Source: UNIVERSITY OF NEBRASKA submitted to
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Sep 1, 2008
Project End Date
Aug 31, 2013
Grant Year
Project Director
Bullerman, L. B.
Recipient Organization
Performing Department
Non Technical Summary
Mold and yeast (fungal) deterioration of foods and agricultural commodities is a serious problem in the U.S. and worldwide. Molds may be a problem in cereal grains, cereal products, oilseeds, nuts and various processed foods such as intermediate moisture food products, natural cheeses, cured and smoked meats, jams, jellies and various refrigerated and frozen foods, as well as livestock feeds and petfoods. Yeasts may spoil salad dressings, cottage cheese, fruit juices and juice concentrates. Mold deterioration of these products causes damage that reduces quality, grades and prices, resulting in economic loss. In addition, the presence of molds in these commodities poses hazards to human and animal health. A serious hazard associated with mold growth in foods and feeds is the possible production of mycotoxins, substances which are toxic, potentially carcinogenic, and which may adversely affect immune systems. Mold growth with accompanying potential mycotoxin production makes foods and feeds unfit for human or animal consumption. Lactic acid bacteria (LAB), propionic acid bacteria (PAB) and Bacillus pumilus are potential food-grade biological control agents that can be used to prevent growth of mold and yeasts and mycotoxin production in stored acidic and refrigerated foods, stored cereal grains, grains in the field, livestock feeds and petfoods. This project is designed to discover and isolate these antifungal biological control agents and develop them into food grade biological control agents that can be used to prevent mold and yeast spoilage of stored foods, feeds and grains and prevent mycotoxin contamination in these products. The outcomes of this project are expect to be the development of novel, new food and feed perservation systems using freindly, not-toxic bacteria to prevent mold and yeast spoilage and mycotoxin production. The impacts of this will be safer foods and feeds and economic savings.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Goals / Objectives
Lactic acid bacteria (LAB), propionic acid bacteria (PAB) and Bacillus pumilus are potential food-grade biological control agents that can be used to prevent growth of mold and yeasts in stored acidic and refrigerated foods, stored cereal grains and grains in the field. So the overall goal of this project is to develop food grade biological control agents that can be used to prevent mold and yeast spoilage of stored foods, feeds and grains and prevent mycotoxin production and demonstrate their antifungal activity in foods and feeds. Specific Objectives:1.To search for LAB and PAB isolates that have antifungal activity against spoilage and mycotoxigenic molds and yeasts. 2.To study the effects of combining LAB and PAB isolates and strains into mixed cultures on the antifungal activity of the resulting "fermentates." 3.To determine the ability and effectiveness of LAB, PAB, B. pumilus and L. rhamnosus in combination with antimycotic food additives and antifungal naturally occurring phytochemicals to inhibit mold growth and mycotoxin production by selected toxic Aspergillus, Penicillium and Fusarium species. The expected outputs of this project will be the isolation and development of antifungal food grade bacterial agents to be used in preventing mold and yeast spoilage of foods and feeds and prevent mycotoxin production.
Project Methods
Lactic Acid Bacteria (LAB) cultures will be obtained from sources such as probiotic and sourdough cultures and fermented plant foods. Cultures will be evaluated for antifungal activity. Propionic acid bacteria (PAB) obtained from ATCC and commercial culture companies will also be studied for antifungal activity. Pure and mixed LAB cultures and PAB cultures will be screened for antifungal activity against a set of molds and yeasts. Preliminary screening will be carried out by growing the bacterial cultures in modified MRS (mMRS) broth and mixing the fermented mMRS (less bacteria) with Potato Dextrose Broth in 96 well plates, inoculating the wells with the different molds and incubating at room temperature. Presence or absence of mold growth will be determined visually and by measuring the optical density (OD) of the wells. Absence of mold growth will be interpreted as inhibition and defined as antifungal activity. Those cultures, pure and mixed with demonstrated antifungal properties will be studied further. Cultures will be grown in a suitable liquid substrate such as cheese whey, tomato juice broth or other suitable food grade broth substrate to produce a fermented liquid or "fermentate". This fermentate will be mixed with double strength culture broths, both with and without the bacterial cells. These broths will be inoculated with the same mold and yeast species that were previously used to detect antifungal activity. Mold growth will be measured by increase in mold mycelial mass over time. Yeast growth will be determined by optical density measurements and plate counts over periods of time. Propionic acid bacteria produce propionic acid, a known antifungal substance. Lactic acid bacteria produce lactic acid and may co-exist or even stimulate the growth of PAB. These combined LAB and PAB fermentate cultures would be expected to have strong antifungal activity. The most strongly antifungal LAB, PAB and fermentates will be studied in combination with each other and with Bacillus pumilus and Lactobacillus rhamnosus which have been previously studied. These cultures will also be combined with known antimycotic food additives such as potassium sorbate and calcium propionate and naturally occurring substances, with known antifungal properties, such as mustard and ground flaxseed. These combinations will be studied for the ability to inhibit growth and mycotoxin production by known mycotoxigenic molds. Mold growth will be measured by determining mycelial dry weights. Mycotoxin concentrations will be measured using commercially available quantitative ELISA test kits. Quantitation of mycotoxin levels will also be done by high performance liquid chromatography (HPLC). Experimental designs for this work will be developed based on advice from a statistician.

