerated using de Man, Rogosa, and Sharpe agar adjusted to pH 5.2, with anaerobic incubation at 43°C for 72 h. During the 8-h period after inoculation, S. thermophilus and L. delbrueckii ssp. bulgaricus counts in peptone solution at 37°C and 43°C, respectively, were not significantly different in the presence or absence of the aqueous myrrh suspension. Counts of S. thermophilus in yogurt containing myrrh (mean ± SD; 4.96 ± 0.58 log cfu/mL) were not significantly different from those in the control yogurt (4.87 ± 0.39 log cfu/mL). The log counts for L. delbrueckii ssp. bulgaricus in yogurt containing myrrh (5.04 ± 1.44 log cfu/mL) and those of the control (5.52 ± 1.81 log cfu/mL) did not differ, and the counts remained within 1 log of each other throughout 5 wk of storage. The pH of the yogurts containing the aqueous myrrh suspension was not significantly different from that of the control yogurts, and their pH values were within 0.1 pH unit of each other in any given week. Titratable acidity values remained steady around 1.1 to 1.2% lactic acid for both yogurt types throughout the storage period, with no significant differences between them. Yogurt culture bacteria can survive in the presence of a myrrh suspension in yogurt with no significant change in pH or titratable acidity. Therefore, it may be beneficial to add an aqueous myrrh suspension to yogurt.

Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 feeding enhances humoral immune responses, which are suppressed by the antiviral neuraminidase inhibitor oseltamivir in influenza A virus-infected mice.

Antiviral neuraminidase inhibitors, such as oseltamivir, zanamivir, and peramivir, are widely used for treatment of influenza virus infection. We reported previously that oseltamivir inhibits the viral growth cycle, ameliorates symptoms, and reduces viral antigen quantities. Suppressed viral antigen production, however, induces a reduction of acquired antiviral humoral immunity, and increases the incidence of re-infection rate in the following year. To achieve effective treatment of influenza virus infection, it is necessary to overcome these adverse effects of antiviral neuraminidase inhibitors. Feeding of yogurt fermented with Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) OLL1073R-1 is reported to have immune-stimulatory effects on influenza virus infection in mice and humans. In the present study, we assessed the effect of feeding L. bulgaricus OLL1073R-1 yogurt cultures (YC) on local and systemic humoral immune responses, which were suppressed by oseltamivir treatment, in mice infected with influenza A virus. Yogurt culture (1.14 × 108 cfu/0.4 mL per mouse per day) or sterile water (vehicle) was administered by intragastric gavage for 35 d. At d 22, influenza A virus/Puerto Rico/8/34 (H1N1) (PR8; 0.5 pfu/15 µL per mouse) was instilled intranasally, followed immediately by oral administration of oseltamivir (50 µg/100 µL per mouse, twice daily) or 5% methylcellulose (100 µL/mouse) as a vehicle for 13 d. Titers of anti-PR8-specific IgG and IgA in serum and mucosal secretory IgA (S-IgA) and IgG in bronchoalveolar lavage fluid (BALF) were analyzed by ELISA at 14 d after infection. Oseltamivir significantly suppressed the induction of anti-PR8-specific IgG and IgA in serum and S-IgA and IgG in BALF after infection. Feeding YC mildly but significantly stimulated production of PR8-specific IgA in serum, S-IgA in BALF, and IgG in serum without changing the IgG2a:IgG1 ratio. We analyzed the neutralizing activities against PR8 in serum and BALF and found that oseltamivir also reduced protective immunity, and YC feeding abrogated this effect. The immune-stimulatory tendency of YC on anti-PR8-specific IgA and IgG titers in serum and BALF was also detected in mice re-infected with PR8, but the effect was insignificant, unlike the effect of YC in the initial infection.

Prebiotic effects of bovine lactoferrin on specific probiotic bacteria.

