Study On Stability And Acid Hydrolysis Modification Of Phycocyanin

As a natural pigment with health care function,phycocyanin is one of the two natural blue pigments permitted in GB 2760-2014.It is a pigment protein complex formed by the connection of tetrapyrrole chromophore and accessory protein,which has unique color,rich nutrition,antioxidant,anti-tumor,anti-inflammatory and other physiological functions,and has broad development and application prospects.However,phycocyanin is unstable,easy to fade and precipitate in high temperature and acidic environment,which seriously restricts its application in food and cosmetics,especially in acidic food.In this paper,the factors affecting the stability of phycocyanin were investigated in detail,and the methods to improve its stability were explored from two aspects of thermal instability and acid instability,which provided new ideas for the practical application and popularization of phycocyanin.Firstly,starting with the factors affecting the stability of phycocyanin,an evaluation method based on the preservation rate of phycocyanin was established by spectrophotometry.The effects of temperature,p H,light,sugar,preservatives,metal ions and redox agents on the stability of phycocyanin were studied.Temperature greatly affects the stability of phycocyanin,and when the temperature is higher than 50?,the damage to phycocyanin is extremely strong;The preservation rate of phycocyanin in the p H 4?7environment hardly changed significantly,but the preservation rate decreased in the acid and alkali environment,especially in p H 2.5,the color will change from bright blue to green with precipitation.Indoor natural light has little effect on the preservation rate of phycocyanin,while outdoor strong light,especially ultraviolet light,has a strong destructive effect.After ultraviolet irradiation for 3 hours,the color faded obviously,and the preservation rate was only about 50%.Sugar in common food additives can improve the stability of phycocyanin,but preservatives have little effect on it.When the concentration of ethanol is higher than 20%,it will produce flocculent precipitation.Na⁺,Ca²⁺and Mg²⁺in metal ions can enhance the color of phycocyanin to some extent.Cu²⁺,Fe³⁺and Al³⁺can seriously damage phycocyanin,so they should be avoided from contact with it.In a word,the temperature and p H have the greatest influence on the preservation rate of phycocyanin,and phycocyanin should be kept below 40?and p H 4?6 as far as possible,avoiding the environment of strong light.Then,based on the thermal stability of phycocyanin,the effects of polyhydroxy compounds(saccharides and polyols)on the thermal stability of phycocyanin were studied by using kinetic methods and thermodynamic parameters,and the thermal degradation kinetics of phycocyanin was explored.Glucose,fructose,sucrose and maltose all have thermal protection effect on phycocyanin at 65?,and there was a positive correlation with the addition amount,among which glucose and sucrose had the most significant protection effect;Polyols had different effects on the thermal stability of phycocyanin,and xylitol and erythritol could significantly improve the thermal stability of phycocyanin under the condition of low addition amount(mass fraction<10%).The thermal degradation law of phycocyanin accorded with the first-order kinetics(correlation coefficient R²>0.9).Adding sugar and polyol could improve the degradation half-life of phycocyanin at high temperature,especially at 65?.Glucose,sucrose,xylitol and erythritol could increase the t1/2 of phycocyanin from 97.63 to 693.15?693.15?346.57 and 187.34 min respectively,and at the same time increase the activation energy Ea,?H and?G.Combined with fluorescence spectrum analysis,the protective effect of sugar and polyol on phycocyanin under heating may be hydrogen bonding with the accessory protein part of phycocyanin,thus protecting the structural stability of accessory protein.Aiming at the acid stability of phycocyanin,phycocyanin was modified by acid hydrolysis in order to solve the problem of instability and precipitation under acidic conditions.Taking the color and yield of modified phycocyanin by acid hydrolysis as indexes,the acid hydrolysis time,the ratio of phycocyanin to acid and the concentration of hydrochloric acid were optimized by single factor and orthogonal experiments,and the optimum process was determined as follows:acid hydrolysis time 2.5 h,the ratio of phycocyanin to hydrochloric acid amount 1:3,and the concentration of hydrochloric acid 12 mol/L.Under these conditions,the modified phycocyanin can keep ideal bright blue color under acidic conditions(p H 2.5?3.0),with L^*,a^*and b^*values of 82.64?-3.76 and-24.62,respectively,and no precipitation occurs.On this basis,adding polyphenols could enhance and protected the color,among which tannic acid(the ratio of addition amount to acid hydrolysis modified phycocyanin is 1:5)and rosmarinic acid(the ratio of addition amount to acid hydrolysis modified phycocyanin is 1:1)had the best enhancement and protection effects on the pigment solution.The stability of acid hydrolysis modified phycocyanin and its complex with polyphenols was determined.It was found that it could keep bright blue only in acidic environment,and its thermal stability was improved.Even after heating at 80?for 2.5 h,the preservation rate was still about65%,which was less than 10%compared with that of original phycocyanin.The DPPH scavenging rate,total reducing power and ABTS scavenging rate of phycocyanin were improved after acid hydrolysis.Finally,the change of internal structure of acid hydrolysis modified phycocyanin and its complex with polyphenols was characterized.The results showed that acid hydrolysis had little effect on the relative content of amino acids in phycocyanin,and the first limiting amino acid was lysine.The phycocyanin modified by acidolysis has better solubility in acidic solution.After acid hydrolysis,the D₅₀ decreased from 24.46?m to 13.46?m.After adding tannic acid and rosmarinic acid,the particle size of acid hydrolysis modified phycocyanin increased,which indicated that the two polyphenols and acid hydrolysis phycocyanin may be combined by hydrogen bonding or hydrophobic interaction,which made the particle size increase.The microstructure analysis showed that the surface structure of phycocyanin changed from smooth to loose and rough,and the long chain structure became shorter.Polyphenols could polymerize the phycocyanin modified by acid hydrolysis.Fourier transform infrared spectroscopy and circular dichroism analysis showed that the content of?-helix decreased from 79.4%to 57.0%,while the content of?-fold,?-turn and random curl increased,which indicated that the original secondary structure of phycocyanin was destroyed by acidolysis,and the structure of phycocyanin was rearranged and conformationally changed.The existence of tannic acid increased the?-helix content of acid-hydrolyzed phycocyanin and decreased the content of other secondary structures.Rosmarinic acid decreased the?-helix content and increased the?-fold,?-turn and random curl content.This indicated that both polyphenols could interact with acid hydrolysis modified phycocyanin and change its structure in different degrees.