Study On Extraction And Activity Of Polysaccharide From Lemon

Lemon is a dicotyledonous plant belonging to the citrus family Rutaceae.The planting quantity of lemon is only lower than that of orange and tangerine,which has the characteristics of huge annual yield.It contains a variety of active ingredients,making lemon in fresh food,natural flavor,medicine,food processing and other fields have an important position.In this paper,the extraction,purification,physicochemical characteristics,antioxidant activity,antibacterial activity and hypoglycemic activity of lemon polysaccharide（LPs）were studied systematically.The results are as follows:Hot reflux extraction（HRE）,ultrasonic extraction（UAE）,microwave extraction（MAE）and complex enzymolysis extraction（EAE）of LPs were used to optimize the optimum extraction conditions by orthogonal test.The optimum conditions of HRE were reflux temperature 80℃,extraction time 180 min and liquid-solid ratio 100 m L/g.Under these conditions,the yield of LP_HR was 4.20%.The optimal extraction process of UAE was as follows:ultrasonic temperature 45℃,ultrasonic time 40 min,liquid-solid ratio 80 m L/g,ultrasonic power 675 W.Under these conditions,the yield of LP_UA was8.82%.The optimum extraction conditions of MAE were as follows:microwave temperature 80℃,microwave time 60 min,liquid-solid ratio 70 m L/g and microwave power 300 W.Under these conditions,the yield of LP_MA was 6.27%.The optimum extraction conditions of EAE were as follows:liquid/solid ratio 70 m L/g,temperature60℃,time 2 h,p H 4.5,pectinase 600 U/g,cellulase 150 U/g,papain 90 U/g.Under these conditions,the extraction rate of LP_EA was 12.70%.On the basis of EAE orthogonal experiment,LPs was extracted by complex enzyme-assisted ultrasonic extraction（UEE）and complex enzyme-assisted microwave extraction（MEE）.BBD test and response surface method（RSM）were used to optimize the optimal extraction conditions.The optimal extraction parameters of UEE were as follows:ultrasonic temperature 40℃,ultrasonic power 375 W,ultrasonic time39 min,liquid to solid ratio 70 m L/g,p H 4.5,compound enzyme dosage 0.5%（w/w）,compound enzyme activity ratio（pectase:cellulase:papain）2:2:1（U/U）,under this condition,the yield of UEE lemon polysaccharide(LP_UE)was 14.41%.The optimum extraction conditions of MEE were as follows:microwave time 35 min,microwave temperature 50℃,liquid/solid ratio 70 m L/g,power 400 W,compound enzyme dosage0.5%（w/w）,activity ratio of compound enzyme（pectinase:cellulase:papain）2:2:1（U/U）,under these conditions,the yield of MEE lemon polysaccharide(LP_ME)was13.46%.Compared with the other four extraction methods,UEE method and MEE method had the advantages of higher yield and faster extraction rate.Therefore,LP_UEand LP_ME extracted by UEE and MEE methods were finally selected for subsequent separation and purification,and the homogeneous polysaccharide components LP_UE-1,LP_UE-2,LP_ME-1 and LP_ME-2 were obtained.The physicochemical characteristics of LP_HR,LP_EA,LP_UA,LP_UE,LP_MA,LP_ME,LP_UE-1,LP_UE-2,LP_ME-1 and LP_ME-2 were tested as follows:The total sugar content was 80.63%-87.79%,LP_UA was the highest（87.79%±3.24%）in crude polysaccharide,the lowest（80.63%±1.95%）in LP_UE,and the highest（84.19%±1.29%）in purified polysaccharide.LP_ME-1 had the lowest total sugar content（82.38%±3.20%）.Protein content was 0.93%-2.32%,crude polysaccharide had the highest protein content of LP_HR（2.32%±0.22%）,LP_ME had the lowest protein content of 1.88%±0.16%,and purified polysaccharide had the highest protein content of LP_UE-1（1.43%±0.09%）.The protein content of LP_ME-2 was the lowest（0.93%±0.04%）,and there was still a small amount of protein in the purified polysaccharide,indicating that the polysaccharide and protein formed glycoprotein complex.The content of uronic acid was 5.75%-31.89%,the content of uronic acid was the highest in LP_ME（31.89%±2.21%）and the lowest in LP_UA（18.71%±1.04%）.The content of uronic acid in purified polysaccharides was LP_ME-1>LP_UE-1>LP_ME-2>LP_UE-2;LP_ME-1 and LP_UE-1 were acidic polysaccharides.Total flavonoid content was 0.45%-1.46%,total phenolic content was 