Effect Of The Structural Characteristics Of Pectins From Fructus Aurantii On Emulsification Stability

Fructus aurantii,the dried and immature unripe fruit of Citrus aurantium L.,is harvested in from Jiangxi,Sichuan,Hunan and Hubei Provinces,whilst it is known for its ability to regulate the flow of Qi and relieve stagnation and bloating.Fructus aurantii holds a prominent position on the“List of articles that can be used for health food”by the National Health Commission of the People’s Republic of China.It is commonly consumed as a tea in daily life to aid digestion,reduce food retention,and curb appetite.A large number of studies have confirmed that Fructus aurantii contains a variety of bioactive components,such as flavonoids,alkaloids,volatile oils and polysaccharides,which possess activities such as regulating gastrointestinal motility,anti-inflammation,cardioprotection and immunomodulation.The previous studies on Fructus aurantii polysaccharides have primarily focused on monosaccharide composition and linkage,while investigations into the molecular structure,solution conformation and emulsification properties of these polysaccharides remain relatively limited.To the best of our knowledge,there is a lack of publications on the chelating agent-extracted polysaccharides from Fructus aurantii,which hampers the exploration of the“structure-function research”,restricts their application in food industry,and hinders the development of related health food products.Therefore,Fructus aurantii was selected as the raw material,and Fructus aurantii pectins with different structures were obtained by a sequential extraction method involving cold water,hot water,ammonium oxalate,and citric acid.The pectins were then purified using graded alcoholic precipitation.The structural characteristics,solution properties and apparent morphology of Fructus aurantii pectins were analyzed by modern instrumental analysis techniques,and their emulsification properties were compared.The main findings are summarized as follows:（1）The Fructus aurantii water-extracted pectin（FWP）was separated by graded alcoholic precipitation method to obtain the main purified fraction FWP-60.The results showed that FWP-60（M_w,1.20×10⁶ Da）was a homogalacturonan（HG）pectin with66.39%esterification and composed of HG（83.52%）and some rhamnogalacturonan type I（RG-I）pectin.The HG backbone was consisted of→4)-α-Galp A-（1→and→4）-α-Galp A-6-O-Me-(1→in a molar ratio of 1:2.RG-I region was relatively less,while the side chains mainly consisted of arabinan and galactan.The results of the solution parameters showed that the[η]value of FWP-60 was 422.9 m L·g^-1 and the R_h value was 23.3 nm,indicating that FWP-60 was viscous and rigid in 0.1 mol/L sodium nitrate solution.In the rheological characterization,the apparent viscosity of FWP-60 was found to be proportional to the polysaccharide concentration and showed a weak gel characteristic.The SEM results indicated that the apparent morphology of FWP-60 was mainly fine filamentous.（2）The Fructus aurantii hot water-extracted pectin（FHWP）was extracted by using the residue of FWP and separated by graded alcoholic precipitation method to obtain the main purified fraction FHWP-50.FHWP-50 was a highly esterified pectin and the relative weight average molecular weight was 7.81×10⁵ Da.The percentage of esterification and HG domain were 79.10%and 66.09%,respectively.The main chain of FHWP-50 was consisted of→4)-α-Galp A-（1→and→4）-α-Galp A-6-O-Me-(1→in a molar ratio of 1:4.FHWP-50 has a highly branched RG-I structural domain,in which the RG-I main chain accounts for a relatively small proportion of the side chains,including arabinan and galactan side chains.HPSEC-MALLS results showed that the[η]and R_h of FHWP-50 were 182.4 m L·g^-1 and 13.2 nm,respectively,and existed as spherical structure in 0.1 mol/L Na NO₃ solution.The steady-state rheological study showed that FHWP-50 was pseudoplastic fluid and less viscous.In addition,the solid morphology of FHWP-50 showed the presence of small spherical and fine filamentary structures on its surface.（3）A novel Fructus aurantii sourced pectin polysaccharide was isolated from the residue after hot water extraction by using ammonium oxalate solution extraction（FAOP）,with a relative weight average molecular weight of 1.30×10⁶ Da.The methyl esterification of FAOP was 60.36%,and its structure contained about 57.04%HG structural domain,with the main chain consisting of→4)-α-Galp A-（1→and→4）-α-Galp A-6-O-Me-(1→in a molar ratio of 2:3.In addition,the RG-I structural domain was consisted of highly branched arabinan and galactan neutral sugar side chains.The[η]and R_h of FAOP were 355.2 m L·g^-1 and 21.5 nm,respectively,and existed as a rod-like conformation.Steady-state rheological studies showed that FAOP possessed the characteristics of a pseudoplastic fluid and its viscosity increases with increasing concentration.SEM observation revealed that the solid surface of FAOP was composed of fine filaments twisted into a lattice-like structure and adheres to a small number of lamellar structures.