Document Type

Thesis - Open Access

Award Date


Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

William Gibbons


elicitors, fungal incubation, glyceollins, isoflavones, phytoalexins, soybeans


Continual use of antibiotics in the feed of food animals was viewed a solution to the problem of disease outbreaks in livestock produced in confinement operations. This practice also improved animal performance, likely due to the reduction in sub-clinical infections. Unfortunately, this practice led to a new problem, the development of antibiotic resistant microbes. This increase in antibiotic resistance reduced the direct benefits of antibiotics in animal production. Moreover, as antibiotic resistance spread from animal to human pathogens, this practice created a major public health concern. This led the FDA to enact the Veterinary Feed Directive in 2017 that greatly restricts the use of antibiotics in feed or water for production purposes, limiting them instead to therapeutic uses. The goal of this research thesis was to optimize a process for producing a natural antimicrobial (known as glyceollins) in soybeans, so that the glyceollins-enhanced soybeans (GES) could be used to replace antibiotics in the animal feed industry. Various processing factors for optimization were assessed including seed sterilization, pretreatment, fungal application, moisture maintenance, incubation and germination methods, and the use of filter paper. Possibly the most important variable related to production of glyceollins in soybeans is the variety of soybean seed used and how well it responds to the elicitor or microbe introduced. Therefore, this study also compares several soybean varieties to determine which resulted in the most glyceollins production. Controllable processing factors were individually tested by manipulating a standard production process developed previously by our team. This standard process included: 1) 10 min surface sterilization of soybeans in a 2% Micro-90 cleaning solution, 2) rinse with sterile water, 3) soak in 500 ml of sterile water overnight, 4) drain off water and aseptically dehull soybeans, 5) place 10 dehulled soybeans into Petri dish lined with sterile, moistened filter paper, 6) dropwise inoculate the 10 dehulled soybeans with 100 μl of previously grown inoculum, and 7) incubate plate at 30℃ in the dark for 120 hr. The typical fungal strain used to inoculate soybeans was Trichoderma reesei (NRRL- 3653). T. reesei was prepared by inoculating a flask of glucose yeast extract broth (GYE) an incubating 48 hr at 30℃. Soybean samples were collected daily during incuvation by harvesting all 10 soybeans on a plate, freezing and freeze-drying, and then grinding. Samples were extracted and glyceollins were measured by high performance liquid chromatography (HPLC). Application of the surface sterilant showed no negative effect on germination percentage or rate. Comparison of glyceollins titer between sterile and non-sterile beans showed some increase in the former. Inoculum application method showed the best results for both fungal growth uniformity and production of glyceollins when using the standard dropwise method. Type of inoculant (mycelial or spore) had little to no effect on germination of the seeds. Comparison of incubation length showed the titer of glyceollins typically peaking within the 72 to 120 hr, with glyceollin levels dramatically falling after 144 hr. The use of filter paper had no effect on the inoculums ability to stimulate production of glyceollins in the soybeans. The variety of soybean used accounted for the largest difference in glyceollins levels. The MN01050628 soybean variety reached levels of glyceollins ~2 mg/g at the 96 hr, while the lowest producing variety Kouri peaked at ~0.03 mg/g at 48 hr. Investigation of total isoflavones of the soybean varieties and the precursor of glyceollins, daidzein, was also conducted and were shown to have no direct correlation to the glyceollins produced by the varieties examined. This suggests further study is warranted regarding the genetic makeup of the soybean variety and that variety’s response to a chosen fungal strain or physical elicitor.

Library of Congress Subject Headings

Anti-infective agents.
Antibiotics in animal nutrition.



Number of Pages



South Dakota State University



Rights Statement

In Copyright