Progress 07/01/03 to 06/30/08
Outputs
OUTPUTS: Experiments for this project were conducted by 2 MS and 2 PhD students. At the inception of this project 30 commercial and 9 ATCC Type strains of bifidobacteria were characterized. Biochemical evaluations (lactic to acetic acid ratio and assaying for the presence of fructose-6-phosphate phosphoketolase ) and 16s rDNA PCR revealed that of the 30 commercial strains evaluated, only 20 were bifidobacteria. The 20 strains confirmed as bifidobacteria were further characterized and most were identified as B. animalis ssp. lactis (Bal), in over 50% of the cases, not matching the species designation provided by the supplier. PFGE and RAPD PCR and sequencing of portions of 6-different genes were employed in an unsuccessful attempt to differentiate among commercial and reference strains of Bal. A rapid method for PFGE of bifidobacteria was developed which reduced the time required for PFGE analysis from 3-4 days to less than 24 hours. PFGE was applied to 10 bifidobacterial isolates obtained from the Ukrainian Institute of Meat and Milk and all strains (regardless of the stated identity) were Bal and had PFGE patterns indistinguishable from DSMZ10140. Further strain differentiation work was hampered by lack of a Bal genome, and a project to sequence DSMZ 10140 was begun using "454" rapid sequencing technology. The complete circular genome of DSMZ 10140 was finished in May of 2008 and compared with another strain (Bl-04). Comparison of the 2 genomes further proved the high degree of relatedness of Bal strains. Extensive biochemical analysis of glucose transport in DSMZ 10140 revealed glucose is transported via facilitated diffusion-the first report of facilitated diffusion of a carbohydrate in this genus and one of a limited number of reports of transport via facilitated diffusion in bacteria. Analysis of Bal strains unable to grow on glucose revealed that upon extended incubation mutants able to grow on glucose would arise. Genome analysis has identified a SNP in a putative glucose transport gene that differs in "fast" and "slow" strains. The influence of cold adaptation on cryololerance on two strains of B. infantis was evaluated. Increasing the number of freeze-thaw (F-T) cycles resulted in a significant decline in the number of survivors for both strains and the magnitude of the effect was strain dependent. Results suggested pH of the suspending medium is the most important factor in determining survival during F-T cycles. The conclusion from this work was stability to F-T challenge may be enhanced by preliminary incubation at sub-optimal temperature. Spherulitic starch produced from potato amylose was used in adhesion assays with a number of species of bifidobacteria. Strains that adhered to granular high amylose maize starch also adhered to potato amylose spherulites (PAS). Adhesion to PAS did not seem to enhance survival of the strains in the presence of bile or acid. Portions of this work were presented at the annual meetings of ADSA and IFT as well as to the IDFA's annual Cultured Dairy Products Conference and as part of the annual PSU Cultured Dairy Products Short Course. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Work during this project has resulted in a clearer understanding of the need for molecular-based techniques for identification and differentiation of probiotic bacteria. It is well known that the health effects of probiotic microorganisms are "strain specific". As the market for probiotic products continues to grow, the ability to quickly and accurately determine the number of specific bacteria in a probiotic product as well as the specific identity of each organism will be critical. Also critical will be development of an understanding of the mechanisms by which probiotic bacteria exert their health promoting effect. Since the human large intestine contains a large and diverse population of bacteria, it is important to understand how probiotic bacteria, such as bifidobacteria, can compete. Our work with glucose transport in bifidobacteria has shown that transport is via facilitated diffusion. Perhaps the most significant development over the course of this project has been completing to closure the genome of B. animalis ssp. lactis. The sequence data developed is currently being used to design a SNP-based strategy for identifying and differentiating strains of B. animalis ssp. lactis genes used in clinical and food-based systems. In addition, the genome sequence has been used to develop a DNA-microarray which will be used to study the genome-wide impact of various environmental stressors allowing development of a mechanistic understanding of survival and leading to design of new strategies to enhance survival of probiotic bacteria in food products. In addition this genome sequence will allow identification of molecular markers for rapid and accurate assessment of the identity of the probiotic strains in commercial food products for regulatory and quality assurance purposes. Development of strategies to enhance the survival of probiotic bifidobacteria throughout the shelf life of foods may lead to development of new vehicles for probiotic delivery or allow the use of less resilient strains with better health promoting properties in food.
