Viability and physiological state transitions of Rhizopus oligosporus sporangiospores in tempe starter culture

Abstract

The viability and various physiological characteristics of individual sporangiospores of Rhizopus oligosporus in tempe starter cultures that had been stored for 8, 10, 16 and 30 months were examined by flow cytometry in combination with fluorescent dyes. Besides live, dead, and dormant spores we distinguished a category of sublethally damaged spores. Results indicated that the shelf-life of tempe starters was not limited by the death of spores, but by sublethal damage to spores as well as by dormancy which can be overcome by resuscitation, respiratory activation. During storage, the number of dormant and sublethally damaged spores increased: the longer the starter cultures were stored, the less dormant spores could still be activated. In contrast, the transition from sublethally damaged (spores that are not able to transform cFDA and emit green fluorescence except by activation treatment) to activated spores did not decrease with longer storage. However, after very long (30 months) storage, sublethally damaged spores could still be activated but could not germinate anymore. The shelf-life of spores in tempe starter is related to the physiological state of spores being sublethally damaged; a mechanism of physiological state transitions of R. oligosporus sporangiospores is proposed.

Tempeh: A semicentennial review on its health benefits, fermentation, safety, processing, sustainability, and affordability

“…Throughout the storage period, the number of dormant spores that can germinate decreased (Thanh et al., 2007). Factors that affect the activation and germination of spores include glucose, phosphate, and amino acids; in which L‐alanine, L‐leucine, and L‐isoleucine stimulate, whereas L‐proline inhibited alanine uptake (Thanh et al., 2005).…”

The polyene antimycotics nystatin and filipin disrupt the plasma membrane, whereas natamycin inhibits endocytosis in germinating conidia ofPenicillium discolor

“…An important characteristic of cell death is the loss of plasma membrane integrity and the fluorescent marker TOTO‐1 has been used as a reliable viability marker of fungal spores (Chitarra et al. 2005; Thanh et al. 2007).…”

“…After 6 h of germination, the activity of polyenes was within a similar range (Table 1). An important characteristic of cell death is the loss of plasma membrane integrity and the fluorescent marker TOTO-1 has been used as a reliable viability marker of fungal spores (Chitarra et al 2005;Thanh et al 2007). TOTO-1 is a plasma membrane impermeable cyanine dimer with an affinity to nucleic acids including the DNA of the nucleus and RNA.…”

“…Conidia were subsequently studied with standard fluorescence microscopy (FM) or confocal laser scanning microscopy (CLSM). Viability staining (Thanh et al. 2007) was undertaken with 2 μ mol l −1 TOTO‐1 (Molecular Probes) in 100 μ l MEB at 25°C for 30 min.…”

“…Conidia were subsequently studied with standard fluorescence microscopy (FM) or confocal laser scanning microscopy (CLSM). Viability staining (Thanh et al 2007) was undertaken with 2 lmol l )1 TOTO-1 (Molecular Probes) in 100 ll MEB at 25°C for 30 min. Following dye removal, glass coverslips with the immobilized conidia were placed upside-down onto a thin (<0AE5 mm) layer of 2% agar.…”