Comparison of the antifungal activity of kefir and garlic extract against bread spoilage fungi

Abstract

In previous studies, the antifungal capacity of kefir, an ancient, fermented beverage, was evaluated. In the present work, 6 strains of fungi isolated from spoiled breads were identified and the antifungal activity of garlic extract against these fungi was evaluated and compared with the antifungal activity of kefir. The kefir beverage was prepared by inoculating a total of 3 g of kefir grains in 30 mL of sterile brown sugar aqueous solution (5 g%). It was incubated at 30°C with an orbital shaker at 140 rpm. The sample was taken after 72 h and the cell-free supernatant (CFS) was obtained by centrifugation at 10,000 x g for 10 minutes. It was sterilized by filtration using a 0.22-micron membrane. The garlic extract was prepared by macerating garlic (8 g) in a mixture of ethanol and water in equal parts. After one week, it was centrifuged at 6000 xg for 20 minutes. The fungi isolated were cultivated into Sabouraud medium for 24 h. The identification was realized by molecular techniques: DNA was obtained using a biologic kit; PCR amplification with universal primers were made; DNA concentration in the PCR products was determined using Epoch (Biotek) and the integrity of the samples was evaluated through 1% agarose gel electrophoresis. The PCR products were sent to CERELA (Tucuman-Argentina) for their purification and sequencing. The sequences were edited with Molecular Evolutionary Genetics Analysis (MEGA v7.0) and were analyzed with BLASTn using NCBI databases. The isolated were identified belong the genus Penicillum, Aspergillus and Paecilomyces. The Minimum Inhibitory Concentration (MIC) was determined for each fungi. Serial dilutions of kefir and garlic were prepared. A volume of 300 µL of each dilution was placed into sterile Eppendorfs containing 620 µL of 2x Sabouraund medium. Each tube was inoculated with 620 µL of spore suspension of 1/100 dilution of an original suspension of OD 600 nm 1.4 of each strain. Spores were obtained from a 7-day-old plate on glucose potato agar. Positive controls (inoculated medium without the addition of kefir or garlic) and negative controls (uninoculated medium with the addition of kefir or garlic) were included. They were cultured for 72 h and observed every 24 h. The results showed that garlic extract has greater antifungal activity than kefir. The most sensitive were fungi of the Penicillium genus, while the strains most resistant to them were Aspergillus niger and Paecilomyces variotii. In future, the synergies and stability over time of kefir and garlic extract will be studied, in order to advance their use as natural preservatives in bread.