The Preliminary Study On Production Technology Of Antagonistic Yeasts

With the advance of people’s consciousness on physical health and environmental protection, the biocontrol as a new way to control the postharvest diseases has been considered as an ecologically sound and environmentally friendly approach to control postharvest diseases. Many researches have confirmed that antagonistic yeasts have shown great efficacy and application prospects as an alternative to chemical fungicides in controlling postharvest decay of fruits and vegetables. However, because of the high cost and low efficacy, few commercial antagonistic yeasts products are now available. This article uses the sugarcane molasses as the antagonism yeasts fermentation raw material and carries on the thorough research on the optimization of molasses medium in order to produce cheap and efficient antagonistic yeast bio-control agents and the fermentation condition and vacuum freeze drying condition are also studied preliminary. The main findings are as follows:1. The molasses were pretreated by the method with boiling, adding phosphoric acid or ulfuric acid, potassium ferrocyanide, and activated carbon adsorption, and comparing the impact of different methods on the growth of antagonistic yeasts. The results showed acid method was superior to other treatment and sulfuric acid was the most effective way.2. Different media components (such as cane molasses, nitrogen sources and concentrations, initial pH, phosphate, magnesium sulfate, and sodium chloride) were studied on their influence on the biomass production of H. uvarum, P. guilliermondii and composite of the two antagonistic yeasts by single factor test. The Plackett-Burman design and Response Surface Method were used to optimize the medium components, and optimized medium were as follow:H. uvarum:pretreated molasses:79.9 g·L-1, yeast powder:2.2 g·L-1, initial pH:6.0; P. guilliermondii:pretreated molasses:75.2 g·L-1, yeast powder:2.5 g·L-1, initial pH:5.9; composite antagonistic yeasts:pre-after processing molasses:78.0 g·L-1, yeast powder:2.7 g·L-1; urea:0.03 g·L-1, initial pH:7.0. The optimized medium made the biomass production of H. uvarum, P. guilliermondii and both composite antagonistic yeasts increase by 1.8 times,2.2 times and 1.9 times respectively.3. Through the preliminary studies on the fermentation conditions both in the shake flash system and 10L fermentor, the most suitable fermentation conditions in the shake flask were confirmed and for H. uvarum as follows:inoculation volume of 3%(v/v), liquid volume of 50 mL, rotation speed of 180 r·min-1; for P. guilliermondii as follows: inoculation volume of 2%, liquid volume of 50 mL, rotation speed of 180 r·min-1; both composite antagonistic yeasts:inoculation volume of 5%, liquid volume of 50 mL, rotation speed of 180 r·min-1. In 10 L fermentor, batch fermentation process characteristic curves showed that H. uvarum and P. guilliermondii were able to take advantage of cane molasses, and the cells concentration were up to 1×1010CFU·mL-1 after cultivation for 24 h and had the feasibility of expansion of production. While when the amount of residual sugar were less than 3.0 g·L-1 and pH value below about 4.0, the cell concentration were no longer increased in the culture medium. Therefore, we need control the pH value and the amount of residual sugar in the subsequent experiments through supplement with fresh medium, sucrose and ammonia, etc.4. We studied the conditions of preparaing active dry antagonistic yeasts. The results showed that, for H. uvarum, the optimum compounding of protectant was 10% Sucrose and10% Skim Milk, and freeze-dried survival ratio was 86.0%; for P. guilliermondii, the optimum compounding of protectant was 5% Sucrose and 5% Skim Milk, freeze-dried survival ratio was 81.6%; and the optimum compounding of protectant for both composite antagonistic yeasts was 10% Sucrose and 15% Skim Milk, freeze-dried survival rate can reach 89.9%. After the active dry antagonistic yeasts products were stored at 20℃ and respectively for three months, we found that their activated cells number changed very slightly and maintain the activated cells number of more than 1×1012 CFU·g-1, and the activity of dry antagonistic yeasts decreased not significantly at 4℃ storage conditions; this showed the low temperature is more suitable for storage of dry antagonistic yeasts.