Study Of Whole-cell Catalytic Hydrolysis Of Citrus Flavonoid Glycosides And Its Mechanisms

Citrus is one of the fruit crops mainly used for food processing as well as fresh juice production in the world.The by-products of citrus processing,such as fruit leaves,peels and seeds,are rich in flavonoid active ingredients,which have abundant functional activities such as antioxidant,anti-inflammatory and antibacterial.Citrus flavonoids mainly exist in the form of flavonoid glycosides,which have problems such as poor solubility,low bioavailability and obvious bitterness during application.The structural modification of citrus flavonoid glycosides by glycoside hydrolysis is one of the important methods to solve the above problems.Currently,the hydrolysis of flavonoid glycosides is mainly based on chemical acid hydrolysis and biocatalytic enzymatic hydrolysis,but both of them have some disadvantages.The reaction conditions of acid hydrolysis are harsh,which can easily cause excessive hydrolysis of flavonoid glycosides and produce by-products,reduce the selectivity of the target products and increase the difficulties of subsequent separation and purification.Enzymatic hydrolysis has high selectivity and mild conditions,but the enzyme production process is complicated and costly,which is not conducive to reusability.In view of the above research status,this study explored the use of microbial whole cells to selectively catalyze the hydrolysis of citrus flavonoid glycosides and the characteristics of the hydrolysis in various green solvents such as ionic liquids and natural deep eutectic solvents were also studied.The influence of microbial culture conditions on the hydrolysis reaction of the whole-cell catalyst was further explored,and the mechanism of the Aspergillus niger whole-cell catalytic hydrolysis was revealed through multi-omics analysis.Furthermore,a new route for the stepwise preparation of novel citrus dihydrochalcone sweeteners was established using neohesperidin as a substrate.The main results are as follows:(1)An Aspergillus niger with dual-enzyme activity of α-L-rhamnosidase and β-Dglucosidase was successfully screened from 13 kinds of microorganisms including 4 bacteria,3 yeasts and 6 molds under different culture conditions,and prepared as whole-cell catalyst.The hydrolysis of hesperidin by Aspergillus niger whole cells was investigated using hesperidin as a typical citrus flavonoid glycoside substrate.The reaction time of 24 h,the substrate-tocatalyst mass ratio of 1:2,the reaction temperature of 40℃ and the reaction pH of 5.0 were determined by studying the optimal reaction conditions for hydrolysis of hesperidin.Under these reaction conditions,the substrate conversion can reach over 90%.The hydrolyzates of hesperidin were identified as hesperetin-7-O-glucoside and hesperetin by RP-HPLC and HRMS technologies.The addition of different sugars to the reaction system can adjust the reaction balance,thereby regulating the ratio of the two hydrolysates.The Aspergillus niger whole-cell catalyst had good operational stability and pH stability,were good,which can be applied to scale-up reactions.Additionally,the whole-cell catalyst had a broad substrate spectrum,which can catalyze the hydrolysis of various flavonoid glycosides,such as naringin,neohesperidin and rutin.(2)This study explored the application and effect of green solvents in the catalytic hydrolysis of Aspergillus niger whole cells.The results showed that the solubility of hesperidin in 5[choline][amino acid]ionic liquids with choline hydroxide as cation and glycine,proline,arginine,histidine and serine as anions were higher than that of aqueous solution,and the highest was up to 45.6 times.Both[choline][glycine]and[choline][serine]ionic liquids can be applied to the whole-cell catalytic hydrolysis of hesperidin,and the conversions were 37.5%and 25.3%.To further expand the types of green solvents,dimethyl carbonate,diethyl carbonate,and 2-methyltetrahydrofuran were used in the efficient whole-cell catalytic hydrolysis of hesperidin,and the conversions were 93.2%,90.8%and 76.3%,respectively.Furthermore,28 natural deep eutectic solvents were successfully synthesized,of which choline chloride/ethylene glycol,choline chloride/propanetriol and choline chloride/neopentyl glycol were applied to the reaction systems with the highest substrate conversions of 96.86%,100%and 100%respectively.The excessive deep eutectic solvent(>60%)in the reaction system reduced the efficiency of the whole-cell catalytic hydrolysis,which was mainly due to the conformation and activity of the enzyme requiring sufficient water support.The excessive deep eutectic solvent not only increased the difficulties of substrate transport,but also caused the leakage of nucleic acid and protein,destroyed the structure of enzyme protein and led to significant cell death.