Immobilization of whole cells of Lactococcus lactis containing high levels of a hyperthermostable ß-galactosidase enzyme in chitosan beads for efficient galacto-oligosaccharide production.

Galacto-oligosaccharides (GOS) are prebiotic food ingredients that are proposed to stimulate the growth of beneficial gut microorganisms, particularly bifidobacteria. Previously, we developed a method for efficient GOS production using whole cells of Lactococcus lactis containing high levels of a hyper-thermostable ß-galactosidase enzyme from Sulfolobus solfataricus. In this study, a recombinant DNA removal and whole-cell enzyme immobilization process was developed to produce GOS from lactose before removal of the immobilized whole-cell enzyme, which could be reused for subsequent applications. Chitosan was found to be a superior immobilization material compared with alginate, as it retained its bead structure during the high temperature (90°C) used here for GOS production. Prior to immobilization, the recombinant DNA was degraded in the whole cells using UV treatment, resulting in an immobilized whole-cell enzyme that was free of recombinant DNA and with minimum effect on the efficiency of the enzyme. The optimum pH and temperature for GOS synthesis using the chitosan beads was pH = 5.5 and 90°C. The highest GOS production using the chitosan beads occurred with 40% initial lactose resulting in 150 g/L of GOS (tri-oligosaccharides and tetra-oligosaccharides) in addition to di-oligosaccharide GOS products that were not quantified. Notably, the highest lactose conversion rate was found using lower starting lactose concentrations, with more than 60% conversion into tri-oligosaccharides and tetra-oligosaccharides. The immobilized enzyme retained ∼50% activity after 2 cycles of GOS production. In conclusion, the chitosan-immobilized whole-cell enzyme can be used for efficient GOS production that is free of the whole-cell enzyme as well as detectable recombinant DNA.

Production of galactooligosaccharides using a hyperthermophilic ß-galactosidase in permeabilized whole cells of Lactococcus lactis.

Galactooligosaccharides (GOS) are novel prebiotic food ingredients that can be produced from lactose using ß-galactosidase, but the process is more efficient at higher temperatures. To efficiently express the lacS gene from the hyperthermophile Sulfolobus solfataricus, in Lactococcus lactis a synthetic gene (lacSt) with optimized codon usage for Lc. lactis was designed and synthesized. This hyperthermostable ß-galactosidase enzyme was successfully overexpressed in Lc. lactis LM0230 using a nisin-controlled gene expression system. Enzyme-containing cells were then killed and permeabilized using 50% ethanol and were used to determine both hydrolysis and transgalactosylation activity. The optimum conditions for GOS synthesis was found to be at pH 6.0 and 85 °C. A maximum production of 197 g/L of GOS tri- and tetrasaccharides was obtained from 40% initial lactose, after 55 h of incubation. The total GOS yield increased with the initial lactose concentration, whereas the highest lactose conversion rate (72%) was achieved from a low lactose solution (5%). Given that a significant proportion of the remaining lactose would be expected to be converted into disaccharide GOS, this should enable the future development of a cost-effective approach for the conversion of whey-based substrates into GOS-enriched food ingredients using this cell-based technology.

Factors influencing the stability of freeze-dried stress-resilient and stress-sensitive strains of bifidobacteria.