0.16%-0.42%,total flavonoid content of LP_ME was the highest（1.46%±0.13%）,polyphenol content of LP_ME was the highest（0.42%±0.04%）,and total flavonoid content of LP_EA was the lowest（0.93%±0.08%）.The content of polyphenols in LP_MA was the lowest（0.16%±0.04%）,and the content of total flavonoids in LP_MA was LP_ME-1>LP_UE-1>LP_UE-2>LP_ME-2,total polyphenol content was LP_ME-1>LP_ME-2>LP_UE-1>LP_UE-2;LP_UE-2 showed that a small amount of total flavonoids and polyphenols formed complex with polysaccharide.The solubility of LP_UE-1 was up to 98.29%±1.55%.All the polysaccharide components had good solubility,and the lower the molecular weight,the higher the solubility.The average molecular weights of LP_HR,LP_EA,LP_UA,LP_UE,LP_MA and LP_ME were 180.28,97.96,132.07,52.93,148.86,51.93 k Da;The average molecular weights of LP_UE-1,LP_UE-2,LP_ME-1 and LP_ME-2 were 5.80,43.27,7.26 and 48.79 k Da.LP_UE-1 was composed of glucuronic acid（Glc A）,galacturonic acid（Gal A）,glucose（Glc）,galactose（Gal）and xylose（Xyl）,and its molar ratio was 19.4:23.9:5.3:23.1:26.2;LP_UE-2 was composed of rhamnose（Rha）,Glc A,Gal A,Glc,Gal and Xyl with molar ratio of 3.1:1.5:35.5:10.9:13.2:36.2.LP_ME-1 is composed of Rha,Glc A,Gal A,Glc,Xyl and Ara with molar ratio of 0.2:2.1:35.6:39.7:10.2:9.7.LP_ME-2 was composed of Rha,Glc A,Gal A,Glc and Gal,and the molar ratio was 1.9:3.7:43.2:31.8:15.2,indicating that the monosaccharide composition of polysaccharides extracted by different extraction methods had no significant difference,which mainly affected the molar ratio..Infrared spectrum analysis（FT-IR）:Four kinds of purified LPs located near3432-3415 cm^-1 all contained strong absorption peaks,which were tensile vibration of hydroxyl group（O─H）or N─H.The alkyl C─H tensile vibration is located near2926-2922 cm^-1,which is the characteristic absorption peak of polysaccharide.It is C─H asymmetric tensile vibration near 2860-2850 cm^-1.The absorption peaks near1735-1730 cm^-1 indicated that all four LPs contained a certain amount of methylated uronic acid.Near 1637-1617 cm^-1 the characteristic absorption peak of the C═O asymmetric stretching vibration of the─COO,showed that carbonyl in LPs,both contain uronic acid.The absorption peak at 1636 cm^-1 may also be due to the presence of the amino band of the protein.The characteristic absorption peak near 1420-1384cm^-1 was due to the contraction vibration of C─O in─COOH,and the absorption peak at 1420 cm^-1 indicated galacturonic acid in LPs.Near 1384-1380 cm^-1for the C═O symmetric stretching vibration of─COO;Near 1260-1254 cm^-1 for C─O─C stretching vibration absorption peak,moreover also may be due to the S═O the tensile deformation vibration;Three strong absorption peaks in 1200-1010 cm^-1 were pyranoside;The characteristic absorption peak in the fingerprint region was 950-750cm^-1,and the strong absorption peak around 780 cm^-1 was inferred to be the asymmetric contraction of the C─O─C skeleton vibration of glucopyranose ring.The absorption peak at about 840 cm^-1 was the sign ofα-heterotropic isomer,suggesting thatα-D-glucopyranoid was the main component of polysaccharide.Absorption peaks around 951 cm^-1 reveal the presence of furan rings.The peaks in the range of 750-500cm^-1 may be the O─H surface bending and vibration of alcohol or phenol,FT-IR showed that none of the four LPs produced new structural peaks,and no new functional groups were produced in different extraction methods and separation and purification processes.The protein,uronic acid,polyphenol and other components determined in the composition analysis were reflected in FT-IR.CD analysis showed that the polysaccharides existed orderly in the form of irregular curl in aqueous solution,and the asymmetry was strong.TG tests showed that all samples had similar TG-DTG curves,and LP_UE-2 had the highest thermal stability.The shorter the side chain,the more uniform the distribution and the more difficult the decomposition.These small thermal stability differences may be caused by different chemical compositions and structures produced by different extraction methods.The triple helix structure