Production And Characteristics Of Main Product Produced By Aureobasidium Pullulans

Pullulan,also known as pullulan polysaccharide,poly（polysaccharide）or pullulan,and made up ofα-1,4 andα-1,6 glucosides alternately.Because of its unique structure,it has a very wide range of applications in food,the chemical industry,medicine,tobacco,cosmetics,environmental protection and fresh preservation of agricultural products.Phenyl ethanol（also known as 2-phenylethanol orβ-phenylethanol）was extracted and separated from pullulan fermentation broth of Aureobasidium pullulans A5 for GC-MS,which was initially found in peaches,pears,strawberries,cocoa and other natural plants).It possesses a soft,pleasant and lasting aroma of roses,so could be used in the preparation of rose-type food additives and the main raw materials of rose flavor.Therefore,it is also a food spice that can be allowed to be used in our country,so this study also provides a new strain source and certain theoretical basis for the biosynthesis of 2-phenylethanol（2-PE）by biological method.In this disertation,to screen strains which could selectively producing elevated molecular weight and pigment-free pullulan for pullulan industry,and identified,identified the polysaccharide and optimized the fermentation conditions.The aim is to provide a new strain source for industrial pullulan fermentation.The main contents of this study are as follows:1)In this paper,the YPD solid medium was used as the screening medium,chloramphenicol was used as the screening pressure,triphenyl blue was used as the screening indicator.A polysaccharide-producing yeast strain A5 was obtained.Based on the morphology,colony and ITS interval sequence analysis,which was found that the similarity between the yeast strain A5 and Aureobasidium pullulans YM 24528 or PR-23was 99%,respectively,and had a very close relationship.It could be confirmed that the yeast strain A5 belongs to the A.pullulans familiy.2)A preliminary study on the structure of the extracellular products of this strain A5was carried out firstly,and the characterization of structure and determination of molecular weight were carried out by using pullulanase and combined with thin layer chromatography,infrared spectrum analysis,nuclear magnetic resonance spectroscopy and gel permeation pigment.The results showed that the extracellular polysaccharide produced by this strain A5 was the polysaccharide of pullulan and the molecular weight was 63.8 kDa.3)In order to further improve the yield of the extracellular polysaccharide of the strain A5,the culture medium and conditions for pullulan fermentation by the single factor experiment were optimized as follows:maltose 8%（w/v）,yeast 1%（w/v）,peptone2%（w/v）,K₂HPO₄ 0.5%（w/v）,（NH₄）₂SO₄ 0.06%（w/v）,CaCl₂ 0.03%（w/v）,pH 6,inoculation amount 7%（v/v）.Under the optimum culture conditions,the yield of the fermentation tank was 22.9 g/L,and the fermenter fermentation achieved 96.4 g/L.4)While A.pullulans A5 produced pullulan in the fermentation process and a sweet rose like aroma,as well the latter one extracting from the fermentation broth by n-hexane,and then testing by gas chromatography-mass spectrometry,which revealed that is 2-phenylethanol,2-PE itself will have toxic effect on yeast cells,so firstly revealed that pullulan could improve the tolerance of yeast cells to 2-PE.For further improving 2-PE output and achieving the best extractant-three alkyl phosphorus（TRPO）,and then optimized the condition of the extract from fermentation broth of 2-PE,the fermentation conditions were screened and optimized then.Finally,the results showed that A.pullulans A5 producing 2-PE by the medium 1(maltose 2%（w/v）,YNB 0.18%（w/v）,KH₂PO₄0.4%（w/v）,MgSO₄·7H₂O 0.04%（w/v）,K₂HPO₄ 0.5%（w/v）,pH6,4 g/L of L-phenylalanine at the optimum fermentation conditions by fermentor fermentation,and2.6 g/L of 2-PE was obtained.In this paper,the production of pullulan and 2-phenylethanol by fermentation of A5was studied,which provided a theoretical basis for further exploring the co-production of these two products in the same culture medium,and a new strategy for eliminating the toxic effect of 2-PE.