Freeze-drying is a common method for preservation of probiotics, including bifidobacteria, for further industrial applications. However, the stability of freeze-dried bifidobacteria varies depending on the freeze-drying method and subsequent storage conditions. The primary goals of this study were to develop an optimized freeze-drying procedure and to determine the effects of temperature, water activity, and atmosphere on survival of freeze-dried bifidobacteria. To address these goals, a commercially used bifidobacteria strain that is resilient to stress, Bifidobacterium animalis ssp. lactis Bb-12, and a characterized intestinal strain that is more sensitive to stress conditions, Bifidobacterium longum DJO10A, were used. A freeze-drying protocol was developed using trehalose as the cryoprotectant, which resulted in almost no loss of viability during freeze-drying. Resuscitation medium, temperature, and time did not significantly influence recovery rates when this cryoprotectant was used. The effects of temperature (-80 to 45°C), water activity (0.02 to 0.92), and atmosphere (air, vacuum, and nitrogen) were evaluated for the stability of the freeze-dried powders during storage. Freeze-dried B. animalis ssp. lactis Bb-12 was found to survive under all conditions tested, with optimum survival at temperatures up to 21°C, water activities up to 0.44, and all 3 atmospheres tested. The intestinal-adapted strain B. longum DJO10A was much more sensitive to the different storage conditions, but could be adequately maintained using optimum conditions. These optimum storage conditions included frozen storage, replacement of oxygen with nitrogen, and water activities between 0.11 and 0.22. These results indicated that an optimized storage environment is required to maintain viability of stress-sensitive bifidobacteria strains, whereas stress-resilient bifidobacteria strains can maintain viability over a wide range of storage conditions, which is practical in countries where controlled cold storage conditions may not be readily available.

Comparative analysis of an intestinal strain of Bifidobacterium longum and a strain of Bifidobacterium animalis subspecies lactis in Cheddar cheese.

Bifidobacteria cultures were incorporated into Cheddar cheeses to conduct a comparative analysis between the commercially available strain Bifidobacterium animalis ssp. lactis Bb-12 and the wild-type intestinal isolate, Bifidobacterium longum DJO10A. They were incorporated as starter adjuncts in separate vats and as a mixed culture, and survival through manufacturing and cheese ripening was assessed. For cheese using only Bb-12, the cells may have grown during cheese manufacture as 133% of the inoculum was incorporated into the cheese, resulting in 8.00 log cfu/g. Counts remained high during ripening showing less than 1 log decrease over a 12-mo period. For cheese using a mixed culture of Bb-12 and DJO10A, both strains were incorporated at much lower levels: 3.02 and 1.11%, respectively. This resulted in cheese with 6.00 and 5.04 log cfu/g for Bb-12 and DJO10A, respectively. Bifidobacteria survival rates were low, most likely due to the moisture of the cheese being below 38%. The Bb-12 demonstrated almost 100% viability during ripening. Numbers of DJO10A started to decline after 2 mo of ripening and dropped below the level of detection (2 log cfu/g) after 4.5 mo of ripening. Neither DJO10A nor Bb-12 fortified cheeses produced detectable amounts of organic acids during ripening other than lactic acid, indicating the lack of detectable metabolic contribution from bifidobacteria during cheese production and ripening such as production of acetic acid. To determine if sublethal stresses could improve the viability of DJO10A, 2 more vats were made, 1 with DJO10A exposed to sublethal acid, cold, and centrifugation stresses, and 1 exposed to none of these stresses. Although stress-primed DJO10A survived cheese manufacture better, as 72.8% were incorporated into the cheese compared with 41.1% of the unprimed, the statistical significance of this difference is unknown. In addition, the difference in moisture levels in the cheese cannot be excluded as influencing this difference. However, the rate of decline during ripening was similar for both. After 6 mo of ripening, cell counts in cheese were 4.68 and 4.24 log cfu/g for primed and unprimed cultures, respectively. These results suggest that whereas priming bifidobacteria with sublethal stresses before incorporation in a cheese fermentation may improve the number of viable cells that get incorporated into the cheese, it does not affect viability during cheese ripening.

A survey of undergraduate education in dental implantology in UK dental schools.

The aim of this study was to ascertain knowledge on current teaching of implant dentistry in the undergraduate curriculum of Dental Schools in the UK. Information on the teaching modalities, including year of introduction of implant dentistry into undergraduate curriculum, departments involved in teaching, format of teaching, use of adjunctive teaching aids, and types of implant systems used in undergraduate teaching was collected by means of a questionnaire, which was sent to all undergraduate dental schools in the UK. Based on a 100% response rate, the findings indicate that all dental schools in the UK reported that they included dental implantology in their undergraduate curriculum; however there were marked variations in the content and delivery of the teaching.