Bovine lactoferrin (bLf) is a natural iron-binding protein and it has been suggested to be a prebiotic agent, but this finding remains inconclusive. This study explores the prebiotic potential of bLf in 14 probiotics. Initially, bLf (1-32 mg/mL) treatment showed occasional and slight prebiotic activity in several probiotics only during the late experimental period (48, 78 h) at 37 °C. We subsequently supposed that bLf exerts stronger prebiotic effects when probiotic growth has been temperately retarded. Therefore, we incubated the probiotics at different temperatures, namely 37 °C, 28 °C, room temperature (approximately 22-24 °C), and 22 °C, to retard or inhibit their growth. As expected, bLf showed more favorable prebiotic activity in several probiotics when their growth was partially retarded at room temperature. Furthermore, at 22 °C, the growth of Bifidobacterium breve, Lactobacillus coryniformis, L. delbrueckii, L. acidophilus, B. angulatum, B. catenulatum, and L. paraplantarum were completely blocked. Notably, these probiotics started regrowing in the presence of bLf (1-32 mg/mL) in a significant and dose-dependent manner. Accordingly, bLf significantly increased the growth of Pediococcus pentosaceus, L. rhamnosus, and L. paracasei (BCRC 17483; a locally isolated strain) when their growth was retarded by incubation at 22 °C. In conclusion, bLf showed inconsistent prebiotic activity in the 14 probiotics at 37 °C, but revealed strong prebiotic activity in 10 probiotic strains at 22 °C. Therefore, this study enables determining additional roles of Lf in probiotic strains, which can facilitate developing novel combinational approaches by simultaneously using Lf and specific probiotics.

Production of reuterin in a fermented milk product by Lactobacillus reuteri: Inhibition of pathogens, spoilage microorganisms, and lactic acid bacteria.

We assessed the antimicrobial activity of reuterin produced in vitro in glycerol aqueous solutions in situ by Lactobacillus reuteri ATCC 53608 as part of a fermented milk product against starter (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus), spoilage (Penicillium expansum), pathogenic (Staphylococcus aureus Salmonella enterica ssp. enterica, and Listeria monocytogenes), and pathogen surrogate (Escherichia coli DH5α) microorganisms. We also assayed the influence of cold storage (28 d at 4°C) and reuterin on the color and rheology of the fermented milk product. We obtained maximum reuterin concentrations of 107.5 and 33.97 mM in glycerol aqueous solution and fermented milk product, respectively. Reuterin was stable throughout its refrigerated shelf life. Gram-positive microorganisms were more resistant to reuterin than gram-negative microorganisms. Penicillium expansum and Lactobacillus reuteri ATCC 53608 survived at concentrations up to 10 and 8.5 mM, respectively. Escherichia coli DH5α was the most sensitive to reuterin (0.9 mM). The presence of reuterin did not cause relevant changes in the quality parameters of the fermented milk product, including pH, acidity, soluble solids, color, and rheological aspects (storage and loss moduli and viscosity). This study demonstrated the viability of using Lactobacillus reuteri ATCC 53608 as a biopreservative in a fermented milk product through reuterin synthesis, without drastically modifying its quality parameters.

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.

The bacteriocin bactofencin A subtly modulates gut microbial populations.

The diverse and dynamic microbiota of the gastrointestinal tract represents a vast source of bioactive substances. These include bacteriocins, which are antimicrobial peptides with the potential to modulate gut populations to impact positively on human health. Although several gut-derived bacteriocins have been isolated, there remain only a few exceptional studies in which their influence on microbial populations within the gut has been investigated. To facilitate such investigations, in vitro faecal fermentation systems can be used to simulate the anaerobic environment of the colon. In this instance, such a system was employed to explore the impact of bactofencin A, a novel broad spectrum class IId bacteriocin produced by gut isolates of Lactobacillus salivarius, on intestinal populations and overall microbial diversity. The study reveals that, although bactofencin A is a broad spectrum bacteriocin, it has a relatively subtle influence on intestinal communities, with a potentially positive impact on anaerobic populations such as Bacteroides, Clostridium and Bifidibacterium spp. The strategy taken is an important first step in investigating the merits of using bactofencin A to manipulate the gut microbiota in a beneficial way for health.

Whey protein isolate improves acid and bile tolerances of Streptococcus thermophilus ST-M5 and Lactobacillus delbrueckii ssp. bulgaricus LB-12.

