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Bifidude: Lab Investigation of the Bifidobacteria-Supporting Soy-Free Non-Dairy Infant Formula

06/05/2012

RE: LAB INVESTIGATION OF BIFIDUDE (A SOY-FREE,LACTOSE-FREE, BIFIDOGENIC INFANT FORMULA)

Dear Potential Backers, Bifidude has made its project proposal available online for anyone to review. Currently, the project is hosted at FundaGeek, a crowdfunding site for technical innovation and scientific research. The objective of Bifidudes FundaGeek project is to raise funds for the laboratory investigation of the unique infant formula that would include a couple of microbial and animal assays. Bifidude is an independent infant formula label designed to complement breastfeeding and in cases where breastfeeding is absent, would be the best choice to replace the role of breast milk in selectively growing Bifido strains in the infant gut. It is soy-free and non-dairy and has a unique indigestible component similar to human milk oligosaccharides. The goal of the lab investigation is to get the study published in order to proceed to human testing and developing the formula for future mass feeding programs in poverty-stricken areas in the Philippines where child malnutrition is prevalent. The ambitious aim of Bifidude is to make a dent in improving the problem of child malnutrition and child mortality through its economical and scientifically-feasible alternative infant formula. It would be greatly appreciated if you take interest in making the laboratory investigation of Bifidude a reality. It would be a great step towards helping alleviate the global problem of under-5 child undernutrition and under-5 child mortality. Please send me an email if you require further information or if you have questions concerning this proposal. Thank you,

Donna Longstocking/Donna Estampador Creator/Starving Scientist Bifidude Iloilo City 5000 Philippines donnapuff@gmail.com bifidude@gmail.com

Bifidude: Lab Investigation of the Bifidobacteria-Supporting Soy-Free Non-Dairy Infant Formula

Bifidude: Lab Investigation of the Bifidobacteria-Supporting Soy-Free Non-Dairy Infant Formula

Public Access

Date: June 5, 2012

Donna Longstocking Creator Bifidude Iloilo City 5000 Philippines bifidude@gmail.com

Bifidude: Lab Investigation of the Bifidobacteria-Supporting Soy-Free Non-Dairy Infant Formula

Bifidude: Lab Investigation of the Bifidobacteria-Supporting Soy-Free Non-Dairy Infant Formula

Project Abstract
Bifidude, through FundaGeek, is seeking to crowdfund its laboratory investigation project to establish the formulas bifidogenic activity with the objective of publishing the study in order to proceed to the formulas human testing and product development for future mass feeding programs in poverty-stricken areas in the Philippines where child malnutrition is prevalent. The objective is to perform the microbial and animal assays by October of this year and publish the research by the end of the year. Bifidudes formula is based around the biology of the gut microbiota and the study could give a fresh perspective on how infant formula design is approached.

Statement of Need
Child death and child malnutrition is a global problem. By just improving the state of child health, death of millions of children can be prevented. Bifidude targets to improve child health without increasing the need for resources. The formula is made of cheap and local ingredients designed to provide optimum nutrition and with its unique mixture of long-chain fructooligosaccharides, it will greatly benefit early infant health. Nourishing early infant health equates to a child NOT dying before reaching the age of five, improved child growth, improved cognitive development, and other enhanced health outcomes. But before Bifidude can be used by infants, it has to undergo first a couple of experiments to establish its bifidogenic activity and has to be assessed for safety and suitability. And that is the main goal of the FundaGeek project. Bifidude needs your help in getting funding to establish the basis of its benefits for early infant health.

Project Description
Although the use of a germ-free mouse model is desired, the author lacks the facilities and the experience to deliver and breed or house germ-free mice in a germ-free environment. For the experiment, Turnbaughs (Turnbaugh, et.al) humanized mouse gut microbiota model has been modified. Five to seven week old male mice will be initially colonized with a freshly voided fecal sample from a healthy human infant. The sample will be immediately kept in an anaerobic environment. An aliquot of the sample will be frozen, a second aliquot will be diluted in reduced phosphate-buffered saline and an aliquot of this dilution will be introduced via gavage into each of 15 recipient mice. The mice will be maintained on a standard low-fat, plant polysaccharide-rich diet. Fecal samples will be collected from the mice one day, one week, and one month after colonization. At this point, 10 of the mice will be switched to a different diet. Five will be switched to an infant formula containing probiotics (Nestle NAN most probably will be used) and five will be switched to Bifidude (soy-free, non-dairy, with a fructooligosaccharides as indigestible component). Mice from the three groups will be kept on their respective diets for two additional months, with weekly fecal sampling, and at which point they will be sacrificed.