（4）An acid-soluble pectin FCAP with a relative weight average molecular weight of 1.05×10⁶ Da was obtained from the residue after ammonium oxalate extraction by using citric acid solution at p H 2.5.The esterification of FCAP was 73.14%and both HG and RG-I domains were present in its structure.The main chain of HG was consisted of→4)-α-Galp A-（1→and→4）-α-Galp A-6-O-Me-(1→in a molar ratio of1:3.The RG-I domain was consisted of 4.18%main chain structure and 31.89%side chain structure,which was mainly composed of 28.67%arabinan and 3.22%galactan.The[η]and R_h of FCAP were 303.1 m L·g^-1 and 21.9 nm,respectively,and existed as globular structure or irregularly curled chain conformation.When the concentration of FCAP was 0.5%,the apparent viscosity of FCAP showed a typical Newtonian fluid with a positive correlation between solution concentration and viscosity.The solid morphology of FCAP was consisted of lamellar,filamentary and spherical structures.（5）The emulsification abilities of FWP-60,FHWP-50,FAOP and FCAP were analyzed by comparing the morphology,particle size,potential and stability of the emulsions.The rule of the influence between structural characteristics of Fructus aurantii pectin and emulsification stability was investigated.The results showed that the emulsions prepared by FWP-60 had better emulsification stability compared with the other three pectins.Comparative analysis of the structures of the four Fructus aurantii pectins revealed that FWP-60 had the highest content of HG structural domain and the lowest content of branched chains of RG-I structural domain,and this structure could reduce the spatial interactions between emulsion droplets.Therefore,the stability of pectin emulsions was mainly influenced by the ratio of HG to RG-I and the length of RG-I side chains.In order to optimize the emulsification ability of FHWP-50,FAOP and FCAP,different concentrations of calcium ions were added to the emulsions.The results showed that the induction of calcium ions could significantly enhance the stability of the emulsions prepared by FAOP,and the best stability of the emulsions was achieved when the calcium ion concentration was 5 m M.The results of laser confocal showed that the addition of calcium ions promoted the formation of the"egg box"structure of FAOP.Comparing the droplet size,zeta potential,rheological properties,distribution pattern and stability of the emulsions prepared from pectin,it was found that the mechanism of calcium ions affecting the stability of the emulsions may be related to the degree of particle aggregation in the emulsions.Therefore,FAOP could be used as a novel alternative to emulsion formulations or stabilizers in food,medical and industrial fields.In summary,Fructus aurantii pectins in the present study were composed of HG pectin and highly branched RG-I pectin.And cold-water extraction was used to prepare pectins containing～80%or more HG structural domains,while hot water,chelating agent and acid extraction were gradually used to obtain pectins more easily with a larger percentage of RG-I structural domains,and RG-I pectins were characterized by a shorter main chain and a large number of arabinan and galactan side chains.The HG and RG-I pectins obtained by the four extraction methods had similarity in the types of structural domains and were all highly esterified pectins.The influence of structural characteristics of the four pectins on their emulsion stability was further investigated,and it was found that FWP-60 pectin could obtain emulsions with best stability.The stabilization mechanism may be related to the larger characteristic viscosity and the ratio of HG to RG-I and shorter RG-I side chains.The RG-I region with short side chains could provide better spatial stability,which made the emulsion droplets keep relatively constant distance from each other.Therefore,the presence of linear HG and a small amount of RG-I structural domains in Fructus aurantii pectin was conducive to the creation of physically stable emulsion.The best emulsion stability was obtained when 5 m M of calcium ions were added to FAOP,which may be due to the fact that the addition of calcium ions affected the degree of particle aggregation in the emulsion.As a result,the addition of calcium ions helps to improve the stability of FAOP emulsions.