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Antifungal Activity of Lactic Acid Bacteria Against Molds Isolated from Corn and Fermented Corn Paste

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Antifungal Activity of Lactic Acid Bacteria Against Molds Isolated from Corn and Fermented Corn Paste American Journal of Microbiological Research, 2016, Vol. 4, No. 4, 90-100 Available online at http://pubs.sciepub.com/ajmr/4/4/1 ©Science and Education Publishing DOI:10.12691/ajmr-4-4-1 Antifungal Activity of Lactic Acid Bacteria against Molds Isolated from Corn and Fermented Corn Paste Leopold Ngoune Tatsadjieu1,*, Roger Tchikoua2, Carl Moses Mbofung Funtong3 1Department of Food Engineering and Quality Control, University Institute of Technology, University of Ngaoundere, Cameroon 2Department of Microbiology, University of Yaounde I, Cameroon 3Department of Food Sciences and Nutrition, National High School of Agro-Industrial Science, University of Ngaoundere, Cameroon *Corresponding author: [email protected] Abstract A total of 336 molds were isolated from dried corn, soaked corn and fermented corn paste. The macroscopic and microscopic studies of fungal growth in the following identification media, grouped all the 336 molds into 21 strains. The strains belonged mainly to 4 fungal genera: Aspergillus, Fusarium, Penicillium and Rhizopus. In addition, the aflatoxinogenic strains were dominant and were mostly isolated from Maroua (63 strains of Aspergillus flavus). Moreover, the antifungal activity of 53 Lactic acid bacteria (LAB) isolated from the samples was performed against 21 fungal strains. After a screening test, 06 were selected for their potent antifungal activity and were identified as Lactobacillus brevis (2 isolates), Lactobacillus buchneri (1 isolate), Lactobacillus cellobiosus (1 isolate) and Lactobacillus fermentum (2 isolates). During the antifungal tests in solid medium, most of the LAB inhibited the growth of molds but Lactobacillus brevis G25 (80 ± 0.5 mm) and Lactobacillus cellobiosus (82 ± 0.1 mm) had the greatest antifungal activities after 48 hours against Aspergillus carbonarius G23 and Aspergillus carbonarius G24. However, the antifungal activity was more efficient in liquid medium and Lactobacillus brevis G11 and Lactobacillus fermentum N33 totally inhibited the growth of the 21 molds tested in liquid medium. Thus organic acids were identified as substances responsible for the antifungal activity of the LAB. These results show the possibility of exploiting some of these LABs as starters to fight against spoilage molds in fermented corn paste. Keywords: antifungal activity, fermented corn paste, lactic acid bacteria, molds Cite This Article: Leopold Ngoune Tatsadjieu, Roger Tchikoua, and Carl Moses Mbofung Funtong, “Antifungal Activity of Lactic Acid Bacteria against Molds Isolated from Corn and Fermented Corn Paste.” American Journal of Microbiological Research, vol. 4, no. 4 (2016): 90-100. doi: 10.12691/ajmr-4-4-1. seeking and demanding for products without chemical preservatives which still maintain good shelf life and 1. Introduction safeness. Due to these drawbacks, the concept of biopreservation is gaining popularity. This concept refers Several molds are known for their ability to grow on a to extended shelf life and enhanced safety of foods by wide range of foods and to alter their nutritional and growth of the natural or added microflora and their organoleptic qualities. The most frequent genera antimicrobial products [6]. implicated in food contamination include: Aspergillus, Among the microorganisms which can potentially be Penicillium and Fusarium [1]. Their capacity to produce used as biopreservatives, Lactic Acid Bacteria (LAB) toxic secondary metabolites or mycotoxins is a serious represent a good example and are considered, Generally health danger worldwide [2]. In Africa, there is ample Recognized As Safe (GRAS) and have traditionally been evidence of the direct and negative effects of aflatoxins on used in food and animal feed, sauerkraut and silage. A human health through increased incidence of liver cancer number of studies have focused on inhibition of fungi and because of its potential synergistic effect on hepatitis growth using LAB and their preserving effect relates B [3]. Also, studies have pointed out the capacity of some mainly to the formation of organic acids, hydrogen mycotoxins to alter normal immune function when present peroxide, competition for nutrients and production of in food at levels below observable over toxicity [4]. antimicrobial substances [7]. In respect of the dangers which molds and their Although the activity of LAB varies with ecosystems, it mycotoxins represent, some food preservation methods, would be good that a number of studies be carried such as addition of benzoic acid and sorbic acid, used as worldwide in order to discover new and active LAB. antifungal in food. These acids are usually added in form In the foregoing, the aim of this study was to determine of water-soluble salts (sodium benzoate and potassium the antifungal activity of LAB strains isolated from corn, sorbate). However, their consequences such as resistance fermented paste and to determine the origin of this activity of the species, the residual level in food, remain the major with the hope that they can be applied as biopreservatives. problems of their use [5]. Moreover, consumers are 91 American Journal of Microbiological Research temperature. Then, they were placed directly on Dichloran 2. Materials and Methods 18% Glycerol Agar (DG18). This method permits recovery of the fungi actually growing in the particles. 