Publications
- Briczinski, E. P., A. T. Phillips, and R. F. Roberts. 2008. Transport of glucose by Bifidobacterium animalis subsp. lactis occurs via facilitated diffusion. Applied and Environmental Microbiology. 74(22) 6941-6948
- Gevorgyan, A., L. Tmanova, and R. F. Roberts. 2008 . Effect of cold adaptation on cryotolerance of two strains of bifidobacteria. Journal of Agrobiology and Ecology.
- Briczinski, E. P. and R. F. Roberts. 2007. Characterizing Probiotic Microorganism. In: Sarela M, editor. Functional Dairy Products Volume II: Woodhead Publishing Limited. Abington Hall, Abington, Cambridge, UK. 560 pgs.
- Briczinski, E. P. and R. F. Roberts 2006. "Technical note: a rapid pulsed-field gel electrophoresis method for analysis of bifidobacteria." Journal of Dairy Sci. 89(7): 2424-7.
- Gevorgyan, A. 2003. Effects of cold adaptation on cryotolerance of Bifidobacterium infantis. M. S. Thesis. The Pennsylvania State University, University Park, PA. 107 pp.
- Briczinski, E. P. 2007. Characterization of strain of Bifodobacterium animals ssp. lactis from commercial starter culture manufacturers-A study of glucose transport. Ph.D. Dissertation. The Pennsylvania State University, University Park, PA. 274 pp.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Experiments were conducted over this period in number of areas related to the project objectives. All of the experiments conducted were done by graduate students working towards MS or Ph.D. degrees. Ten isolates of Bifidobacterium spp were obtained from the Ukrainian Institute of Milk and Meat. Using PCR-based analysis isolates were confirmed as members of the genus Bifidobacterium. However, all isolates were determined to be strains of B. animalis ssp. lactis. Further analysis showed all the isolates had identical Pulsed Field Gel Electrophoresis (PFGE) patterns. Extensive biochemical analysis of glucose transport in B. animalis ssp. lactis DSMZ 10140 revealed transport via facilitated diffusion. Based on analysis of the preliminary genome sequence and biochemical assay, DSMZ 10140 does not have a functional glucose PEP-PTS system. Analysis of strains of B. animalis ssp. lactis unable to grow on glucose as the sole carbon source revealed that upon extended incubation
mutants able to grow on glucose would arise. Transport, via facilitated diffusion as measured using radiolabelled glucose was increased in the "glucose fast" derivatives. Work is underway to identify the genetic basis for this mutation. A new medium "YES" was evaluated for growth of B. animalis ssp. lactis. This medium uses soy oligosaccharides as the carbon source. YES supported better growth of strains of B. animalis ssp. lactis then MRS or Liver Lactose medium. Research is currently underway to evaluate the utility of YES medium for enumeration of bifidobacteria in yogurt in the presence of other lactic acid bacteria. Preliminary research has been conducted to design a qPCR (rtPCR) system for rapid and simultaneous estimation of the populations of bifidobacteria, lactobacillus and streptococcus in yogurt. At this writing, primers and probes have been developed and tested for cross reactivity with DNA isolated from the target organisms. Currently we are developing cell extraction
and lysis for the yogurt matrix. Spherulitic starch produced from potato amylose was used in adhesion assays with a number of species of bifidobacteria. Strains that adhered to granular high amylose maize starch also adhered to potato amylose spherulites (PAS). Adhesion to PAS did not seem to enhance survival of the strains in the presence of bile or acid. Strategies for encapsulation of bifidobacteria in the PAS are being developed. Using a combination of high throughput 454 sequencing and PCR based gap closing the complete genome of DSMZ 10140 has been sequenced and is currently assembled into 3 contigs. Preliminary annotation of the sequence has been conducted. The most striking feature of the genome is that a significant portion (perhaps up to 40% ) is related to carbohydrate metabolism. In addition to formal experimental work, I have presented a lecture on probiotic strain identification at the International Dairy Foods Association Cultured Dairy Products Conference and included
material developed in this research in the Penn State Cultured Dairy Products Short Course.
PARTICIPANTS: Over the past 12 months this project has supported 4 graduate students (1 Ph.D. and 3 MS) as well as provided opportunities for 3 undergraduate research associates. Additional PSU collaborators have included Dr. Greg Ziegler, Dr. Ed Dudley and Dr. Stephan Schuster. External collaborators have included Dr. Jim Steele-University of Wisconsin, Dr. Jeff Broadbent-Utah State University and Dan Merenstein, MD-Georgetown University Medical Center.
TARGET AUDIENCES: The target audience for this project is multi-fold and includes the consuming public in that we are developing methods to test the potency and identity of probiotic bacteria used in food, the dairy products processing industry in that we are establishing methods to enhance survival of rpobiotic bacteria during processing and the starter culture industry in that our techniques for strain differentiation and the sequence of the B. animalis ssp. lactis genome will add to knowledge that can be used to optimize production and survival of these important microbes.