Acid tolerance and bile tolerance are important probiotic characteristics. Whey proteins contain branched-chain amino acids, which play a role in muscle building and are popular among athletes. Increasing emphasis is being placed on diets containing less carbohydrate, less fat, and more protein. The effect of incremental additions of whey protein isolate (WPI) on probiotic characteristics of pure cultures is not known. The objective of this study was to determine the influence of added WPI on acid tolerance and bile tolerance of pure cultures of Streptococcus thermophilus ST-M5 and Lactobacillus bulgaricus LB-12. The WPI was used at 0 (control), 1, 2 and 3% (wt/vol). Assessment of acid tolerance was conducted on pure cultures at 30-min intervals for 2h of acid exposure and bile tolerance at 1-h intervals for 5h of bile exposure. Use of 1, 2, and 3% WPI improved acid tolerance of Strep. thermophilus ST-M5 and Lb. bulgaricus LB-12. The highest counts for acid tolerance of Strep. thermophilus ST-M5 and Lb. bulgaricus LB-12 were obtained when 3% WPI was used. Use of 2 and 3% WPI improved bile tolerance of Strep. thermophilus ST-M5 and Lb. bulgaricus LB-12 over 5h of bile exposure. The use of WPI is recommended to improve acid and bile tolerance of the yogurt culture bacteria Strep. thermophilus ST-M5 and Lb. bulgaricus LB-12.

Chemistry and bioactivity of an artificial adenosylpeptide B(12) cofactor.

Artificial influence: We describe a semi-artificial adenosylpeptide B(12) that behaves as a cofactor in B(12)-dependent enzymatic reactions and demonstrate that the peptide backbone influences its chemical properties and modulates its bioactivity in vitro and in vivo. Inhibition of the growth of L. delbrueckii is demonstrated, thus providing a potentially powerful approach for the development of antibacterial and antiproliferative compounds.

Effect of human defensins on lactobacilli and liposomes.

AIMS: To study the effect of human ß-defensins (HBD-1 and HBD-2) on lactobacilli membranes as well as on liposomes prepared from purified bacterial lipids. METHODS AND RESULTS: Lactobacillus delbrueckii subsp. bulgaricus CIDCA 331 and Lact. delbrueckii subsp. lactis CIDCA 133 were grown in Man, Rogosa, Sharpe broth for 16 h at 37 °C. After being washed, micro-organisms were treated with 0.1-10 µg ml(-1) of HBD-1 and HBD-2 (30 min, 37 °C). Bacterial damage was determined by flow cytometry after propidium iodide staining. In parallel experiments, release of carboxyfluorescein from liposomes prepared from bacterial lipids was determined fluorometrically (excitation 485/20 nm, emission 528/20 nm) in the presence of HBD-1, HBD-2 or Nisin. Exposure of lactobacilli to HBD-2 resulted in a significant membrane permeabilization being Lact. delbrueckii subsp. bulgaricus CIDCA 331 the most susceptible strain. Liposomes prepared with lipids from strain CIDCA 133 were destabilized neither by HBD-1 nor by HBD-2, whereas liposomes derived from strain CIDCA 331 were susceptible to HBD-2 but not to HBD-1. Effect of defensins was strongly inhibited in the presence of NaCl, and the activity increased in water. CONCLUSIONS: Results reported in the presented work indicate that lipid composition of bacterial membranes lead to a different interaction with cationic peptides such as defensins. SIGNIFICANCE AND IMPACT OF THE STUDY: The results represent an advance in the understanding of the differential effect of HBDs on micro-organisms. Differences in susceptibility to anti-microbial peptides could modify the fate of micro-organisms after the interaction with host's cells.

Effects of Roundup(®) and glyphosate on three food microorganisms: Geotrichum candidum, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus.

Use of many pesticide products poses the problem of their effects on environment and health. Amongst them, the effects of glyphosate with its adjuvants and its by-products are regularly discussed. The aim of the present study was to shed light on the real impact on biodiversity and ecosystems of Roundup(®), a major herbicide used worldwide, and the glyphosate it contains, by the study of their effects on growth and viability of microbial models, namely, on three food microorganisms (Geotrichum candidum, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus) widely used as starters in traditional and industrial dairy technologies. The presented results evidence that Roundup(®) has an inhibitory effect on microbial growth and a microbicide effect at lower concentrations than those recommended in agriculture. Interestingly, glyphosate at these levels has no significant effect on the three studied microorganisms. Our work is consistent with previous studies which demonstrated that the toxic effect of glyphosate was amplified by its formulation adjuvants on different human cells and other eukaryotic models. Moreover, these results should be considered in the understanding of the loss of microbiodiversity and microbial concentration observed in raw milk for many years.

