Accumulation Characteristics And Stability Of Phycoerythrin In Thermophilic Cyanobacteria

As early as the 70 s of the last century,natural pigments have entered people’s lives.As a pink natural pigment,phycoerythrin is found in red algae,cryptoalgae,and cyanobacteria.Due to its bright color,anti-inflammatory,antioxidant and free radical scavenging functions,phycoerythrin has good application prospects in food,health care products,medicine,cosmetics,biological reagents and other fields.However,due to the poor thermal stability and photostability of phycoerythrin obtained by ordinary strains,it is easy to fade and discolor,which limits its application.The research object of this project is thermophilic cyanobacteria collected in the hot spring area of Ganzi area of western Sichuan,which is a kind of photosynthetic prokaryotes living in extreme thermal environments,and its growth environment is likely to give its phycoerythrin better thermal stability and other characteristics,thereby providing new ideas for expanding the source and application of phycoerythrin.In addition,studies have shown that light conditions have a significant effect on the pigment accumulation of cyanobacteria.Therefore,the effects of different photoperiod,light intensity and light color on the phycoerythrin accumulation of three thermophilic cyanobacteria were explored,and the effects of different phycoerythrin purification methods on their yield and purity were tested.In addition,the stability of phycoerythrin of thermophila under different temperatures,pH values,light,storage and food additives was identified,which provided an experimental basis and technical reserve for its application to food addition.In this paper,eight strains of thermophilic cyanobacteria were obtained through strain screening,and three representative strains(E412,B334 and B212)were identified in detail.By morphology and molecular biology(phylogenetic analysis based on the sequence of 16 S r RNA),B212,B334 and E412 were identified as Stanieria cyanosphaera B212,Leptothermofonsia sichuanensis B334 and Leptothermofonsia sichuanensis E412.The photoadaptability study of phycoerythrin synthesis by thermophila showed that the light intensity of 600 Lx and the cultivation of thermophilic cyanobacteria with periodic changes such as 16 h light/8 h dark and 12 h light/12 h dark were more conducive to the accumulation of phycoerythrin,and the wavelengths of 454 nm(blue light)and 511 nm(green light)had a promoting effect on the accumulation of phycoerythrin of thermophilic cyanobacteria compared with ordinary incandescent lamps,and inhibited the accumulation at a wavelength of 654 nm(red light).The experimental results showed that E412 reached the maximum accumulation of phycoerythrin under the light period of 16 h light/8 h dark,intensity of 600 Lx and wavelength of 454 nm(blue light),so that the crude amount of phycoerythrin of thermophila cyanobacteria reached 21.92 mg/g DCW.The stability results showed that phycoerythrin of thermophila phycoerythrin could maintain good stability when it was in an environment between 60 ℃ and pH between 4-7.It can be stored for a long time at low temperature conditions of 4 ℃ and below;It has excellent lightfastness under light,especially ultraviolet light,but outdoor sunlight with high intensity is not conducive to maintaining its physiological activity;Appropriate concentrations of potassium sorbate,sucrose and sodium chloride are conducive to the preservation and color protection of phycoerythrin of thermophila,and show its outstanding stability under different temperature,pH value,light,storage conditions and food additive treatment.Protein structure homologous modeling showed that amino acid residue substitution occurred in E412 phycoerythrin at six positions compared with two strains of marine cyanobacteria,which may be the mechanism that makes E412 phycoerythrin more stable.In this project,the three thermophilic strains of phycoerythrin had better stability than the phycoerythrin of other medium and low temperature algae,and had better tolerance to medium and low temperature strains of phycoerythrin under high temperature,acidic environment and ultraviolet lamp irradiation,and their phycoerythrin was more stable when stored at low temperature or added with potassium sorbate,sucrose and sodium chloride.The results of the extraction and purification process showed that secondary freeze-thaw and ultrasonic disruption could increase the amount of phycoerythrin extracted by thermophila cyanobacteria,and the phycoerythrin salting out of thermophilic cyanobacteria was suitable for ammonium sulfate with a saturation of 25 %,and the fractional salting out could significantly improve the purification effect compared with the first level of salting.Phycoerythrin 23.24 mg/g DCW was finally extracted by freeze-drying,mortar grinding,phosphate buffer swelling,secondary freeze-thaw and ultrasonic disruption,which was 30%higher than that of the crude extraction method.Using the fractional salting out of ammonium sulfate with a saturation of 25 %,the final phycoerythrin purity was 2.68,which was higher than that of food grade and close to pharmaceutical grade.The purification products of SDS-PAGE analysis showed that the thermophila E412 phycoerythrin contained three subunits,α,β and γ,all with molecular weights between 10-25 kDa.The results found that photoperiod,light intensity and light color had different degrees of effects on the phycoerythrin accumulation of the three strains of thermophilic cyanobacteria.From the perspective of temperature,pH value,light,storage conditions and food additives,it was found that the phycoerythrin of thermophila was more stable than that of medium and low temperature strains.Through the comparison of phycoerythrin purification methods,secondary freeze-thaw,ultrasonic crushing,and fractional salting out of ammonium sulfate can improve its purification quality,and while ensuring a higher level of phycoerythrin accumulation in thermophilic cyanobacteria,phycoerythrin with high purity and good stability is obtained in a cost-effective way.It lays the foundation for the development of thermophilic cyanobacterial resources,and also provides the corresponding basis and data basis for the subsequent utilization and industrial production of thermophilic cyanobacterial phycoerythrin.