Studies On Nisin Production Using Distiller's Grain From High Gravity Alcohol Fermentation

Distiller's grain includes abundant nutrient substance. As a result, it is important that how to make good use of distiller's soluble and make it transform into the product with high additive value. This paper was studied on Nisin production by using distiller's solubles as main medium.A high Nisin-producing strain was directionally screened from 75 samples of fresh milk. It was preliminarily identified as Lactococcus Lactis Subsp. Lactis WX3129 by the experiments of assaying morphological character and physiological and biochemistry feature.The conditions of determining Nisin activities by Oxford Plate Assay System were studied in this paper. A few variables were evaluated. The optimal determining conditions were obtained: adding quantity of a monolayer medium 15ml, the suspension concentration of Micrococcus luteus 109 cfu/ml, the adding quantity of Oxford Plate 100μl, agar concentration 1%, the concentration of Na2HPO4 1％, and the pH of the medium adjusted to 7.0.The medium was optimized by single factor and the orthogonal experiments on the basis of the centrifugate of distiller's grains. The optimal composition was obtained as follows: sucrose 0.6, peptone 0.1, yeast extract 0.2, K2HPO4?3H2O 0.2, NaCl 0.2, MgSO4?7 H2O 0.02, centrifugate of distiller's grains 100 ml. Then, on the basis of the optimized distiller's grain culture medium, the optimal fermentation conditions were obtained: initial pH 7.5, inoculation volume 5%, culture temperature 30 ℃, 50 ml medium in 250 ml shake flask. The effect of pH control on Nisin production was studied. The results showed that, when pH was maintained at constant pH 6.0, 6.5 and 7.0, Nisin activities in fermentation broth were increased by 26.3%, 49.5%, 32.4% respectively, compared to those without pH control.The influence of dilution rate(D) on Nisin production was investigated during continuous fermentation. The result showed that when dilution rate was 0.1 h-1 and 0.2 h-1, Nisin activity was steadily about 2600 IU/ml, reducing 21.8% compared to batch fermentations. Nevertheless, the volumetric productivities were higher than batch cultures. And the volumetric productivity was the highest for dilution rate of 0.2 h-1, increasing by 2.61 fold compared to batch cultures.The pH adsorption method was used to extract and purify the fermentation product from strain WX3129. Identification of urea sodium dodecyle sulfate polyacrylamide gel electrophoresis and reverse phase high pressure liquid chromatography revealed that the fermentation product is Nisin.