Ecophysiological approaches to production and formulation of the biocontrol yeast Pichia anomala

To produce commercial biocontrol agents (BCAs) successfully, it is important that cheap and economic substrates are used which support high numbers of good quality inoculum. Production of formulations conserving ecological competence and shelf-life should also be ensured. With this in mind, studies focusing on yeast ecophysiology were conducted to produce and formulate ecologically competent P. anomala cells for controlling spoilage of moist cereal grain. The liquid culture systems used were synthetic, nutrient yeast dextrose broth (NYDB), and a complex (industry byproduct, cane molasses) media. Manipulation of cultural conditions by means of imposing water-stress with several solute additions to the media had an impact on yield, cell water potentials (Ψc), viability and endogenous sugar/polyol accumulation. Glucose addition resulted in higher yeast yield (6.15 and 3.4 mg cell ml-1 medium for NYDB and molasses, respectively). Water activity (aw) modifications of the media resulted in modification of Ψc so that Ψc ≤ Ψw (medium water potential). The change in yeast Ψc was attributed to the intracellular accumulation/synthesis of polyols, mostly glycerol and arabitol and sugars, mostly trehalose. In molasses-based medium cells accumulated/synthesized trehalose [32 mg g-1 fresh weight (f.w.) yeast cell]. Higher amounts of endogenous trehalose (up to 140 mg g-1 f.w. yeast cell) were retained intracellularly when modified yeast cells were isotonically washed compared to those subjected to hypo-osmotic shock by washing with water. The pattern was similar for endogenous arabitol. Trehalose retention doubled and quadrupled, and arabitol increased by 65 and 100% in proline and NaCl treatments respectively. The molasses control medium gave high [>1010 colony forming units (CFU) ml-1] cell viability, which was further increased by addition of NaCl and proline (≈ 3x1010CFU ml-1).

Fluidised bed drying of yeast cells showed that drying at 50oC for 20 min resulted in high cell viability (67%) and low moisture content (7%). Osmoprotection and several carriers and adjuvants affected viability and moisture content. Cotton seed flour (CSF) + 10% skimmed milk (SM) resulted in the highest cell protection (74%) during the drying process, with a final moisture content of about 5% and this was easy to resuspend. Storage stability of the formulation was 50% at 4oC and ambienta temperatures for up to 150 days. P. anomala cells grown in NaCl ii modified molasses-media, when osmoprotected, retained four times more trehalose and resulted in significantly increased survival after drying and storage stability for 150 days. When SM + sucrose at 10% (w/v) was used as a protective solution, P. anomala cells were highly resistant to freezing, thawing and freeze drying processes. Storage stability at 4oC of freeze dried P. anomala cells was particularly high (>86%) over a period of 150 days while storage at 22oC resulted in a rapid decrease in cell viability to <35% over a period of 30 days. Osmoprotection using post-harvest isotonic washing treatment had no effect on storage stability. P. anomala produced high β-(1-3)-glucanase (>2.2 activity units), low chitinase (<0.9 activity units) and β-glucosidase (<3 nmol 4-nitrophenol min µg protein of specific enzyme activity)-1-1 amounts. The role of the first hydrolase in biocontrol activity is possibly important while that of the other two is not clear for P. anomala. In lab-scale bioassays using wheat grain under aerobic conditions, populations of P. verrucosum 22625 were significantly reduced by formulated P. anomala cells at both 0.93 and 0.95 aw levels while OTA production was significantly reduced at 0.93 aw only.