Robert F. Roberts

Comparison of the Complete Genome Sequences of Bifidobacterium animalis subsp. lactis DSM 10140 and Bl-04

Bifidobacteria are important members of the human gut flora, especially in infants. Comparative genomic analysis of two Bifidobacterium animalis subsp. lactis strains revealed evolution by internal deletion of consecutive spacer-repeat units within a novel clustered regularly interspaced short palindromic repeat locus, which represented the largest differential content between the two genomes. Additionally, 47 single nucleotide polymorphisms were identified, consisting primarily of nonsynonymous mutations, indicating positive selection and/or recent divergence. A particular nonsynonymous mutation in a putative glucose transporter was linked to a negative phenotypic effect on the ability of the variant to catabolize glucose, consistent with a modification in the predicted protein transmembrane topology. Comparative genome sequence analysis of three Bifidobacterium species provided a core genome set of 1,117 orthologs complemented by a pan-genome of 2,445 genes. The genome sequences of the intestinal bacterium B. animalis subsp. lactis provide insights into rapid genome evolution and the genetic basis for adaptation to the human gut environment, notably with regard to catabolism of dietary carbohydrates, resistance to bile and acid, and interaction with the intestinal epithelium. The high degree of genome conservation observed between the two strains in terms of size, organization, and sequence is indicative of a genomically monomorphic subspecies and explains the inability to differentiate the strains by standard techniques such as pulsed-field gel electrophoresis.

Development of a growth medium suitable for exopolysaccharide production by Lactobacillus delbrueckii ssp. bulgaricus RR

Complex media are commonly used in studies examining exopolysaccharide production by Lactobacillus delbrueckii ssp. bulgaricus. However, quantification of exopolysaccharide in complex medium can be complicated by interference due to carbohydrate polymers contained in media components. This study was undertaken to identify components of MRS, a common medium for cultivation of L. delbrueckii ssp. bulgaricus, that interfere with exopolysaccharide quantification, to develop a medium for production of exopolysaccharide that provides for growth of L. delbrueckii ssp. bulgaricus strain RR similar to MRS, and to demonstrate exopolysaccharide production by L. delbrueckii ssp. bulgaricus RR grown in the newly developed medium. Phenol-sulfuric acid determinations were conducted on uninoculated MRS broth with and without yeast extract, beef extract and proteose peptone #3. These three ingredients accounted for 94% of the total background exopolysaccharide-equivalent in MRS broth. Based on these results, a semi-defined medium (SDM) providing minimal interference was developed using yeast nitrogen base and Bacto casitone. Growth of L. delbrueckii ssp. bulgaricus RR at 42 degrees C in semi-defined medium and MRS was evaluated, and generation times did not differ significantly (0.94 h in MRS and 0.85 h in SDM). Exopolysaccharide production by L. delbrueckii ssp. bulgaricus RR during growth in semi-defined medium was evaluated at 30 and 40 degrees C. The rate of exopolysaccharide production was lower at 30 degrees C (8.04 (mg/l-h) than at 40 degrees C (11.95 (mg/l-h), but the maximal concentration of exopolysaccharide produced was similar at both temperatures.

Estrone and 17β-estradiol concentrations in pasteurized-homogenized milk and commercial dairy products.

Some individuals fear that estrogens in dairy products may stimulate growth of estrogen-sensitive cancers in humans. The presence of estrone (E(1)) and 17beta-estradiol (E(2)) in raw whole cows milk has been demonstrated. The objectives of this study were to determine if pasteurization-homogenization affects E(2) concentration in milk and to quantify E(1) and E(2) concentrations in commercially available dairy products. The effects of pasteurization-homogenization were tested by collecting fresh raw milk, followed by pasteurization and homogenization at 1 of 2 homogenization pressures. All treated milks were tested for milk fat globule size, percentages of milk fat and solids, and E(2) concentrations. Estrone and E(2) were quantified from organic or conventional skim, 1%, 2%, and whole milks, as well as half-and-half, cream, and butter samples. Estrone and E(2) were quantified by RIA after organic solvent extractions and chromatography. Pasteurization-homogenization reduced fat globule size, but did not significantly affect E(2), milk fat, or milk solids concentrations. Estrone concentrations averaged 2.9, 4.2, 5.7, 7.9, 20.4, 54.1 pg/mL, and 118.9 pg/g in skim, 1%, 2%, and whole milks, half-and-half, cream, and butter samples, respectively. 17Beta-estradiol concentrations averaged 0.4, 0.6, 0.9, 1.1, 1.9, 6.0 pg/mL, and 15.8 pg/g in skim, 1%, 2%, whole milks, half-and-half, cream, and butter samples, respectively. The amount of fat in milk significantly affected E(1) and E(2) concentrations in milk. Organic and conventional dairy products did not have substantially different concentrations of E(1) and E(2). Compared with information cited in the literature, concentrations of E(1) and E(2) in bovine milk are small relative to endogenous production rates of E(1) and E(2) in humans.