2.1. Collection of Samples Plates were incubated at 25°C for 5 to 7 days. For the fermented corn paste, the isolation of the molds was The preparation of fermented corn paste in the Sudano- carried out according to the method described by Nkwe et Guinean (Ngaoundere) and Sudano-Sahelian (Maroua and al. [9]. These strains were kept in distilled water at 4°C Garoua) zones of Cameroon was done principally by prior to further identification. soaking of dried corn, milling soaked corn and fermentation of corn paste for 3 to 5 days (Figure 1). The 2.3. Molds Identification sampling was performed at different stages of preparation of fermented corn paste. Sampling of dried corn, soaked To identify the different molds isolated from dried corn, corn and fermented corn paste were randomly obtained. soaked corn and fermented corn paste samples obtained They were collected in January 2012 during the dry from the 03 cities under study, the method elaborated by season. For the cities of Maroua and Garoua, 9 samples of Pitt and Hoking [8] was used. The fungal strains were dried corn, 9 samples of soaked corn and 9 samples of inoculated onto three media. The three medium were CYA fermented corn paste were collected. While in town of (Czapek Yeast Extract Agar), MEA (Malt Extract Agar) Ngaoundere, 11 samples of dried corn, 11 samples of and YES (Yeast Extract Sucrose Agar). After 7 days of soaked corn and 11 samples of fermented corn paste were incubation at 25°C, the macroscopic and microscopic collected. Each sample weighed 250g with the exception observations were used to evaluate parameters such as the of fermented corn paste which weighed 100g. The soaked diameter of the growth, the color of the aerial parts of the corn and fermented corn pastes were introduced in sterile mycelium, conidial heads, pigmentation of the base of the plastic bags and preserved at -18°C prior for analysis. mycelium, time of growth and the surface texture of the Grains of dried corn were packed in sterile craft papers colonies. and preserved in a dry environment. 2.4. Determination of the Aflatoxigenic Molds Isolates of Aspergillus and Penicillium were cultured on Malt Extract Agar (MEA), Czapek Yeast Extract Agar (CYA), Yeast Extract Sucrose Agar (YES) and Coconut Milk Agar (CMA) at 25°C and 30°C for 7 days and detected by fluorescence of the medium under ultraviolet light [10]. 2.5. Isolation of Lactic Acid Bacteria LABs were isolated in different samples according to the method of Voulgari et al. [11]. Briefly, 1 g of the various samples (dried corn, soaked corn and fermented corn paste sample) was homogenized in a stomacher blender for 1 min with NaCl solution (0.85%). The suspension was tenfold serially diluted and appropriate dilutions were plated onto Man Rogosa Sharp (MRS) agar. Plates were incubated at 37oC for 48 hr. After this time, colonies were purified by streaking on the same medium. Only Gram-positive, non-spore forming and catalase- negative bacterial isolates were selected and stored in glycerol (40%) at -20°C for further investigation. 2.6. Biochemical Identification of Selected Lactic Acid Bacteria The most active isolates were characterized by their carbohydrate fermentation profile using the API 50 CH strips and the API 50 CHL medium according to the Figure 1. General process used for the preparation of the fermented corn paste manufacturer’s instructions (API system, Bio-Merieux, France). Strains were tentatively identified into species 2.2. Evaluation of Contamination of Samples level using APIDENT (version 2.0, Bio-Merieux). by Molds 2.7. Screening of Lactic Acid Bacteria To evaluate sample contamination, molds were isolated This analysis was carried out according to the method from dried corn and soaked corn by the direct plating of Lind et al. [12]. Each isolated LAB strain was tested on technique as described by Pitt and Hoking [8]. Ten grains mold isolates on MRS medium. To perform this test, the of corn per sample were disinfected in aqueous solution of LAB were inoculated by streaking in Petri dishes sodium hypochlorite (NaOCl) for one minute at room containing MRS agar. The preparation was incubated for American Journal of Microbiological Research 92 24 h at 37°C. After this time, the preparation was covered treated respectively with proteinase K (20 mg/mL, with 5 mL of Potato Dextrose Agar (PDA) medium Eurobio, France), trypsin (40 U/mg, Merck) and α- containing the moulds spores (106spores/mL). The whole chymotrypsin (40-60 U/mg) from bovine pancreas. The preparation was incubated for 48 h at 25°C. The presence reaction of catalase and various proteases were stopped by of a zone of inhibition permits to highlight the active incubating the fractions for 10 min at 65°C in a water bath.
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