Faculty PI: Michael L. Dunn (NDFS)
Co-PI: Frost M. Steele (NDFS)
Proposal Approved: Fall 2010
Work Completed: January 2013

Evaluation of how well the academic objectives of the proposal were met
This was an intensive project, which turned out to be a great opportunity for our students. We had one graduate student and four to five undergraduate students working on it at any given time. Due to attrition in the lab, we had about 16 undergraduates who were involved across the course of the entire project. Students learned the nuances of a very challenging multi-day, microbial assay for folic acid, and also learned many other laboratory skills and became quite proficient in microbial enumeration and isolation. The importance of laboratory methods validation across multiple analysts became readily apparent, and students were involved in looking for and minimizing analyst-to-analyst (or student-to-student) differences in techniques. It was hard, painstaking work, but our student eventually managed to produce accurate and reliable results as a group. Some of the students were also able to travel go Guadalajara, Mexico to collect samples and run analyses. They were thus able to participate in a cultural experience and learning, in addition to their work in the lab.

Evaluation of the mentoring environment
Our research team was organized with the two faculty PIs and Stephen Adolphson, a graduate student, working closely with all the undergraduate students to provide safety and proficiency training for all of the techniques used. One undergraduate student served as the lead student, coordinating the efforts and schedules of all of the other undergraduates on the team. We held weekly lab meetings and reviewed methods and discussed data, and determined objectives for the coming week. Everyone participated, and a different student was asked to take minutes and distribute them each week. Part of the project involved travel to Mexico to collect samples, and to run assays at a lab in Guadalajara. We ended up taking two trips to Mexico, and Dr. Dunn accompanied the students each time. During one trip we were able to visit the research labs of one of the largest micronutrient premix providers in the world. They allowed us to use their facilities to conduct a portion of our research. The students benefited from observing the workings of a commercial analytical/research lab.

List of students who participated and what academic deliverables they have produced or it is anticipated they will produce
The following students were involved at different stages of the project:

• Stephen Adolphson (Grad Student)
• (Undergraduates)
  • Curtis Park
  • Brandon Jahner
  • Shintaro Pang
  • Eric Engstrom
  • Carly Stone
  • Jonathan Kershaw
  • Grace Park
  • Brayden Willis
  • Adam Beus
  • Brian Coffman
  • Jason Demmerly
  • Dallin Hardy
  • Jason Kim
  • Junwoo Kim
  • Christopher McNeil
  • Scott Whitlock

This project should result in two publications, as described below.

Description of the results/findings of the project
This was a large project which actually had two distinct sub-projects within it. One aspect of the project involved collecting masa from six small, traditional, tortilla mills in Guadalajara. Then the unique microflora present in the masa from the different mills was enumerated and characterized. This study of the native microflora of nixtamalized corn masa produced in these traditional mills was published in the International Journal of Food Microbiology, with graduate student Stephen Adolphson as the first author.

Adolphson, Stephen J., Dunn, Michael L., Jefferies,Laura K., Steele, Frost M. 2013. Isolation and Characterization of the Microflora of Nixtamalized Corn Masa, International Journal of Food Microbiology, doi: 10.1016/j.ijfoodmicro.2013.05.010

Lactic acid bacteria and aerobic mesophiles were the predominant organisms isolated, with both groups having counts ranging from 104 to 107 CFU/g across all mills. Coliforms were present at about 102 CFU/g in most mills, while yeast and mold were nearly absent. Streptococcus bovis and Lactobacillus oris were isolated from all mills, and were the most prevalent organisms representing 43% and 17% of all lactic acid bacteria isolated, respectively. Streptococcus bovis was also isolated on the aerobic tryptic soy plates and was the most prevalent species representing 19% of the total organisms from these aerobic plates. There was a diversity of organisms present, but a few key organisms predominated.

A second paper is currently ready for submission discussing the potential role of bacteria in the significant degradation of folic acid during manufacture of micronutrient fortified tortillas from these traditional mills. Previous research from our lab has demonstrated between 70-80% loss of added folic acid, most of which occurs in the wet masa prior to baking. It was postulated that folate requiring lactic acid bacteria might be the cause of this loss. In this MEG-funded study, folic acid losses of up to 79.1% were again seen as corn masa dough was incubated at an elevated temperature. However, these losses occurred even in sterile, irradiated masa, and were not due to the natural bacteria present in the masa. It appears that a time-temperature dependent chemical degradation is taking place in the high moisture masa, when held at significantly elevated temperatures typical of those generated during mechanical milling with volcanic mill stones, as used in Mexico. It is hoped that these results will be published in the same journal.

Description of how the budget was spent
Student wages, both graduate and undergraduate, took up the bulk of the funds (approximately $12,000). Supplies for analyses cost about $5000 and travel to Mexico (Faculty and 3 students to Guadalajara, Mexico. Two trips) was about $3000.