Impacts
Work over this period has resulted in a clearer understanding of the need for molecular-based techniques for identification and differentiation of probiotic bacteria. It is well known that the health effects of probiotic microorganisms are "strain specific". As the market for probiotic products continues to grow, the ability to quickly and accurately determine the number of specific bacteria in a probiotic product as well as the specific identity of each organism will be critical. Also critical will be development of an understanding of the mechanisms by which probiotic bacteria exert their health promoting effect. Since the human large intestine contains a large and diverse population of bacteria, it is important to understand how probiotic bacteria, such as bifidobacteria, can compete. Our work with glucose transport in bifidobacteria has shown that transport is via facilitated diffusion. This information will be useful in understanding the ecology of bifidobacteria
in the intestine and in designing methods for isolation and enumeration of bifidobacteria from probiotic products. This is the first report of facilitated diffusion of a carbohydrate in this genus and one of a limited number of reports of transport via facilitated diffusion in bacteria. Research is currently underway to evaluate the utility of "YES" medium for enumeration of bifidobacteria in yogurt in the presence of other lactic acid bacteria. The most significant development over the last 12 months has been the near complete sequencing of the B. animalis ssp. lactis genome. The sequence data developed will be used to design microarrays for identifying and studying expression of B. animalis ssp. lactis genes required for survival of various environmental stressors allowing development of a mechanistic understanding of survival and leading to design of new strategies to enhance survival of probiotic bacteria in food products. In addition this genome sequence will allow identification
of molecular markers for rapid and accurate assessment of the identity of the probiotic strains in commercial food products for regulatory and quality assurance purposes.
Publications
- Roberts, R. F. and E. P. Briczinski. 2007. Characterizing Probiotic Microorganisms. Functional Dairy Products Volume II. Vol. II. M. Sarela, ed. Woodhead Publishing Limited, Abington Hall, Abington Cambridge, CB21 6AH, UK. pp 359-384.
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Progress 01/01/06 to 12/31/06
Outputs
A rapid method for Pulsed Field Gel Electrophoresis of bifidobacteria was developed. This method reduced the time required for PFGE analysis from 3-4 days to less than 24 hours. The developed PFGE technique was applied to reference strains of a variety of Bifidobacterium spp. obtained from culture collections as well as from commercial sources and performed as well as the "traditional" longer method. In addition, the technique was used to characterize strains of Bifidobacterium obtained from the Ukrainian Institute of Meat and Milk. As a prelude to development of a multilocus sequence typing method for B. animalis ssp. lactis, efforts were begun to sequence the genome of B. animalis ssp. lactis DSMZ 10140 using "454" rapid sequencing. At the present time we have achieved 20X coverage of the genome in a total of 51 contigs. Gap filling and annotation are proceeding simultaneously. Homology with the published B. longum genome is quite low. We have already sequenced a
putative glucose permease from 4 strains of B. animalis ssp. lactis. To further explore glucose metabolism in B. animalis ssp. lactis, transport assays were conducted using C14 labeled glucose. Rates of transport, via glucose permease, could not explain slow growth of some B. animalis strains when glucose was employed as a substrate. Efforts are underway to determine if B. animalis ssp. lactis has a glucose specific PTS system. Conditions have been developed for production of starch-based spherulites from amylose on a gram-basis. The spherulites formed have been evaluated using microscopy and laser light scattering. A method is under development to quantitatively evaluate adherence of bifidobacteria to the manufactured spherulites.
Impacts
Since probiotic effects of bifidobacteria are strain specific, techniques for rapid characterization to the strain level are important to regulatory agencies, food manufacturers and starter culture manufacturers. The information regarding repression of glucose metabolism when cells are grown on lactose is particularly important because media using glucose as a carbon source may not be suitable for isolation of bifidobacteria from cultured dairy products. If starch spherulites can enhance survival of bifidobacteria in food and in the gut, this will increase the number of tools available to assure probiotics are viable when consumed and can survive transit through the intestinal tract. Evaluation of strains from other countries may result in additional strains with novel traits for use in the US. Elucidation of the total genome sequence of B. animalis ssp. lactis will allow us and other researchers to probe more important and fundamental questions about the physiology
of these important probiotic organisms.
Publications
- Briczinski, E. P. and Roberts, R. F. 2006. Technical note: a rapid pulsed-field gel electrophoresis method for analysis of bifidobacteria. J Dairy Sci 89(7): 2424-7.
