Isolation And Identification Of Red Pigment Producing Strain And Study On The Properties Of Red Pigment

With the development of society,pigment has been widely used in daily life,and its safety has been paid more and more attention.Synthetic pigments are restricted in application due to their chemical harmfulness,so the research and development of natural pigments have great significance.There are many kinds of pigments produced by microorganisms,which are not restricted by resources,environment and space.As a potential pigment resource bank,it has a broad prospect in the industrial production of natural pigments.In the process of culture of Amauroderma rude in our laboratory,we stumbled upon a strain that could secrete a large amount of red pigment,named it as EFOZ-01,the classification,screening,stability of red pigment,optimization of fermentation conditions for producing red pigment and purification and identification of pigment were studied in order to find a production strain that could be used as a natural pigment.The main research results as follows:1)The strain was identified by classical morphological characteristics and molecular biological analysis.The mycelium of this strain has transverse septum,and the conidium has multiple branches,producing small asymmetrical stalks in the shape of broom,while the conidium is oval in shape with blue-green color.It was preliminarily determined that this strain belongs to Penicillium fungi.By using ITS sequence analysis,the evolutionary tree was constructed,and the homology between the bacterium and Penicillium was determined to be 99%,belonging to Penicillium purpurogenum.In combination with characteristics of morphology and molecular biology analysis,named it as Penicillium purpurogenum EFOZ-01.2)The effects of carbon source,nitrogen source,inorganic salt ion and fermentation medium inoculum,fermentation time,temperature,loading volume and initial p H on the production of red pigment by EFOZ-01 were determined by single factor experiment.The best carbon source for producing red pigment is sucrose,and the best nitrogen source is sodium nitrate.The addition amount of inorganic salt ions is K₂HPO₄0.4 g/L,Mg SO₄0.2 g/L,KCl 0.3 g/L,Fe SO₄·7H₂O 0.004 g/L,The optimal fermentation conditions were:10%inoculum,5 d fermentation time,28?fermentation temperature,100 m L/250 m L loading,and p H 7.0.Based on the single-factor experiment,the inoculum,fermentation time,fermentation temperature and carbon source were selected for the orthogonal experiment with four factors and three levels.The optimal conditions were determined as follows:8%inoculum,5 d fermentation time,and 28?fermentation temperature,the carbon source was sucrose.3)Using the fermentation broth of the strain producing red pigment as raw material,the red pigment was extracted by organic solvent extraction method,and the characteristic spectrum curve of the red pigment was measured.The characteristic absorption peak of the red pigment was determined at 510 nm.At the same time,the properties of red pigment were studied.The results showed that the pigment was soluble in organic solvents such as methanol,ethanol and n-butanol,and also soluble in phosphate buffer,easily soluble in organic solvents such as methanol and ethanol;The red pigment has good stability in neutral,weak acid and weak alkaline environment;The red pigment has good stability under the condition of avoiding light,but under the condition of light,it has a certain degree of influence on the red pigment;The pigment has a certain heat resistance,in the room temperature environment,under high temperature conditions,the red pigment will have more losses.This pigment has good stability on oxidants,reducing agents,most metal ions,preservatives,food additives and vitamins,among which V_Ehas certain enhancement effect on red pigment,and it should be noted that Fe³⁺has certain adverse effect on the stability of red pigment.4)The crude pigment extract was separated and purified by solid phase extraction,high performance liquid chromatography?HPLC?and liquid-mass chromatography?LC-MS?.The molecular weight of the red pigment has been preliminarily determined to be 364.1,it may be a new substance that has not been reported in the current natural products database.Its specific structure needs to be determined by NMR.