Comparative genomic analysis of the gut bacterium Bifidobacterium longum reveals loci susceptible to deletion during pure culture growth.

BACKGROUND: Bifidobacteria are frequently proposed to be associated with good intestinal health primarily because of their overriding dominance in the feces of breast fed infants. However, clinical feeding studies with exogenous bifidobacteria show they don't remain in the intestine, suggesting they may lose competitive fitness when grown outside the gut. RESULTS: To further the understanding of genetic attenuation that may be occurring in bifidobacteria cultures, we obtained the complete genome sequence of an intestinal isolate, Bifidobacterium longum DJO10A that was minimally cultured in the laboratory, and compared it to that of a culture collection strain, B. longum NCC2705. This comparison revealed colinear genomes that exhibited high sequence identity, except for the presence of 17 unique DNA regions in strain DJO10A and six in strain NCC2705. While the majority of these unique regions encoded proteins of diverse function, eight from the DJO10A genome and one from NCC2705, encoded gene clusters predicted to be involved in diverse traits pertinent to the human intestinal environment, specifically oligosaccharide and polyol utilization, arsenic resistance and lantibiotic production. Seven of these unique regions were suggested by a base deviation index analysis to have been precisely deleted from strain NCC2705 and this is substantiated by a DNA remnant from within one of the regions still remaining in the genome of NCC2705 at the same locus. This targeted loss of genomic regions was experimentally validated when growth of the intestinal B. longum in the laboratory for 1,000 generations resulted in two large deletions, one in a lantibiotic encoding region, analogous to a predicted deletion event for NCC2705. A simulated fecal growth study showed a significant reduced competitive ability of this deletion strain against Clostridium difficile and E. coli. The deleted region was between two IS30 elements which were experimentally demonstrated to be hyperactive within the genome. The other deleted region bordered a novel class of mobile elements, termed mobile integrase cassettes (MIC) substantiating the likely role of these elements in genome deletion events. CONCLUSION: Deletion of genomic regions, often facilitated by mobile elements, allows bifidobacteria to adapt to fermentation environments in a very rapid manner (2 genome deletions per 1,000 generations) and the concomitant loss of possible competitive abilities in the gut.

The effect of a range of disinfectants on the dimensional accuracy and stability of some impression materials.

Disinfection of dental impressions should be considered as a routine procedure in dental surgeries and dental laboratories. Disinfectants can have deleterious effects on some properties of impression materials. The aim of this study was to evaluate the dimensional accuracy and dimensional stability of a model dental stone, reproduced from five commonly used impression materials (Aquasil soft putty/Aquasil Ultra LV; Aquasil Monophase; Aquasil Ultra Heavy; Impregum F and Provil putty/Provil Light CD wash) retained by their adhesives in acrylic resin trays and exposed to three disinfectant solutions (Perform ID; Haz-Tabs and MD 520). Two hundred models were used to investigate the effect of the three disinfectants on the dimensional accuracy of the five impression materials. Five impressions were taken for each impression material for each disinfection treatment group. Measurements were carried out using a High Precision Reflex Microscope. All materials demonstrated a percentage change in dimensions when subjected to no disinfection when compared to the brass master die and all materials demonstrated a percentage change in dimension when subjected to the different disinfection procedures. The results of this study have demonstrated that for all of the materials investigated, the changes in dimensional stability were small in the order of microns. These changes may however be of clinical significance for procedures requiring a high degree of accuracy, for example fixed prosthodontics. The materials respond differently depending on the disinfectant used and it may therefore be appropriate that manufacturers recommend the use of particular disinfectants for their products in order to ensure optimum dimensional accuracy and stability.

An in vitro investigation of the effect of some analgesics on human enamel.

