## 3 Results and Discussion
### Fermentation Kinetics of Potential Biopreservation Compounds Produced in GPE Broth
Response Surface, Model Validation and Optimum Conditions for the Production of Biopreservation Compounds  
The interactive effect of the four independent parameters (fermentation time, pH, temperature and total sugar concentration) on production of biopreservation compounds is represented in a 3D plot (Figure 4). An interdependence of fermentation time, temperature and sugar concentration on the production of biopreservation compounds was observed. It was noted that the optimal conditions for production of biopreservation compounds were both pH and temperature dependent. Therefore, optimal production was only obtained at pH lower than 5 and temperature between 15 and 25 \({}^{\circ}\)C for all three yeasts strains studied (Figure 4a-1,b-1,c-1). These conditions typify mild and low cost conditions, as these bioreactor conditions would be easily maintained. Previously, [PERSON] et al. [12] showed that temperature and pH had a significant effect on cellular growth subsequent to the production of biopreservation compounds. The optimal production time for the biopreservation compounds ranged between 16 and 24 h for \(C\). _pyralidae_ Y1117, and between 16 to 28 h for \(P\). _kluyveri_ Y1125 and Y1164 (Figure 4a-2,b-2,c-2). Variations in sugar concentration of the production medium had an insignificant effect on biopreservation compound production. However, a combinatorial effect of fermentation period and sugar concentrations of the GPE broth on production of biopreservation compounds was noted when both conditions (viz. 'prolonged fermentations in higher sugar must' and'shorter fermentations in lower sugar must') were responsible for higher growth inhibition activity (Figure 4a-3,b-3,c-3). It was then economically realistic to consider the use of lower sugar containing broth (diluted GPE broth to 150 g L\({}^{-1}\)), which still showed the highest growth inhibition activity rapidly (\(\pm\)24 h) for \(C\). _pyralidae_ Y1117 (Figure 4a-3). Contrarily, \(P\). _kluyveri_ Y1125 (Figure 4b-3) and Y1164 (Figure 4c-3) seemed to have higher physiological requirements that were characterized by longer fermentation periods and higher sugar requirements. The significance of fermentation parameters such as pH, temperature and carbon source for production of biopreservation compounds was also highlighted for _Lactobacillus_ and _Saccharomyces_ species [25; 26; 27]. In this study, the determination of the optimum conditions for production of biopreservation compounds in a GPE medium was done based on the desirability function. Insignificant variations in biopreservation compound production were noted when both 'low sugar concentration-short fermentation time' and 'high sugar concentration-prolonged fermentation time' settings were compared for all strains (Table S3). Three different quadratic models (\(Y_{1}\), \(Y_{2}\) and \(Y_{3}\)) were generated to explain the production of biopreservation compounds in GPE medium. Based on the analysis of variance for each model, it was found that all models were significant with a probability value of <0.05. The lack of fit value 0.013, 0.080 and 0.014 corresponding to \(C\). _pyralidae_ Y1117, \(P\). _kluyveri_ Y1125 and \(P\). _kluyveri_ Y1164, respectively, implied that the models developed were significant (Table S3). From the ANOVA analyses, the significance of the models was observed by the values of the lack of fit obtained, which confirmed a good predictability of the models. It was observed that the predicted regression coefficient of 0.9721, 0.8385 and 0.9927 was in reasonable agreement with the adjusted regression coefficient of 0.9897, 0.9787 and 0.9972 for \(C\). _pyralidae_ Y1117, \(P\). _Pluyveri_ Y1125 and \(P\). _kluyveri_ Y1164, respectively, which further confirmed a good predictability of the models to observed experimental data. Table S4 clearly illustrates the comparison of the experimental and the predicted response values of each model developed for the production of potential biopreservation compounds by each yeast.