| Oligomeric proanthocyanidins are polyphenolic compounds widely existing in many plants,it can be divided into A-type and B-type.B-type proanthocyanidins are formed by carbon-carbon bond between monomers,while A-type proanthocyanidins are linked by both carbon-carbon bond and ether oxygen double bond and rare in nature.Unlike B-type proanthocyanidins,A-type proanthocyanidins have strong antioxidant activity,anti-inflammatory activity and cardiovascular protection and other physiological effects,but also have a unique inhibition of Escherichia coli and other bacteria adhesion to urinary tract cell,which can prevent and treat urinary tract infection.A-type proanthocyanidins from food sources are mainly found in cranberry and its products,while cranberry is only produced in North America,Northern Europe and parts of Da Hinggan Ling Prefecture of China,with limited in yield and high price.Therefore,it is a scientific problem with theoretical value and promising application prospect to transform anthocyanins from Lonicera caerulea berry and other raw materials into A-type proanthocyanidins by using anthocyanins widely existing in berries though precise regulation of processing technology and parameters.In this paper,anthocyanins and epicatechin were transformed into A-type proanthocyanidins dimers in the simulation system,the reaction conditions,reaction mechanism and physiological effects were explored,the transformation method were established and optimized,and the results were applied to the processing of Lonicera caerulea berries,which provide new methods and ideas for the high-value utilization of berries rich in anthocyanins.The main results are as follows:(1)To prepare various A-type proanthocyanidins with different structures and establish detection methods.Since there is no commercial standard of A-type anthocyanin epicatechin,five A-type anthocyanin-epicatechin dimers were synthesized from anthocyanin and epicatechin by nucleophilic addition reaction.These A-type anthocyanins-epicatechin dimers were separated and pured by Sephadex LH-20 gel column and semi-preparative liquid phase.The structure and purity of A-type anthocyanins-epicatechin dimers were identified by MALDI-TOF MS.The products were A-type Cy-Ec,Dpd-Ec,Cy-3glc-Ec,Cy-3gal-Ec and Pt-3rut(pC)-5glc-Ec,and the purity of standard products were more than 95%.An UPLC-MS/MS method was developed for the simultaneous qualitative and quantitative detection of five A-type proanthocyanidins dimers in the mixed system.Under the optimal detection conditions,the detection limit was 0.0265–0.0571 mg/L,the recovery rate was 85.32%–93.43%,and the precision was 8.74%–12.67%.This method has the advantages of simple pretreatment,short detection time,and the recovery rate and precision can meet the analysis requirements.Among the five common commercial berry concentrated juice,only cranberry concentrated juice contains A-type proanthocyanidins dimer,PAC-A2.(2)The key factors of A-type procyanidins transformation under simulated conditions and the determination and optimization of control conditions and its reaction mechanism were studied.The components of anthocyanins in four kinds of berry extracts were identified,the reaction substrates of the simulation system were determined,and the simplified reaction system of A-type proanthocyanidins was established.Through the screening of O2,pH value,reaction temperature,treatment time,substrate concentration and other factors,identificaiton of the existence of target components in the products by HPLC-MS,and reaction conditions of the production of A-type proanthocyanidins in the simulation system were optimized.The optimal reaction conditions were obtained.The results showed that blueberry extract reacted with epicatechin to form A-type Cy-EC and Dpd-EC,which were formed by direct condensation of two deglycoside products cyanidin(cyanidin and delphinin)and epicatechin;A-type Cy-3glc-Ec?Cy-3gal-Ec and Pt-3rut(pC)-5glc-Ec were detected in the reaction of Lonicera caerulea berry,black chokeberry,and Lycium ruthenicum Murray extract with epicatechin,and formed by direct nucleophilic addition of anthocyanins and epicatechins,respectively.(3)Preparation of concentrated juice of Lonicera caerulea berry with high A-type proanthocyanidins content.By optimizing the epicatechin concentration,pH value,concentration pressure,selecting the temperature lower than the simulation system and prolonging the concentration time,and monitoring the changes of A-type proanthocyanidins,anthocyanins,polyphenols,5-HMF and browning index,the high content of A-type proanthocyanidins of Lonicera caerulea berry juice was prepared.The optimal preparation conditions were as follows:epicatechin 0.66 g/L,pH 2.8–3.1,vacuum degrees 200 mbar,the heating temperature 80?and the heating time 100 min.The production of A-type proanthocyanidins reached the level of laboratory technology after the scale of preparation experiment was expanded 20 times.The content of A-type anthocyanin epicatechin dimer in high A-type procyanidins concentrated juice was 620.85±33.09 mg/L,which was significantly higher than that in commercially available concentrated juice.There was no significant difference between the content of A-type proanthocyanidin in high A-type procyanidins Lonicera caerulea berry concentrated juice and that in cranberry concentrated juice,but the content of total proanthocyanidins was lower than that in commercially available cranberry concentrated juice.The contents of total anthocyanins,total phenols,total flavonoids,ascorbic acid,iron reduction capacity and oxidation free radical absorption capacity of high A-type procyanidin Lonicera caerulea berry concentrated juice were higher than those of cranberry concentrated juice.(4)The efficacy of A-type anthocyanin-epicatechin dimers and Lonicera caerulea berry concentrated juice with high content of A-type proanthocyanidins on prevention of urinary tract infection in vitro.Compared with the reaction substrates(anthocyanins and epicatechins),in the low concentration range(25,50,100,250 and 500?mol/L)without affecting the growth of E.Coli,A-type Cy-3glc-Ec?Cy-3gal-Ec and Pt-3rut(pC)-5glc-Ec significately inhibited the formation of E.coli biofilm,destroyed the mature E.coli biofilm,inhibited the adhesion of E.coli to bladder epithelial cells,and there was no significant difference with PAC-A2.Moreover,A-type Cy-3glc-Ec?Cy-3gal-Ec and Pt-3rut(pC)-5glc-Ec significantly reduced the hydrophobicity of E.coli surface,inhibited the secretion of IL-8 and the induction activity of NF-?B p65 in bladder epithelial cells stimulated by E.coli.At the same time,the ability of prevention of urinary tract infection of the Lonicera caerulea berry concentrated juice with high A-type proanthocyanidins content was significantly higher than that of the commercially available Lonicera caerulea berry concentrated juice,and there was no significant difference between the Lonicera caerulea berry concentrated juice and cranberry concentrated juice.These results indicated that three A-type anthocyanin-epicatechin dimers and high A-type procyanidin content of Lonicera caerulea berry concentrated juice showed good potential to prevent urinary tract infection.In conclusion,the results of this study provide a new idea for the efficient transformation of A-type proanthocyanidins,and provide a theoretical basis for its application in food;the development of concentrated juice products rich in A-type proanthocyanidins from Lonicera caerulea berry juice provides a new idea for the comprehensive processing of fruits and vegetables and the development of functional food. |
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