Bifidude: Lab Investigation of the Bifidobacteria-Supporting Soy-Free Non-Dairy Infant Formula

To isolate strains from fecal samples, samples will be kept in a sterile bag containing peptone water (0.1%) added to 0.25% L-cysteine. Samples will be homogenized and diluted in peptone water with Lcysteine. Each sample will be spread in MRS agar containing 0.25 L-cysteine and incubated at 37C for 48h in an anaerobic chamber containing an atmosphere of 85% N2, 10% H2, and 5% CO2. One of the things being considered after growing the samples in MRSc, is to transfer them to a modified MRSc broth (pH3.0) with 0.3% bile salts. But this has to be tested first if B. longum/infantis will be able to tolerate this media. Its supposed to simulate the micro-environment of human gut. Reference strains of bifidobacteria (B. longum, B. longum infantis) will be used to test the quality of the medium to be used. For reference species, the author is targeting to contact the STELA Center in Laval University, Canada because according to some related article, they donate to independent researches. To identify the isolates, the option is phenotypic characterization for now but on the lookout for institutions willing to help out in genotypic identification in their laboratories. For phenotypic characterization, each strain isolated will be cultured on MRSc agar (MRS agar + 0.25% L-cysteine). Inoculated plates will be incubated at 37C for 24-48h. Strains will be observed with a microscope (1000x) after staining with methylene blue. Only strains with the Y phenotypic form will be selected for identification. Selected colonies will be purified by repeated streaking on MRSc agar and identified by API 50 CH kit (however, still researching for a cheaper alternative). Prior to use, strains will be activated in MRSc broth at 37 for 24h. After centrifugation at 6000 t min for 5 mins, cells will be washed twice with PBS (phosphate saline buffer) and will be suspended in 2ml of PBS added to 0.25% L-cysteine. API 50 CH kits will be inoculated with the tested strains. API kits will be covered with mineral oil and will be subject to incubation at 37C for 24-48h in anaerobic conditions. After incubation, the acidification of the wells will lead to a change of coloration from yellow to purple.

References Gootenberg DB, Turnbaugh PJ. 2010. Humanized animal models of the microbiome. Journal of Animal Science, 2010-3371. Turnbaugh PJ, Ridaura VK, Faith JJ, Rey FE, Knight R, Gordon JI. 2009. The effect of diet on the human gut microbiome: a metagenomic analysis in humanized gnotobiotic mice. Science Translational Medicine, 11, 1(6):6ra14. Mai V, Draganov PV. 2009. Recent advances and remaining gaps in our knowledge of associations between gut microbiota and human health. World Journal of Gastroenterology, 15, (1): 8185. Mnard O, Butel M-J, Gaboriau-Routhiau V, Waligora-Dupriet A-J. 2008. Gnotobiotic mouse immune response induced by Bifidobacterium sp. strains isolated from infants. Applied Environmental Microbiology, 74, (3): 660666. Costalos C, Kapiki A, Apostolou M, Papathoma E. 2008. The effect of a prebiotic supplemented formula on growth and stool microbiology of term infants. Early Human Development, 84, (1):45-9.

Bifidude: Lab Investigation of the Bifidobacteria-Supporting Soy-Free Non-Dairy Infant Formula

Zinedine, A. 2007. Isolation and characterization of strains of Bifidobacteria with probiotic proprieties in vitro. World Journal of Dairy & Food Sciences, 2, (1): 28-34. Moro G, Minoli I, Mosca M, Fanaro S, Jelinek J, Stahl B, Boehm G. 2002. Dosage-related bifidogenic effects of galacto- and fructooligosaccharides in formula-fed term infants. Journal of Pediatric Gastroenterology and Nutrition, 34, 291295. Livina V, Peiffera I, Hudaulta S, Rochatb F, Brassartb D, Neeserb J-R, Servina AL. 2000. Bifidobacterium strains from resident infant human gastrointestinal microflora exert antimicrobial activity. Gut, 47, 646652.

Goals and Objectives

The goal of Bifidudes FundaGeek project is to investigate the ability of the infant formula to support specific Bifidobacteria growth in the gut of a humanized infant mouse model. This will basically establish Bifidudes bifidogenic activity that could be the basis for further research projects and health program outcomes like feeding programs (which is the main goal). For this project, the main objectives include: 1. Isolating and identifying the presence of bifidobacteria strains in fecal samples. 2. Comparing the number of colonies to estimate the density of microorganisms within the three types of fecal samples (standard diet versus milk formula with probiotics diet versus Bifidude diet).

Timeline
Activities September 2012: October 2012: October to December 2012: December 2012: December 2012 to February 2013: 100% Funding from FundaGeek sponsors. Obtain lab equipment and gears. Set up Bifidude laboratory. Begin animal inoculation. Scheduled fecal sampling and strain isolation. Phenotyping and other microbiologic assays. Prepare research results and submit the study to an online journal.

Budget
The project goal of $4000.00 would cover for the purchase of an animal care system (the MCIE 14-Cage System), a bench-top incubator, a bench-top autoclave, a digital compound microscope, reference bacteria, microbial identification kits, supplementary lab equipments like a Softouch forceps (for mice handling), additional petridishes, petridish holder, inoculating loops, test tubes, test tube holders, culture media and culture media ingredients.

Bifidude: Lab Investigation of the Bifidobacteria-Supporting Soy-Free Non-Dairy Infant Formula

Open to Collaborate
Since I am working on a budget and minimal equipment, I am open to do collaborative work with people who can donate their time and laboratory space to perform genotypic identification of my isolates. <insert big smiley here>

Author Information
Donna Estampador (also Donna Longstocking in various online networks), the Bifidudette, is a registered pharmacist in the Philippines and holds a Bachelors Degree in Pharmacy from the University of San Agustin, Iloilo, Philippines.