of LPs samples was analyzed by the fruit red test,and it was found that LP_UE-1 and LP_ME-1 had triple helix structure,while LP_UE-2 and LP_ME-2 did not.This phenomenon may be due to the destruction of intramolecular and intermolecular hydrogen bonds of LPs and the change of triple helix structure by long-term heat,microwave or ultrasonic treatment.XRD tests showed that the four LPs had three clear and sharp narrow diffraction peaks,indicating that LPs was a semi-crystalline polymer with low crystallinity.In vitro antioxidant evaluation was conducted by 1,1-diphenyl-2-picrohydrazine（DPPH·）,superoxide anion radical（·O₂^-）,2,2-diazo-di（3-ethyl-benzothiazol-6-sulfon-ic acid）diamiammonium salt（ABTS⁺·）and hydroxyl radical（·OH）scavenging experiments.The test results showed that all polysaccharides had high antioxidant activity.It is dose-dependent on the concentration.When the concentration reached 5.0mg/m L,the DPPH·clearance rate of LP_ME was the highest（71.87%）,while the DPPH·clearance rate of LP_HR was the lowest（66.63%）.When the concentration reached 5.0 mg/m L,the clearance rates of LP_UE-1,LP_UE-2,LP_ME-1 and LP_ME-2 to DPPH·were 69.99%,64.16%,64.43%and 60.16%,respectively.When the concentration reached 5.0 mg/m L,the·OH clearance rate of LP_ME was the highest（79.57%）,while the·OH clearance rate of LP_HR was the lowest（59.46%）.When the concentration reached 5.0 mg/m L,the clearance rates of LP_UE-1,LP_UE-2,LP_ME-1 and LP_ME-2 to·OH were 66.76%,58.97%,72.18%and 60.74%,respectively.When the concentration reached 5.0 mg/m L,the clearance rate of·O₂^-by LP_ME was the highest（93.89%）,and the clearance rate of·O₂^-by LP_MA was the lowest（85.36%）.When the concentration reached 5.0 mg/m L,the clearance rates of LP_UE-1,LP_UE-2,LP_ME-1 and LP_ME-2 to·O₂^-were 83.22%,9.23%,42.48%and 31.16%,respectively.When the concentration reached 5.0 mg/m L,the ABTS⁺·clearance rate of LP_UE was the highest（56.74%）,and the ABTS⁺·clearance rate of LP_HR was the lowest（42.25%）.When the concentration reached 5.0 mg/m L,the clearance rates of LP_UE-1,LP_UE-2,LP_ME-1 and LP_ME-2 to ABTS⁺·were 40.37%,36.81%,38.97%and 7.23%,respectively.Data analysis and comparison showed that the free radical scavenging ability of LP_UE and LP_ME was stronger,indicating that microwave and ultrasonic treatment degraded polysaccharide,and the natural hydrogen bond of polysaccharide was broken.After degradation,the specific surface area of polysaccharide in contact with free radicals increased,which was able to provide hydrogen atoms better.Therefore,the antioxidant activities of LP_UE extracted by UEE and LP_MEextracted by MEE were better.The addition of complex enzymes also further reduced the molecular weight of polysaccharides,increased the structure of the branch chain,increased the number of reduced ends,and increased the side chain groups,which contributed to the improvement of the activity.The activity of the four LPs after separation and purification was reduced to some extent,which may be due to the reduction of the contents of polyphenols,flavonoids,proteins,uronic acid and other components,and the weakening of the synergistic effect,and finally led to the decrease of the activity.The results showed that the complex of polysaccharide and other components could effectively improve the antioxidant activity of polysaccharide.Inhibition ofα-glucosidase andα-amylase by LPs showed that LP_UE-1,LP_UE-2,LP_ME-1 and LP_ME-2 showed a dose-dependent inhibitory effect onα-glucosidase andα-amylase.At the concentration of 2.0 mg/m L,The inhibition rates of LP_UE-1,LP_UE-2,LP_ME-1 and LP_ME-2 onα-glucosidase were 87.82%,90.61%,94.63%and 96.60%,respectively.At the concentration of 2.0 mg/m L,the inhibition rates of LP_UE-1,LP_UE-2,LP_ME-1 and LP_ME-2 onα-amylase were 86.71%,79.75%,92.66%and 91.28%,respectively,indicating that LPs could be used asα-glucosidase inhibitor andα-amylase inhibitor in functional foods.In the test of bacteriostatic ability,LPs had a good inhibitory ability to Staphylococcus aureus and a certain inhibitory ability to Escherichia coli,and all LPs had no inhibitory ability to Candida albicans.