A combination of direct viable count and fluorescence in situ hybridization for specific enumeration of viable Lactobacillus delbrueckii subsp.bulgaricus and Streptococcus thermophilus.

AIMS: We have developed a direct viable count (DVC)-FISH procedure for quickly and easily discriminating between viable and nonviable cells of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains, the traditional yogurt bacteria. METHODS AND RESULTS: direct viable count method has been modified and adapted for Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus analysis by testing different times of incubation and concentrations of DNA-gyrase inhibitors. DVC procedure has been combined with fluorescent in situ hybridization (FISH) for the specific detection of viable cells of both bacteria with specific rRNA oligonucleotide probes (DVC-FISH). Of the four antibiotics tested (novobiocin, nalidixic acid, pipemidic acid and ciprofloxacin), novobiocin was the most effective for DVC method and the optimum incubation time was 7 h for both bacteria. The number of viable cells was obtained by the enumeration of specific hybridized cells that were elongated at least twice their original length for Lactobacillus and twice their original size for Streptococcus. CONCLUSIONS: This technique was successfully applied to detect viable cells in inoculated faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: Results showed that this DVC-FISH procedure is a quick and culture-independent useful method to specifically detect viable Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus in different samples, being applied for the first time to lactic acid bacteria.

Technological characterization and survival of the exopolysaccharide-producing strain Lactobacillus delbrueckii subsp. lactis 193 and its bile-resistant derivative 193+ in simulated gastric and intestinal juices.

The capacity of lactic acid bacteria to produce exopolysaccharides (EPS) conferring microorganisms a ropy phenotype could be an interesting feature from a technological point of view. Progressive adaptation to bile salts might render some lactobacilli able to overcome physiological gut barriers but could also modify functional properties of the strain, including the production of EPS. In this work some technological properties and the survival ability in simulated gastrointestinal conditions of Lactobacillus delbrueckii subsp. lactis 193, and Lb. delbrueckii subsp. lactis 193+, a strain with stable bile-resistant phenotype derived thereof, were characterized in milk in order to know whether the acquisition of resistance to bile could modify some characteristics of the microorganism. Both strains were able to grow and acidify milk similarly; however the production of ethanol increased at the expense of the aroma compound acetaldehyde in milk fermented by the strain 193+, with respect to milk fermented by the strain 193. Both microorganisms produced a heteropolysaccharide composed of glucose and galactose, and were able to increase the viscosity of fermented milks. In spite of the higher production yield of EPS by the bile-resistant strain 193+, it displayed a lower ability to increase viscosity than Lb. delbrueckii subsp. lactis 193. Milk increased survival in simulated gastric juice; the presence of bile improved adhesion to the intestinal cell line HT29-MTX in both strains. However, the acquisition of a stable resistance phenotype did not improve survival in simulated gastric and intestinal conditions or the adhesion to the intestinal cell line HT29-MTX. Thus, Lb. delbrueckii subsp. lactis 193 presents suitable technological properties for the manufacture of fermented dairy products; the acquisition of a stable bile-resistant phenotype modified some properties of the microorganism. This suggests that the possible use of bile-resistant derivative strains should be carefully evaluated in each specific application considering the influence that the acquisition of a stable bile-resistant phenotype could have in survival ability in gastric and intestinal conditions and in technological properties.

Bergamot essential oil nanoemulsions: antimicrobial and cytotoxic activity.

