Study On The Directional Transformation Of Mulberry Red Pigments Catalyzed By Microfluidic Aqueous Two-Phase System

Cyanidin-3-O-glucoside?C₃G?and cyanidin-3-O-rutinoside?C₃R?are the main components of mulberry red pigments,accounting for approximately 30%and 60%of the total amount,respectively.C₃G has the functions of blood glucose reducing,liver and viscera protection.It is widely used in baked and health products.However,there is currently a lack of high purity C₃G products.In this paper,novel microfluidic aqueous two-phase systems were constructed to prepare high purity C₃G by specific hydrolysis of C₃R with the aim of the directed transformation of glycosyl groups in mulberry red pigments.The main research contents are as follows:?1?The biotransformation system and the qualitative analysis methods of mulberry red pigments were constructed.The results showed that two main components of C₃G and C₃R were detected in“Da Shi”mulberries using HPLC-PDA-ESI-MS/MS and HPLC-UV methods.The contents of C₃G and C₃R were 4.72±0.98 mg/100g and 7.86±1.33 mg/100g.The homogeneous system with?-L-rhamnosidase?enzyme concentration 36.07 mg/m L?was reacted under p H 5,temperature 45?and substrate concentration 0.086 mg/m L for 1h.The C₃R conversion and C₃G purity reached 62.92±0.79%and 75.29±0.78%,indicating that enzymatic hydrolysis of of C₃R to C₃G was feasible.But it showed substrate inhibition.Therefore,on the basis of ultrasound-assisted"ethanol/ammonium sulfate"aqueous two-phase system?ATPS?,the feasibility of enzyme catalysis was explored.The ATPS was composed of 27.12%ethanol,18.10%ammonium sulfate,15%mulberry juice?C₃R concentration 0.11 mg/m L?and 39.78%water according to the mass fraction.The C₃R conversion and C₃G purity by the ATPS were 74.41±0.85%and 86.47±1.49%,which were11.49%and 11.18%higher than homogeneous system.It showed that the ATPS was suitable for bioconversion in mulberry red pigments.?2?The“ethanol/ammonium sulfate”ATPS with immobilized enzyme for catalytic reaction separation and coupling was constructed to transform mulberry red pigments.The results showed that multi-walled carbon nanotubes were selected from 9 kinds of nanomaterials to prepare immobilized?-L-rhamnosidase.The enzyme activity of immobilized enzyme was 914.31±1.39 U/mg and the enzyme immobilization capacity was4 g/g.The ATPS with immobilized enzyme was composed of 27.12%ethanol,18.10%ammonium sulfate,15%mulberry juice?C₃R concentration 0.11 mg/m L?,4.24%immobilized?-L-rhamnosidase and 35.54%water according to the mass fraction.The system was reacted under p H 5,45°C for 1 h.The C₃R conversion and C₃G purity reached71.68±0.94%and 82.42±1.04%.The optimal substrate concentration in this system was0.11 mg/m L,which was 1.28 times that of the homogeneous system.The C₃R conversion rate and C₃G purity of the ATPS with immobilized enzyme were 8.76%and 7.13%higher than homogeneous system.The immobilized?-L-rhamnosidase could be reused 7 times,indicating that the constructed ATPS with immobilized enzyme for catalytic reaction separation and coupling was efficient.?3?A new process of W/W microdroplet system with enzyme in dispersed phase was designed for the conversion in mulberry red pigments.The results showed that when 17%PEG?20 k Da?flow rate was 0.2-1.0?L/min and 15%Dex?500 k Da?flow rate was0.058-0.09?L/min,the diameters of W/W microdroplets were ranged from 3.41±1.34?m to 43.74±11.68?m.When the flow rate of PEG was 0.40?L/min and the flow rate of Dex was 0.074?L/min,the system was reacted under p H 5,45?and substrate concentration0.007 mg/m L for 2.8 min,the C₃R conversion and C₃G purity reached 53.79±0.98%and68.14±1.38%.Compared with conventional reactors,the enzyme catalysis time of the W/W microdroplet system was shortened from 1 h to 2.8 min,which took only 1/20 of the original.It indicated that the W/W microdroplet system had a high catalytic rate and the enzyme in dispersed phase was conducive to continuous production.?4?A new process of microfluidic aqueous two-phase system?MATPS?with immobilized enzyme under parallel condiction was used for the conversion in mulberry red pigments.The results showed that:when ammonium sulfate flow rate was 14.50?L/min and ethanol flow rate was 10?L/min,the system was reacted under p H 5,45?and substrate concentration 0.008 mg/m L for only 8.6 s.The C₃R conversion and C₃G purity reached 68.66±1.63%and 80.78±1.59%,respectively.The immobilized?-L-rhamnosidase could be reused 9 times,maintaining a relative activity of over 50%.The reaction time of the MATPS was 7/3000 and 1/20 of the conventional reactors and the W/W microdroplet system.The stability of the immobilized enzyme was improved and the technology of MATPS was higher.In conclusion,novel microfluidic aqueous two-phase systems for enzyme catalysis were successfully constructed to biotransform mulberry red pigments.Around enzyme and substrate,reaction and separation,the biocatalytic mechanisms at the microscale were comprehensively analyzed.It provided new ideas for efficient bio-manufacturing of mulberry red pigment with high purity of C₃G and the development of new products.