Utilization of cheddar cheese containing nisin as an antimicrobial agent in other foods

Cheddar cheese made with nisin-producing lactococci contained between 400 and 1200 IU of nisin per gram of cheese. Cultures used were Lactococcus lactis ssp. cremoris JS102, a nisin-producing transconjugant developed in the laboratories of Dr. L.L. McKay and Lactococcus lactis ssp. lactis NCDO 1404 obtained from the National Collection of Food Bacteria, Reading, England. Pasteurized process cheese spreads with 53% and 60% moisture and 0, 301 and 387 IU nisin/g were manufactured and inoculated with 2000 spores of Clostridium sporogenes PA 3679 during manufacture. The heat process did not reduce nisin activity in the cheese spreads. The spreads were incubated at 22 degrees and 37 degrees C for 90 days. Spoilage was detected by the presence of gas and/or odor in the packages. The shelf-life of the nisin-containing cheese spreads was significantly greater than that of the control cheese spreads at the lower temperature at both moisture levels, whereas the keeping quality of the higher moisture cheeses at the higher temperature was not significantly different. Club cheese or cold pack cheese spreads with moisture levels of 44% and 60% and 0, 100 and 300 IU nisin/g were made. These cold processed cheese spreads were inoculated with 1000 cfu per g of Listeria monocytogenes V7, Staphylococcus aureus 196E and spores of C. sporogenes PA 3679. Heat shocked spores of PA 3769 at the same number were added to separate lots of the cheese spread. The cold pack cheese spreads were incubated at 23 degrees and 37 degrees C for up to 8 weeks. Samples were taken weekly and analyzed for surviving organisms. Significant reductions in numbers of the non-sporeforming test microbes were noted at both temperatures, at both moisture levels and both levels of nisin. Heat shocking the spores was needed to show reduction in numbers during the storage of the cold pack cheese spreads. The data obtained in this study suggest that the use of nisin-containing cheese as an ingredient in pasteurized process cheese or cold pack cheese spreads could be an effective method of controlling the growth of undesirable microorganisms in these processed foods.

In vitro assessment of the cytotoxicity of nisin, pediocin, and selected colicins on simian virus 40-transfected human colon and Vero monkey kidney cells with trypan blue staining viability assays.

Gram-positive bacterial bacteriocins (nisin and pediocin) and gram-negative bacterial bacteriocins (colicins [Col] E1, E3, E6, E7, and K) were evaluated for cytotoxicity against cultured simian virus 40-transfected human colon (SV40-HC) and Vero monkey kidney (Vero) cells. Bacteriocin-treated cells were assessed for viability by trypan blue staining. Monolayers of SV40-HC and Vero cells were cultured in tissue culture plates (35 degrees C, 10% CO2 in humidified air) with the use of Dulbeccos modified Eagles medium supplemented with 10% (vol/vol) calf serum. Actively growing cells in the log phase (ca. 10(4) cells per ml) were treated with individual partially purified bacteriocin preparations at 170, 350, and 700 activity units per ml. Duplicate culture plates for each bacteriocin treatment and untreated controls were withdrawn after 16, 32, and 48 h of incubation. Cells were dissociated with trypsin and treated with trypan blue and were then counted in a hemocytometer with the use of a phase-contrast microscope. Viability assays indicated dose-dependent toxicity for some bacteriocins. Nisin, pediocin, and Col E6 were the most cytotoxic bacteriocins; SV40-HC cells demonstrated greater sensitivity than Vero cells did. Some bacteriocins can be toxic to mammalian cells; therefore, bacteriocins intended for use as biopreservatives must be evaluated for toxicity to mammalian cells and for other toxicities. Col E1, Col E3, Col E7, and Col K demonstrated little toxicity at the activities tested, indicating that they are safe and thus have potential for use as food biopreservatives.

Strain-Specific Genotyping of Bifidobacterium animalis subsp. lactis by Using Single-Nucleotide Polymorphisms, Insertions, and Deletions

ABSTRACT Several probiotic strains of Bifidobacterium animalis subsp. lactis are widely supplemented into food products and dietary supplements due to their documented health benefits and ability to survive within the mammalian gastrointestinal tract and acidified dairy products. The strain specificity of these characteristics demands techniques with high discriminatory power to differentiate among strains. However, to date, molecular approaches, such as pulsed-field gel electrophoresis and randomly amplified polymorphic DNA-PCR, have been ineffective at achieving strain separation due to the monomorphic nature of this subspecies. Previously, sequencing and comparison of two B. animalis subsp. lactis genomes (DSMZ 10140 and Bl-04) confirmed this high level of sequence similarity, identifying only 47 single-nucleotide polymorphisms (SNPs) and four insertions and/or deletions (INDELs) between them. In this study, we hypothesized that a sequence-based typing method targeting these loci would permit greater discrimination between strains than previously attempted methods. Sequencing 50 of these loci in 24 strains of B. animalis subsp. lactis revealed that a combination of nine SNPs/INDELs could be used to differentiate strains into 14 distinct genotypic groups. In addition, the presence of a nonsynonymous SNP within the gene encoding a putative glucose uptake protein was found to correlate with the ability of certain strains to transport glucose and to grow rapidly in a medium containing glucose as the sole carbon source. The method reported here can be used in clinical, regulatory, and commercial applications requiring identification of B. animalis subsp. lactis at the strain level.