The sale of over-the-counter pain relief medication has increased dramatically in recent years, and typically amounts to several hundred thousands of pounds per year in the UK. Many soluble analgesic preparations contain citric acid, and it has been suggested that these formulations may cause dental erosion. The aim of this study was to investigate the effect of some over-the-counter analgesics on tooth surface loss from human enamel. Six commonly available analgesics were chosen for this study and the effect of immersing unerupted human enamel was examined using non-contact optical profilometry. Two of the six analgesics investigated caused no detectable erosion (Boots soluble aspirin and Anadin Extra). Three caused statistically significant enamel erosion, but this was very slight and is thought to be clinically insignificant (Alka Seltzer, Panadol and Solpadeine). Only one analgesic caused possible potentially clinical significant enamel erosion. Further studies are needed to determine whether Aspro causes clinically significant enamel erosion.

An in vitro investigation of the effect of the addition of untreated and surface treated silica on the transverse and impact strength of poly(methyl methacrylate) acrylic resin.

Silica is a commonly used filler in dental materials and as a reinforcing agent in industry. The aim of this study was to further investigate the effect of the addition of untreated and a novel surface treated silica on the transverse bend and impact strength of acrylic resin denture base material. It was hypothesized that the silica/resin composite materials would have an improved flexural and impact strength than the conventional heat-cured acrylic resin. Three types of untreated and two of treated silica powder were used in this study. The range of percentages used were 1%, 0.5%, 0.2%, 0.1%. The treated particles were coated with hexamethyldisilazane or dimethyldichloridesilazane. Conventional heat cured acrylic resin was used as a control. The modulus of rupture for all groups of acrylic resin containing silica was significantly lower than for the control. The modulus of elasticity was not significantly greater than the control group. For the impact strength statistical analysis revealed a significant difference between the groups. There was a nonsignificant increase in the impact strength for specimens compared to the control. In conclusion the addition of silica to poly(methyl methacrylate) denture base materials did not produce a significant improvement in the transverse bend or impact strength compared to conventional heat-cured acrylic resin. The incorporation of untreated and surface treated silica cannot be recommended as a method of reinforcement.

Lesion of thalamic centromedian--parafascicular complex after chronic deep brain stimulation.

A patient with PD who exhibited disabling tremor and prominent dyskinesia underwent deep brain stimulation (DBS) of the left thalamic ventral intermediate nucleus. The electrode migrated and was replaced but with suboptimal clinical response. Two years later, postmortem analysis found the second electrode tip had entered the thalamic centromedian-parafascicular complex. There was a small thalamotomy and cell loss exceeding that found in PD. Thalamic damage may occur in association with DBS for PD.

Screening of intestinal microflora for effective probiotic bacteria.

Increasing consumer awareness of health-promoting intestinal bacteria has fueled the addition of viable probiotic bacteria as functional ingredients in certain foods. However, to effectively market the enhanced attributes of these foods, the added probiotic bacteria need to have scientific credibility. The scientific rationale for using many of the strains of probiotic bacteria currently on the market is weak. Furthering the current understanding of what features a bacterium needs to have for effective probiotic functionality will enable the selection of strains with a more credible scientific rationale. To screen for effective strains, one must understand the microbial diversity in the intestines of healthy individuals. The advent of molecular tools has greatly enhanced our ability to accomplish this. These tools comprise genetic fingerprinting, specific probes, molecular speciation, and techniques for the in situ analysis of specific microbial groups in the intestine. This review will detail these scientific approaches and how their impact will improve criteria for selection of probiotic bacteria.

Isolation, characterization, and influence of native, nonstarter lactic acid bacteria on Cheddar cheese quality.

