Potato dextrose agar (pda)

Mycelial discs of 2 mm-diameter from the 7-day revitalized cultures on potato dextrose agar (PDA, Sigma Aldrich, Darmstadt, Germany) were taken and transferred to PDA medium in a combination of each antagonist isolate with a pathogen on opposite sides of a Petri dish (85 mm diameter). The controls were grown in monocultures. Cultures were incubated at 25 ± 2 °C, 12 h/12 h night/day for 5 days. Each culture was performed in three replicates. The radial growth of the colony size was measured with a ruler every 24 h, and measurements were used to calculate the colony growth rate (mm/day). Inhibitory effect of antagonists on pathogen was estimated as the percentage reduction in pathogen growth in the presence of the antagonist, in accordance with the formula: (Rc − R)/Rc × 100 where R_c is the pathogen's mycelial growth in the control, and R is the pathogen's mycelial growth in the co-culture^{16 (link)}.

AC inoculum was prepared by retrieving the fungal culture (maintained on a potato-dextrose-agar [PDA; Merck KGaA, Darmstadt, Germany] and stored at 4°C), streaking on PDA medium and incubating at 38°C for 2 days. After being dislodged from the PDA, the fungal mycelia was diluted in 200 ml of sterilized distilled water and further used to prepare the fungal starter.
About 200 g of sterilized dry cassava pulp (87.5% dry matter) was inoculated with the suspension of fungal mycelia and then thoroughly mixed. After aerobic incubation at room temperature for 4 days, the inoculation starter was enumerated based on the colony counting method. The fungal starter produced was eventually used to ferment the cassava pulp for the in vivo experiment.

Ten grams of the cut apple samples were homogenized with 90 mL deionized water, using a homogenizer (CK-100, Chemist Scientific Corp. New Taipei City, Taiwan) and diluted with 0.1% peptone water, to obtain the microbial count. An appropriate dilution of 1.0 mL were made with 9 mL peptone water. Aerobic counts were determined on sterile disposable Petri dishes plate count agar (PDA, Merck, Darmstadt, Germany). They were incubated at 37 °C for 48 h. Molds and yeast were estimated on the potato dextrose agar (PDA, Merck, Darmstadt, Germany) and incubation conditions were 37 °C for 72 h. Each microbial count was obtained from the mean of three determinations and was expressed as log CFU/g (Song et al., 2013) [33 (link)].

The fungal pathogen Fol strain Fo-To-S-V-1 used in this study was obtained from the culture collection from the Iranian Research Institute of Plant Protection. The fungus was maintained on a potato dextrose agar (PDA, Merck, Germany) slant at 4 °C and was sub-cultured onto a fresh PDA plate at 27 °C for 7 days for further tests.
Strain KR2-7 was maintained on nutrient agar (NA, Merck, Germany; with a 0.3% beef extract, 0.5% peptone, and 1.5% agar) plate with a periodic transfer to a fresh medium. For long-term storage, it was kept at −80 °C in lysogeny broth (LB, Merck, Germany) with 20% glycerol (v/v).

The accelerated shelf-life test of dark chocolates was designed based on [40 ]. Freshly produced chocolates were heat-sealed in polyethylene bags and kept in two different incubators (Binder BD-53, Binder GmbH, Tuttlingen, Germany) maintained at 20 and 30 °C with a relative humidity level of 80% for 3 weeks. The microbial analysis was performed on the chocolates on days 1, 6, 8, 13, 15, and 21 of storage. Plate count agar (PCA, Merck, Frankfurt, Germany) and potato dextrose agar (PDA, Merck, Frankfurt, Germany) were used to enumerate the total viable count and yeast and mould count, respectively. The dark chocolate sample (10 g) was crushed and homogenised with sterilized peptone water (90 mL, Merck, Frankfurt, Germany) using a stomacher (Interscience Bag Mixer, Saint Nom, France). Subsequently, the homogenised solution was subjected to a 10-fold serial dilution with sterilized peptone water (Merck, Frankfurt, Germany). An aliquot of appropriate dilution was then inoculated onto the PCA (Merck, Frankfurt, Germany) and PDA (Merck, Frankfurt, Germany) plates using the pour plate technique. After incubation (37 °C, 48 h), the grown colonies were then counted, and the counts were reported as colony forming units per gram of the chocolate sample (CFU/g).