Progress 09/01/08 to 08/31/13

OUTPUTS: This research studied the organic acid profile of Lactobacillus plantarum, the factors influencing the production of active compounds by these bacteria, the stability of the antifungal compounds produced and their effect on mold growth by a variety of spoilage and toxic mold spp. The organic acids produced included lactic, acetic, pyruvic, 3-phenyllactic, p-hydroxyphenyllactic and pyroglutamic acids. The production of the antifungal compounds by the bacteria was increased with the incubation time up to 96 hours and by incubation temperatures of 30 and 35C. During bacterial growth, the best results were obtained when the pH was maintained at 7.0. Cultures grown under agitation gave higher antifungal activity than those grown under static conditions, with 10% oxygen saturation favoring the production of antifungals. The bacterial supernatant lost activity at pH 4.0 or higher; while temperature and digestive enzymes did not affect the activity. When the bacterial supernatant was fractionated, the presence of 3-phenyllactic, 3-hydroxyphenyllactic and pyroglutamic acids in some of the active fractions were identified by HPLC-PDA and HPLC-MS. The Lactobacillus plantarum isolates that were studied were obtained from fermented plant products, including sauerkraut and pickled cucumbers. Isolates from these foods were the most inhibitory against the spoilage and toxigenic molds tested, showing a broad spectrum of antifungal activity. The bacterial cultures had inhibitory activity against Fusarium, Penicillium and Aspergillus species. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

The outcomes of this research were to demonstrate the antifungal activity and potential of lactic acid bacteria involved in fermentation of plant/vegetable food products. The antifungal bacteria were identified as Lactobacillus plantarum and pure isolates were obtained. These isolates are now available for further development into commercial antifungal food additives. In this respect the work has resulted in the potential for preventing mold spoilage and improving food safety.


  • Bianchini, Andreia, 2010. Antifungal activity of lactic acid bacteria. Ph.D. Dissertation. University of Nebraska-Lincoln, Lincoln, NE

Progress 10/01/08 to 09/30/09

OUTPUTS: Lactic acid bacteria (LAB), probiotic cultures, propionic acid bacteria (PAB), herbs and spices were studied for naturally occurring antifungal activity. Chemical antimycotic food preservatives were also evaluated in the presence of the naturally occurring antifungal activity of the biological substances to observe any additive or synergistic effects. Thirty LAB isolates were obtained from fresh cucumbers and a home-made fermented pickle brine, and three LAB and PAB cultures from culture collections were screened for antifungal activity against Penicillium roqueforti using a 96-well plate assay. The most inhibitory isolates were idientified by RAPD-PCR and 16s rDNA as Lactobacillus graviea, Lactobacillus plantarum and Weisella ghanensis. Growing two ispolates together in mixed cultures produced greater antifungal effect than growing the isolates alone in individual cultures. Preculturing PAB in MRS broth followed by LAB fermentation of the same broth gave almost 100% inhibition of mold growsth. The presence of mustard enhanced the antifungal activity of individual LAB and mixed LAB cultutes. Potassium sorbate and sodium propionate at low concentrations, 0.025% and 0.5% respectively, enhanced the antifungal activity of LAB and PAB cultures. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Thwe dwork reported was a preliminary study on the new project. the outcome of this work is that it serves to identify the direction that further studies can take to develop new antifungal preservative systems.


  • Kam, P.V. 2009. Antifungal properties of lactic acid bacteria, propionic acid bacteria and natural and chemical preservatives. M.S. Thesis. University of Nebraska, Lincoln, NE.