Bergamot essential oil (BEO) is well-known for its food preservation activity, as well as anticancer efficacy. However, the poor BEO water solubility and deriving low bioaccessibility have limited its wider applications. The incorporation in nanoemulsions of BEO and its refined fractions was investigated to enhance its dispersibility in water to promote its antimicrobial activity, tested against Escherichia coli, Lactobacillus delbrueckii, and Saccharomyces cerevisiae, and its cytotoxicity already at low concentrations. Different nanoemulsion formulations were tested based on food-grade ingredients, which were characterized in terms of hydrodynamic diameter and polydispersity index, and physical stability. The antimicrobial activity against all the tested micro-organisms was observed to be higher for BEO in its initial composition, than the light fraction, richer in d-limonene, ß-pinene, and γ-terpinene, or the heavy fraction, richer in linalyl acetate and linalool. Remarkably, the use of BEO nanoemulsions notably enhanced the antimicrobial activity for all the tested oils. BEO exhibited also a measurable cytotoxic activity against Caco-2 cells, which was also enhanced by the use of the different nanoemulsions tested, in comparison with free oil, which discourages the direct use of BEO nanoemulsions as a food preservative. Conversely, BEO nanoemulsions might find use in therapeutic applications as anticarcinogenic agents.

In vitro evaluation of yoghurt starter lactobacilli and Lactobacillus rhamnosus GG adhesion to saliva-coated surfaces.

AIM: The aim of the study was to evaluate the adhesion of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus rhamnosus strain GG to saliva-coated surfaces in vitro. METHODS: Fifteen radiolabeled dairy L. delbrueckii subsp. bulgaricus strains and L. rhamnosus GG were tested for their ability to adhere to saliva-coated hydroxyapatite beads and polystyrene microtiter plates and the radioactivity was measured by liquid scintillation counter. The effects of lysozyme on the adhesion of lactobacilli and of pretreatment with lactobacilli on the adhesion of Streptococcus sanguinis were also assessed. RESULTS: All strains tested adhered to saliva-coated surfaces but with significantly different binding frequencies. The adhesion of the L. delbrueckii subsp. bulgaricus strains remained lower in comparison to L. rhamnosus strain GG. One L. delbrueckii subsp. bulgaricus strain showed binding frequency comparable to S. sanguinis. Lysozyme pretreatment of the samples significantly increased lactobacillus adhesion to saliva-coated surfaces. CONCLUSION: The present results showed significant variations in the adhesion capacity of the Lactobacillus strains studied. Adhesion to oral surfaces is of primary importance for bacterial colonization in the mouth. Only one of the L. delbrueckii subsp. bulgaricus dairy starter culture strains investigated had a high adhesion percentage. This strain might then be considered for further investigations in the oral environment.

Genome shuffling of Lactobacillus delbrueckii mutant and Bacillus amyloliquefaciens through protoplasmic fusion for L-lactic acid production from starchy wastes.

Current study was focused on the development of a non-fastidious lactic acid producing strain having better growth rate, low pH tolerance and good productivity by genome shuffling of a mutant strain of Lactobacillus delbrueckii NCIM 2025 and an amylase producing non-fastidious Bacillus amyloliquefaciens ATCC 23842. After the third cycle of the protoplast fusion, lactic acid production by few fusants was monitored and the best fusant was selected for further studies. Optimization of the important process parameters for lactic acid production was conducted using Plackett-Burman design and response surface methodology. Selected fusant could utilize the liquefied cassava bagasse starch directly with minimum nutrient supplementation for lactic acid production. During validation, 40g/L of lactic acid was obtained ( approximately 96% conversion of starch to lactic acid) by using fusant inoculum (3%, v/v) from 83g/L cassava bagasse (starch content 50% w/w) supplemented with yeast extract and peptone (0.2% each, w/v) and the buffering agent (2% CaCO3, w/v).

In vitro anti-HIV and -HSV activity and safety of sodium rutin sulfate as a microbicide candidate.

Sodium rutin sulfate (SRS) is a sulfated rutin modified from the natural flavonol glycoside rutin. Here, we investigated its in vitro anti-HIV and -HSV activities and its cytotoxic profile. Fifty percent inhibitory concentration (IC(50)) values of SRS against HIV-1 X4 virus IIIB, HIV-1 R5 isolates Ada-M and Ba-L were 2.3+/-0.2, 4.5+/-2.0 and 8.5+/-3.8 microM with a selectivity index (SI) of 563, 575 and 329, respectively. Its IC(50) against primary R5 HIV-1 isolate from Yunnan province in China was 13.1+/-5.5 microM, with a SI of 197. In contrast, unsulfated rutin had no activity against any of the HIV-1 isolates tested. Further study indicated that SRS blocked viral entry and virus-cell fusion likely through interacting with the HIV-1 envelope glycoprotein. SRS also demonstrated some activity against human herpes simplex virus (HSV) with an IC(50) of 88.3+/-0.1 microM and a SI of 30. The 50% cytotoxicity concentration (CC(50)) of SRS was >3.0 mM, as determined in human genital ME180, HeLa and primary human foreskin fibroblast cells. Minimum inhibitory concentration of SRS for vaginal lactobacilli was >3.0 mM. These results collectively indicate that SRS represents a novel candidate for anti-HIV-1/HSV microbicide development.

