| Objective:Taking honey from different sources as the research object,the antibiotic residue,the content of total flavonoids and flavonoid components and antibacterial property of honey were investigated for exploring the quality evaluation method of honey.Methods:（1）.The honey samples were treated with rapid sample pretreatment technique（Qu ECh ERS method）,then the residues of 49 antibiotics in honey were analyzed by UPLC-MS/MS method.（2）.The standard curve was established by UV-Vis spectrophotometry with quercetin as control to determine the content of total flavonoids in honey from different honey sources;（3）.The flavonoids in honey were purified by D101macroporous adsorption resin column,and 12 flavonoids in honey from different honey sources were analyzed by UHPLC-MS/MS at multiple reaction monitorings（MRM）mode.（4）.Honey without antibiotic residues was selected as the sample,and the antibacterial properties of different sources and 81%sugar solution against six strains were analyzed by plate method and double dilution method under different mass fractions.Finally,the correlation analysis among the content of total flavonoids,flavonoid components and the antibacterial property of honey was preliminarily studied.Results:（1）.The mass concentration of 49 antibiotics in the range of LOQ～200 LOQ(μg·kg-1)had a good linear relationship,the correlation coefficient（r）of 49 antibiotics were all greater than 0.99.The matrix effect（n=6）of each compound ranged from11.8%to 79.6%.Although the matrix interference was large and reduced the detection limit and quantitative limit of the instrument,it could basically meet the requirements of the national standard.The recovery rates were 73.5%～124.7%,except for erythromycin A,which was 49.1%when the concentration was 11.0μg·kg-1.The reproducibility were good,and the RSDs were as follows:low concentration 1.7%～13.5%,medium concentration 0.8%～17.0%,high concentration 0.6%～12.2%.49 compounds were stable within 24 h.Among the 25 batches of honey,7 batches of antibiotic residues were detected.The main residual antibiotics were:salafloxacin,trimethoprimethamine,norfloxacin,trimethoprimethamine,enroxacin,ofloxacin,difloxacin and chloramphenicol.Norfloxacin and ofloxacin were detected with the highest frequency.The residue of antibiotics in one batch exceeded the standard,and the content of chloramphenicol in one sample was as high as 168.0μg·kg-1.Except for the banned components of chloramphenicol and sulfamethoxil were detected in sample 19,the rest were all within the limit range and met the national standard,with a failure rate of 4%.（2）.When UV method was used to measure the total flavonoids,the presence of sugar had a great influenceon the total flavonoids content.For example,the detection result of the total flavonoids content in 81%sugar solution was 47.99μg·g-1,the matrix effect（n=6）was 56.1%,and the recovery was 84.4%.Through the analysis of the total flavonoid content of honey from different honey sources,it was found that the total flavonoid content of honey from different honey sources varied greatly,among which Leonurus content was the highest,followed by codonopsis and Lycium honey,acacia honey was generally low.The results showed that the adulteration of honey could be preliminarily identified by the detection results of the total flavonoids content.The total flavonoids content of honey from the same honey source had a relatively fixed interval value,and it was likely to be adulterated when it was less than 45μg·g-1（Approximately 75%sugar）,or be fake honey when it was even lower.（3）.The mass concentration of catechin,epicatechin,narirutin,rutin,morin,myricetin,naringenin,quercetin,genistein,luteolin,apigenin,wogonin,pinocembrin,chrysin and galangin in the LOQ～10-250 LOQ range had a good linear relationship,and the correlation coefficient（r）were all greater than 0.99.The recovery rates were 70.6%,59.3%,83.0%,87.8%,83.4%,81.1%,93.3%,89.9%,86.1%,95.9%,89.8%and 93.3%,respectively.The matrix effect（n=5）ranged from 94.3%to 115.7%.Allthe compounds werestable for 24 hours.Although the types of flavonoid components were similar among different honey sources,but the content distribution was different For example,Locust honey was rich in chrysin and quercetin,followed by pinocembrin and luteolin,with a small amount of naringenin,kaempferol and apigenin;citrus honey was rich in quercetin and kaempferol,supplemented by naringenin,pinocembrin and chrysin,with a small amount of luteolin,apigenin and galangin;canola flower honey was rich in kaempferol and quercetin,with a small amount of chrysin,terpinein,naringenin and genistein.（4）.The antibacterial activity of different honey sources on 6 pathogenic bacteria including Escherichia coli,Staphylococcus aureus,Salmonella paratyphi B,Pseudomonas aeruginosa,Candida albicans and Aspergillus nigerwere investigated,and it was found that:（1）Antimicrobial activity had a great degree of consistency in different nectar sources of honey against different strains,and the results could be used to evaluate the quality of honey.（2）Escherichia coli,Candida albicans and Aspergillus niger had the characteristic of high osmotic pressure tolerance which could be used to distinguish whether the fake honey was mixed with high concentration of syrup.But whether it couldbe used to effectively distinguished the specific amount of mixing neededtobefurthertested.（3）Staphylococcusaureus,Pseudomonas aeruginosa,Salmonella paratyphi B were sensitive to hyperosmotic.Salmonella paratyphi Bcould distinguish the true from the fake by its bactericidal effect in the case of 1 time dilution,other pathogenic bacteria could not distinguish the true from the fake at the dilution of 1 time,but it could reflect the quality of honey from different nectarsources by further dilution.（4）Due to the complex and changeable antibacterial mechanism of honey and the different sensitivity to different antibacterial factors,the antibacterial property test of a single strain might not be able to effectively distinguish the quality of honey.It was necessary to comprehensively analyze the antibacterial property of two or more strains.（5）The results of antibacterial activity of honey could be used to distinguish the quality differences of honey from the same source.Conclusion:Through the comprehensive analysis of 49 kinds of antibiotic residues,the determination of total flavonoids,different flavonoid constituents and antibacterial research in honeys,we found that the combination of the four methods could be used for the preliminary identification of true or fake honey and honey source,and the evaluation of honey quality grade from the aspects of safety and effectiveness.Based on the initial screening of antibiotic residues,the analysis of flavonoids in honey by UHPLC-MS/MS could be used to identify the source of honey and evaluate the quality of honey combined with the determination results of total flavonoids.The total flavonoids and flavonoid components of honey from the same nectar source were similar.Therefore,we can judge the true,false and inferior condition of honey by the content of total flavonoids or flavonoid components and the antibacterial activity of honey from the same nectar source. |