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Progress 01/01/05 to 12/31/05
Outputs
Twenty commercial strains of Bifidoabacterium lactis were analyzed by Pulsed Field Gel Electrophoresis (PFGE) and Randomly Amplified Polymorphic DNA PCR (RAPD). No differences were detected in the commercial strains using the molecular techniques. As part of the PFGE characterization work a rapid method was developed for this analysis that reduced the time, from cells to gel, from 4 days to less then 24 hours. This work has been submitted to the Journal of Dairy Science. A MLST (multi locus sequence typing) approach using house keeping genes and non-coding intergenic spacer regions is being explored for differentiation of the commercial strains. Additional characterization of the strains included carbohydrate utilization and enzyme profile analysis. The commericial strains exhibited only slight differences in carbohydrate utilization patterns with the most notable difference being in the metabolism of glucose. Further experiments revealed that lactose may serve as a
repressor of glucose metabolism and experiments are underway to determine the exact effect. A project was begun to determine if bifidobacteria can bind to starch spherulites and if binding to the spherulites produced from a variety of starches can enhance survival of bifidobacteria in yogurt. In addition, melts of starch ready to form spherulites are being explored as a means of encapsulating bifidobacteria. Since the spherulites serve as a prebiotic and can reach the colon this synbiotic approach may prove valuable. Seven strains of Bifidobacterium spp used in dairy products produced in Ukraine were obtained from the National Academy of Agricultures Institute of Milk and Meat and were characterized by PCR and speciated. As expected most of the strains were misidentified by the host institution and have been reclassified as Bifidobacterium lactis. Analysis of these strains using molecular techniques to determine the relatedness to strains used in the US is ongoing.
Impacts
Since probiotic effects of bifidobacteria are strain specific, techniques for rapid characterization to the strain level are important to regulatory agencies, food manufacturers and starter culture manufacturers. The information regarding repression of glucose metabolism when cells are grown on lactose is particularly important because media using glucose as a carbon source may not be suitable for isolation of bifidobacteria from cultured dairy products. If starch spherulites can enhance survival of bifidobacteria in food and in the gut, this will increase the number of tools available to assure probiotics are viable when consumed and can survive transit through the intestinal tract. Evaluation of strains from other countries may result in additional strains with novel traits for use in the US.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
Fourteen strains of bifidobacteria were acquired from two different culture collections and thirty-two commercial strains of bifidobacteria were acquired from five different starter culture suppliers. Initial identification of strains as belonging to the genus Bifidobacterium was performed by confirming the presence of fructose-6-phosphate phosphoketolase, an enzyme characteristic of bifidobacteria metabolism, and a PCR-based technique using primers targeting a Bifidobacterium-specific sequence of 16S rDNA. The results from both the enzyme assay and PCR were in agreement for all strains; twenty-two of the commercial strains were identified as belonging to the genus Bifidobacterium, while eleven of the commercial strains were not Bifidobacterium. Further characterization of these strains of bifidobacteria has been performed, including Gram-staining and observation of cellular morphology, assays for gas production during glucose fermentation, assays for the presence of
catalase, assays for production of urease, and assays for the production of indole. All bifidobacteria were Gram-positive rods and were negative for gas production and catalase, and indole and urease production, which is typical of this group of bacteria. The identities of commercial Bifidobacterium strains were established at the species-level using species-specific primers with PCR developed through previous work by Matsuki et al. and Ventura et al. ATCC and DSMZ strains were included as positive and negative controls for verifying species identities. Only four commercial strains of Bifidobacterium were correctly identified by their suppliers at the species level. The other eighteen strains, representing eight different species of bifidobacteria according to their suppliers, were identified by PCR as B. lactis. These results suggest that starter culture companies need to employ more accurate methods to correctly identify the genus and species of their strains. Subsequent research
has focused on methods to differentiate strains of bifidobacteria at the strain level. Additional phenotypic analyses performed included patterns of enzyme activity and carbohydrate fermentation ability (API ZYM and API 50 CHO kits). Only the results for fermentation of glucose differed among the commercial strains of B. lactis. Ten of the twenty strains of commercial B. lactis did not ferment glucose within the forty-eight hours of the assay. Nucleic-acid based methods have also been applied to the differentiation of these strains of B. lactis. A method was developed to apply pulsed-field gel electrophoresis (PFGE) to Bifidobacterium. PFGE has been performed with eighteen of these strains, comparing restriction patterns of chromosomal DNA digested with either XbaI or SpeI. All eighteen strains displayed identical patterns with both endonucleases, suggesting these strains are highly related. Future research will focus on employing additional enzymes with PFGE, as well as using
