Study Of Fermentation Process Of Biologic Preservative Natamycin

The parameters of agar diffusion method that influenced natamycin bioassay were determined. No.62 strain was screened as high yield strain by agar block method with S.gilvosporeus ATCC 13326. Under strict quality controlling of seed, the fermentation process in shaking flask was optimized. Fed-batch fermentation was applied to improve the yield of natamycin based on the studies of batch fermentation metabolic process in 5L auto-fermentor.The yield of natamycin and the diameter square of inhibitory zone took on a good linear relation after the determination of parameters of agar diffusion method. The indicator strain was cultivated at 29 on a rotary shaker at 170r/min for 48h until the concentration was 108/mL. The upper and the lower layer of double test plate whose diameter was 90mm were both prepared with 10mL medium and the upper agar consisted of 10% indicator strain. The volume of tested fermented broth was 70 1. The bioassay was performed at 29 and the diameters of inhibitory zone were recorded after 12h.One high yield strain No.62 was screened from different colony shape by agar block method and shaking flask fermentation. The colonial shape of the strain No.62 displayed white plump spore, a straw hat shape, homocentric round and regular edge. The yield of natamycin in shaking flask was Ig/L.The seed quality controlling was studied. The slope was cultivated at 29 for 5d and the conservative time in the refrigerator(4 ) was not more than 60d. The concentration of inoculation spore suspension was 108spores/mL. The inoculums were transferred to fermentation by 2% of seed volume when seed was cultivated 24h-28h.The optimum medium and fermentation conditions for natamycin production were demonstrated through single factor test and orthogonal design test. The optimum medium consisted of (g/L): glucose 40, soya peptone 19.5 and yeast extract powder 4.5. The pH of the medium was adjusted to 7.5 and inoculum volume was 2%. A 500mL flask containing 70mL medium was cultivated at 29 for 96h and the agitation speed was 200r/min. The yield of natamycin was up to 1.5g/L under the optimized conditions.The feeding process of fed-batch fermentation was determined based on the studies of batch fermentation course in 5L auto-fermentor. The initial glucose concentration was 40g/L. 50% glucose was added by pulsed feeding on the interval of 6h when remained glucose decreased below 20g/L, then the glucoseconcentration was maintained about 20g/L. During the whole fermentation course, the total amount of feeding glucose was 20~25g/L. Under the optimum conditions of fed-batch fermentation, the yield of natamycin was 2.2g/L that was improved by 37%. At the same time, the solution of 2mol/L NaOH was added when pH decreased below 5.3 to maintain pH 5.3-5.4 until the end of fermentation, so the yield of natamycin was improved to 2.4g/L.