To determine whether adventitious nonstarter lactic acid bacteria (NSLAB) might affect cheese flavor and quality, we studied a population of NSLAB present in 30 premium quality Cheddar cheeses (3-mo ripened) produced at a commercial facility in the United States. DNA fingerprinting analysis with a sensitive strategy for arbitrary priming polymerase chain reaction showed that 75 isolates corresponded to at least 18 distinct nonstarter organisms. According to ribotype database comparisons of representatives from the 18 groups, 9 matched Lactobacillus (closest to paracasei species), 8 matched Streptococcus thermophilus, and 1 matched to a Lactococcus species. This finding indicated that among the 75 NSLAB isolates, Lactobacillus made up 64%, S. thermophilus 32%, and Lactococcus 4%. Isolates representing 11 NSLAB groups were characterized for protease, peptidase, and diacetyl production. Based on this phenotypic analysis, two Lactobacillus isolates were evaluated as adjuncts in Cheddar cheese. All of the NSLAB identified from the adjunct cheese at 3 mo by DNA fingerprinting consisted of the adjunct lactobacilli, showing that the adjunct strains predominated throughout the early stages of ripening. The impact of adjunct lactobacilli was evident after 6 mo when free amino acids significantly increased and sensory scores improved in adjunct cheese as compared with a control cheese. The largest impact was found in adjunct cheese containing a blend of both lactobacilli strains. These results show that certain adventitious NSLAB positively contribute to flavor development.

Identification of a gene cluster encoding Krebs cycle oxidative enzymes linked to the pyruvate carboxylase gene in Lactococcus lactis ssp. lactis C2.

We identified a 14-kb pyruvate carboxylase gene-containing fragment from a lactococcal C2-lambda phage genomic library. Downstream of the pyruvate carboxylase gene-containing fragment, a gene cluster coding for open reading frames displaying extensive homology to citrate synthase, aconitase, and a truncated isocitrate dehydrogenase was identified. However, the truncation was shown to have occurred during the cloning by two noncontiguous Sau3AI fragments ligating together. The lactococcal citrate synthase gene consisted of 1323 bp and encoded a 441-amino acid citrate synthase protein. The lactococcal aconitase gene was 2544 bp and encoded an 848-amino acid protein. Corresponding to the complete citrate synthase gene, citrate synthase activity was detected in Lactococcus lactis ssp. lactis C2. Isocitrate dehydrogenase activity was found to be missing in Lactococcus lactis C2, suggesting that the gene may be incomplete or is not expressed, resulting in a requirement for glutamic acid in lactococci.

Classification of a bacterial isolate, from pozol, exhibiting antimicrobial activity against several gram-positive and gram-negative bacteria, yeasts, and molds.

A bacterial isolate, designated CS93, capable of producing a broad-spectrum antimicrobial compound(s) effective against gram-positive and gram-negative bacteria, yeasts, and molds was isolated from pozol, a fermented maize product. This strain was phenotypically similar to another pozol isolate that was previously designated as Agrobacterium azotophilium by other investigators. By using biochemical, phenotypic, and 16S rRNA sequence analysis, both pozol isolates were identified as members of the genus Bacillus, possibly a variant of Bacillus subtilis. While the antimicrobial compound(s) was initially produced only on a solid medium, parameters were identified for production in broth. The compound(s) was heat stable (121 degrees C for 15 min), exhibited activity over a wide pH range (pH 3 to pH 11), and was inactivated by pronase E. The antimicrobial compound(s) was bactericidal and bacteriolytic against Escherichia coli V517, bacteriostatic against Micrococcus luteus, and fungistatic against Saccharomyces cerevisiae. The inhibitory compound(s) could possibly serve as a food biopreservative.

Characterization of AbiR, a novel multicomponent abortive infection mechanism encoded by plasmid pKR223 of Lactococcus lactis subsp. lactis KR2.

The native lactococcal plasmid pKR223 encodes two distinct phage resistance mechanisms, a restriction and modification (R/M) system designated LlaKR2I and an abortive infection mechanism (Abi) which affects prolate-headed-phage proliferation. The nucleotide sequence of a 16,174-bp segment of pKR223 encompassing both the R/M and Abi determinants has been determined, and sequence analysis has validated the novelty of the Abi system, which has now been designated AbiR. Analysis of deletion and insertion clones demonstrated that AbiR was encoded by two genetic loci, separated by the LlaKR2I R/M genes. Mechanistic studies on the AbiR phenotype indicated that it was heat sensitive and that it impeded phage DNA replication. These data indicated that AbiR is a novel multicomponent, heat-sensitive, "early"-functioning Abi system and is the first lactococcal Abi system described which is encoded by two separated genetic loci.