Spontaneous Lactobacillus delbrueckii phage-resistant mutants with acquired bile tolerance.

Three commercial phage-sensitive strains of Lactobacillus delbrueckii (strains Ab(1), YSD V and Ib(3)) and four spontaneous phage-resistant mutants (strains A(7), A(17), V(2) and I(39)) isolated from them, all with a probiotic potential previously demonstrated were studied for their tolerance of bile salts (ox gall). Minimal Inhibitory Concentrations (MICs) ranged from 0.30% to 0.35% (w/v) of ox gall. These strains were exposed to gradually increasing concentrations of ox gall with the aim of isolating bile resistant derivatives. Stable derivatives able to tolerate up to 0.9% of ox gall were obtained from L. delbrueckii Ab(1), as well as from its spontaneous phage-resistant mutants A(7) and A(17). Random Amplified Polymorphic DNA (RAPD-PCR) analysis revealed a strong genetic homology between the ox gall-tolerant derivatives and their respective non-adapted original strains. These derivatives maintained, in general, the phage resistance phenotype of the non-adapted strains, with only one exception (phage-resistant mutant A(7)). After progressive ox gall adaptation, the phage-resistant mutant A(7) also exhibited progressive reversion of the phage resistance phenotype. The derivative with the highest ox gall-acquired tolerance (A(7)(0.9)) became sensitive to the phage, but derivatives with low (A(7)(0.3)) and intermediate (A(7)(0.6)) ox gall-acquired tolerance retained phage resistance. The technological properties of ox gall derivatives were comparable to those of their respective parent strains. However, the cells of the former were smaller than those of the original strains. Finally, the tolerant derivatives grew faster in the presence of ox gall than the parent strains. Our results demonstrated that it was possible to obtain, by a natural selection strategy, probiotic strains with acquired ox gall-tolerance from three (L. delbrueckii Ab(1) and their phage-resistant mutants A(7) and A(17)) of seven tested strains. Since such derivatives keep both phage resistance and other useful technological properties, they could be used for production of functional foods.

In Vitro and In Vivo Evaluation of Lactobacillus delbrueckii subsp. bulgaricus KLDS1.0207 for the Alleviative Effect on Lead Toxicity.

Lead (Pb) is a toxic contaminating heavy metal that can cause a variety of hazardous effects to both humans and animals. In the present study, Lactobacillus delbrueckii subsp. bulgaricus KLDS1.0207 (L. bulgaricus KLDS1.0207), which has a remarkable Pb binding capacity and Pb tolerance, was selected for further study. It was observed that the thermodynamic and kinetic model of L. bulgaricus KLDS1.0207 Pb binding respectively fit with the Langmuir-Freundlich model and the pseudo second-order kinetic model. Scanning electron microscopy and energy dispersive spectroscopy analysis disclosed that the cell surfaces were covered with Pb and that carbon and oxygen elements were chiefly involved in Pb binding. Combined with Fourier transform infrared spectroscopy analysis, it was revealed that the carboxyl, phosphoryl, hydroxyl, amino and amide groups were the main functional groups involved in the Pb adsorption. The protective effects of L. bulgaricus KLDS1.0207 against acute Pb toxicity in mice was evaluated by prevention and therapy groups, the results in vivo showed that L. bulgaricus KLDS1.0207 treatment could reduce mortality rates, effectively increase Pb levels in the feces, alleviate tissue Pb enrichment, improve the antioxidant index in the liver and kidney, and relieve renal pathological damage. Our findings show that L. bulgaricus KLDS1.0207 can be used as a potential probiotic against acute Pb toxicity.