(3)This study analyzed the effect of microbial culture conditions on the hydrolysis capacity of Aspergillus niger whole-cell catalyst and the composition of hydrolyzates.The results showed that 1.5 g/L yeast extract and 1.5 g/L Trition X-100 were the preferred nitrogen source and surfactant in the medium,which could increase the biomass of Aspergillus niger from 0.025 to 1.79 g/L,and the substrate conversion from 45.39%to 95.23%.The type of inducer determined the catalytic hydrolysis ability of the whole-cell catalyst.When rutin,naringin,hesperidin and neohesperidin were selected as inducers,the conversions of hesperidin were all higher than 90%.In addition,the percentage of hesperidin-7-O-glucoside was 30.4%when rutin was used as the inducer,and the percentages were above 60%when naringin,hesperidin and neohesperidin were used.The reason can be attributed to the difference in the molecular structure of the inducer that determined the induction and synthesis of different enzymes in the Aspergillus niger whole cells.Further exploration of the culture conditions found that the optimal culture time of Aspergillus niger was 48 h,the culture temperature was 35℃,and the culture pH was 5.0.Moreover,the activities of two enzymes in Aspergillus niger whole-cell catalyst changed similarly in temperature and pH.(4)In order to further reveal the mechanism of catalytic hydrolysis,transcriptomics and proteomics were used to analyze the whole cells of Aspergillus niger induced by naringin,sucrose and cultured without inducer.The results of transcriptomics showed that there were 3 genes related to rhamnosidase,14 genes related to glucosidase,and 5 genes related to hydrolase activity in Aspergillus niger that conformed to differential expression patterns and had the potential to produce α-L-rhamnosidase and β-D-glucosidase.The reason can be attributed to the change in Aspergillus niger metabolic pathway due to the addition of inducer,resulting in the imbalance of hydrolase abundance.Moreover,the up-regulation of related genes also led to an increase in hydrolase activity and affinity,making the related hydrolases more likely to hydrolyze O-glycosyl compounds.The results of proteomics showed that Rha254813 with an(α/α)6 barrel structure and Glu258940 with a(β/α)8 barrel structure in Aspergillus niger cells were α-L-rhamnosidase and β-glucosidase,respectively.Both of them were consistent with the typical structure of glycoside hydrolase family.(5)In this study,a "two-cell stepwise transformation" one-pot route for the synthesis of novel citrus dihydrochalcone sweetener was designed using neohesperidin as a substrate.The results showed that under the optimal transformation conditions,neohesperidin can be biotransformed into neohesperidin dihydrochalcone(NHDC)by Yarrowia lipolytica or Rhodotorula glutinis after alkalization,and the yield can reach 83.49%and 71.09%,respectively.NHDC was hydrolyzed by Aspergillus niger whole-cell catalysis,and the hydrolyzates were identified as hesperetin dihydrochalcone-7-O-glucoside(HDC-G)and hesperetin dihydrochalcone(HDC).The addition time of the whole-cell catalyst can directly affect the hydrolysis process,and the optimal addition time was 72 h.The type of inducer and sugar can regulate the reaction efficiency and the composition ratio of the hydrolyzates,and the application of the buffer system can realize the proportion of HDC reaching 100%.This study also further analyzed the physicochemical properties and application feasibility of the synthesized new sweetener.The results showed that the molecular structure of flavonoid glycosides was closely related to water solubility,and the water solubility of HDC-G was 2.5 times that of NHDC.Moreover,the hydrolyzates had vitamin C protective properties,high sweetness,storage stability,temperature stability,pH stability,and wettability similar to the original substrates,which were expected to become novel sweeteners.The feasibility analysis results indicated that the biotransformation synthesis designed in this experiment was expected to replace the traditional dihydrochalcone sweetener preparation based on chemical hydrogenation synthesis.In summary,this research work had high theoretical and practical values.The obtained results not only expanded the application scope of whole-cell catalysis in the structural modification of natural products,but also promoted a new route for the high-value utilization of citrus fruit and vegetable processing waste and the production of sweeteners derived from natural products.