Use of chemical mutagenesis for the isolation of food grade beta-galactosidase overproducing mutants of bifidobacteria, lactobacilli and Streptococcus thermophilus.

A classical chemical mutagenesis protocol was evaluated for increasing beta-galactosidase production by probiotic bacteria to improve their potential to treat symptoms of lactose malabsorption in humans. Two Bifidobacterium species (B. breve and B. longum) and one strain each of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus were tested by a single exposure to two chemical mutagens, ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). To screen for beta-galactosidase (beta-gal) overproducing mutants, optimized EMS and MNNG mutant pots for each strain were plated on BHI agar containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal). Colonies that exhibited a blue color were selected for quantitative beta-gal activities using the o-nitrophenyl-beta-galactoside (ONPG) assay. Seventy-five mutants were obtained out of more than 2 million colonies screened and showed increased beta-galactosidase activities compared with the wild-type strains. EMS gave a higher frequency of beta-gal overproducing mutants than MNNG for three of the four strains, S. thermophilus, B. breve, and B. longum, whereas the frequency of L. delbrueckii ssp. bulgaricus beta-gal mutants was similar with both mutagens. The highest beta-gal increases, when induced during growth in lactose, for mutants of each culture were 137% for L. delbrueckii ssp. bulgaricus; 104% for S. thermophilus; 70% for B. breve; and 222% for B. longum mutants. This food-grade classical approach has the ability to moderately increase beta-gal concentrations in probiotic cultures to improve their potential for treating the symptoms of lactose malabsorption in humans.

Use of a single, triplicate arbitrarily primed-PCR procedure for molecular fingerprinting of lactic acid bacteria.

Arbitrarily primed (AP)-PCR can be used to generate characteristic DNA fingerprint patterns. However, small changes in reaction conditions can cause band irreproducibility. In this study, a single methodology encompassing triplicate reactions, which were intentionally exposed to three different annealing temperatures, enabled bands that were reproducibly generated to be recognized. A single triplicate AP-PCR (TAP-PCR) procedure, using an 18-mer primer, was developed and used to fingerprint representative isolates from the major genera of lactic acid bacteria and Bifidobacterium to the strain level.

Cloning, sequencing, and expression of the pyruvate carboxylase gene in Lactococcus lactis subsp. lactis C2.

A functional pyc gene was isolated from Lactococcus lactis subsp. lactis C2 and was found to complement a Pyc defect in L. lactis KB4. The deduced lactococcal Pyc protein was highly homologous to Pyc sequences of other bacteria. The pyc gene was also detected in Lactococcus lactis subsp. cremoris and L. lactis subsp. lactis bv. diacetylactis strains.

Methods for analysis of the intestinal microflora.

The concept of probiotics has been around for about 100 years. Yet its impact on human nutrition is still an emerging concept. Lack of convincing scientific validation for the efficacy of any ingested probiotic bacterium on intestinal health, has been a major reason for the low impact of probiotics on human nutrition. Obtaining positive scientific validation requires the use of suitable probiotic strains and also the necessary tools to monitor the performance of these bacteria in the intestines of individuals. To date, selection of strains for probiotic purposes has not been based on a scientific directed approach, primarily because it is not yet fully known what specific traits a desirable probiotic strain should possess. Filling this knowledge void will depend largely on furthering our understanding of the human intestinal ecosystem and the functional role of specific bacteria for intestinal health. Traditional approaches for studying this ecosystem have provided a good foundation in this knowledge base. Complementation of the traditional approaches with the emergence of sophisticated molecular tools shows enormous promise for obtaining the necessary insight into the intestinal microflora. This review will cover the traditional methodologies which have been used to analyze the human intestinal microflora. It will also reveal the development of modern molecular approaches for studying the diversity and phylogeny of its flora, and the rapid molecular tools for monitoring the presence of specific strains in the intestine. Finally, it will address the advent of in situ analysis of individual microbial cells, which promises to provide tremendous advances in our understanding of the microflora and their metabolic activities in the human intestine.