randomly amplified polymorphic DNA PCR (RAPD-PCR), to differentiate among these commercial strains
Impacts
Results from the initial characterization of the commercial strains of bifidobacteria suggest that starter culture companies need better techniques to identify Bifidobacterium at the levels of both genus and species. Traditional approaches to strain characterization, usually including phenotypic characterization, are insufficient to correctly identify species of bifidobacteria. Molecular techniques for identifying microorganisms based on nucleic acid sequences have recently been developed that are more objective, less time-consuming, and are more sensitive than the traditional phenotypic analysis. However, PFGE, noted for its great discriminatory power, was not a useful tool in differentiating among a group of commercial bifidobacteria strains. Molecular techniques must be developed that have the ability to differentiate among a group of closely related strains.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
A collection of thirty commercial and 9 ATCC Type strains of bifidobacteria was obtained and characterized using morphological, biochemical and genetic methods. All strains were Gram-positive rod shaped bacteria, however the typical bifurcated morphology was observed only rarely. Biochemical evaluations included determination of the lactic to acetic acid ratio and assaying for the presence of fructose-6-phosphate phosphoketolase (F6PPK) a marker enzyme considered definitive for the genus Bifidobacterium. Finally, strains were probed by PCR for the presence of a genus specific 16s rDNA fragment. All ATCC strains produced acetic acid, were positive for F6PPK and were also positive by PCR. Of the 30 commercial strains obtained, only 20 produced acetic acid, possessed F6PPK and were positive by PCR. The remaining strains were negative for both biochemical tests and negative by PCR indicating they were not members of the genus Bifidobacterium. The 20 strains which were
confirmed as members of the genus Bifidobacterium as well as the ATCC type strains were evaluated for ability to grow on a variety of selective and non-selective agars recommended in the literature for enumeration of bifidobacteria. None of the 9 media evaluated proved satisfactory for enumeration of all 29 Bifidobacterial isolates evaluated. None of the selective media proved successful in suppressing the growth of all the non-bifidobacterial isolates. These results suggest two major issues that need to be addressed: 1) starter culture companies need to carefully evaluate the identity of the strains they are providing as bifidobacteria and the second is that there is a need for a suitable medium for recovery, selection and differentiation of bifidobacteria from food products. The influence of cold adaptation on the cryololerance (ability to withstand freeze-thaw challenge) of two strains of Bifidobacterium infantis was evaluated. Cells of B. infantis ATCC 15697 and commercial isolate
BI-4 were inoculated into reinforced clostridial broth (RCB), allowed to reach mid log phase (OD600=0.5), harvested by centrifugation and resuspened in fresh RCB tempered to 20, 25 and 37C and incubated at the respective temperature. Samples were taken at 0, 30, 60, 120 and 240 minutes and frozen at -20C. Viable counts were conducted at after 0, 1, 3, 6 and 9 freeze-thaw cycles, survivor data was normalized to time 0 counts. Increasing the number of freeze-thaw (F-T) cycles resulted in a significant decline in the number of survivors for both strains studied. However, the magnitude of the effect was strain dependent (The ATCC strain was more sensitive to F-T then the commercial strain). Loss in viability as a function of F-T was reduced by incubation at suboptimal temperatures In addition, cells incubated to stationary phase were more sensitive to F-T then cells in the logarithmic phase of growth. However, since the pH of the broth from cells in stationary phase was lower then for
cells in the log phase, the influence of acid on survival must be clarified. These results suggest that stability to F-T challenge may be enhanced by preliminary incubation at sub-optimal temperature.
Impacts
Results developed during this reporting period suggest that suppliers of bifidobacterial cultures need pay close attention to the identity of the cultures provided to food manufacturers. Rapid screening can not be accomplished by simple morphological evaluation. Rather PCR-based or enzyme assay methods should be employed. Our results suggest that simple screening for acetic acid production may be a useful tool in quality assurance. These results also indicate studies undertaken to evaluate various products for the presence or level of bifidobacteria should be interpreted with extreme caution since no selective or differential media evaluated was successful at recovering all bifidobacterial isolates evaluated. Finally preliminary results suggest survival of bifidobacteria during freezing may be enhanced by pre-incubation at sub-optimal growth temperatures which could lead to improved starter preservation techniques.
Publications
- Gevorgyan, A. 2003. Effects of cold adaptation on cryotolerance of Bifidobacterium infantis. M. S. Thesis. The Pennsylvania State University, University Park, PA 150 pp
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