CIP Cleaning of a Pipeline Milking System Using Electrolyzed Oxidizing Water

Electrolyzed oxidizing (EO) water is a technology that electrolyzes a weak sodium chloridesolution into sodium and chlorine, resulting in two solutions alkaline and acid. The goal of thisresearch is to determine if EO water is an acceptable cleaning agent for pipeline milking systems.After constructing 1.5 inch-diameter pipeline milking system, the system was soiled using raw milkthat had been spiked with common raw milk microorganisms. After soiling, the system was rinsedwith warm water and then evaluated in several locations for initial counts. To evaluate the level ofsoiling, the surfaces were evaluated using an ATP bioluminescence method. The milk contactsurfaces were also swabbed for microbial analysis. The pipeline system was then washed with analkaline EO water treatment followed by an acidic EO water treatment. After treatment, theeffectiveness of the treatment was evaluated by ATP bioluminescence and microbiological analysis.First, a 10 min wash with 60C alkaline water followed by a 10 min wash with 60C acid watersuccessfully removed all detectable bacteria and ATP from the non-porous milk contact surfaces.Shorter treatment times (5 and 7.5 min) with EO water were also tested, along with a controltreatment using conventional dairy cleaning chemicals. Using ANOVA, there were no significantdifferences between the EO water treatments and the conventional treatment, however the 5-min EOwater treatment was significantly less effective than the 10-min treatment.

Study to investigate the potential of probiotics in children attending school

Background/Objectives:To determine if consumption of yogurt containing a high dose of probiotic (1 × 1010 colony-forming unit per 100 ml), Bifidobacterium animalis subsp. lactis (B. lactis), decreases absences in children 2–4 years attending daycare/school centers.Subjects/Methods:We conducted a double-blinded, randomized, placebo-controlled, allocation concealment clinical trial in the Washington, DC area. Our active intervention was a strawberry yogurt-based drink supplemented with B. lactis BB-12. The placebo was indistinguishable from the active drink, differing only in absence of the probiotic BB-12.Results:A total of 172 children between the ages of 2 and 4 from the Washington, DC area were enrolled. The primary outcome, missed days of school because of illness per 100 days, was similar in both the active (2.54 days absent/100 school days) and control groups (2.42 days absent/100 school days) (P=0.873).Conclusions:The probiotic-containing yogurt-based beverage studied did not decrease absences because of illnesses in daycare/school for healthy children ages 2–4 years.

The study to investigate the potential benefits of probiotics in yogurt, a patient-oriented, double-blind, cluster-randomised, placebo-controlled, clinical trial

Background:Probiotic functional foods are widely advertised to consumers primarily based on probiotic supplements.Objective:Determine if consumption of yogurt containing a high dose of probiotics improves health in children ages 1–3 years attending daycare/school centers.Subjects/Methods:Double-blinded, randomized, placebo-controlled, allocation concealment clinical trial. Setting: Outpatient participants in the Washington, DC area. Participants: 182 healthy children between the age of 1 and 3 years attending daycare/school at least 3 days a week. Intervention: Active was a strawberry yogurt-based drink supplemented with Bifidobacterium animalis ssp. lactis (B. lactis) BB-12. The placebo was indistinguishable from the active drink, differing only in absence of the probiotic BB-12. Primary objective was to determine if consumption of a probiotic-containing yogurt-based drink decreases absences due to illnesses from daycare for children ages 1–3 years. Secondary was to determine if probiotic-containing yogurt-based drink improves overall parental satisfaction due to decreased absences from work and an overall healthier child.Results:There were no significant differences in the days of missed school per group, with 51.9% in the active group and 47.1% in the placebo group missing at least 1 day of school throughout the study. Additionally, there were no differences in any secondary outcomes among the groups.Conclusions:Consumption of a yogurt-based drink delivering 1010 CFU of Bifidobacterium animalis ssp. lactis (B. lactis) BB-12 per day did not decrease the number of days missed of school due to an illness. Additional independent research on the potential of BB-12 to reduce illness in children needs to be conducted.

CLEANING MILKING SYSTEMS USING ELECTROLYZED OXIDIZING WATER

Electrolyzed oxidizing (EO) water is a novel cleaning and disinfecting agent, produced by separating a weak sodium chloride solution into alkaline and acidic components. A pilot-scale pipeline milking system was soiled using raw milk inoculated with common microorganisms. The milking system was then washed with alkaline EO water followed by acidic EO water. After cleaning, the effectiveness of the EO water treatment was evaluated by ATP bioluminescence and microbiological analysis. A 10 min wash with 60°C alkaline EO water followed by a 10 min wash with 60°C acid EO water successfully removed all detectable bacteria and ATP from the non-porous milk contact surfaces. Shorter treatment times (5 and 7.5 min) with EO water were also evaluated, along with a control treatment using conventional dairy cleaning chemicals. There were no significant differences between the 10 min and 7.5 min EO water treatments and the conventional treatment. In a longer-term soiling-washing simulation, only the 7.5 min EO water treatment was evaluated after ten soiling and cleaning cycles, and it was