| Honey is a natural sweet substance produced by bees collecting nectar or secretions and sucking the excreta of honey-source insect on living plant.Bees combined it with their special substances secreted by themselves to transform,deposit,dehydrate,store and remain in the hive until it matures.Honey contains sugar,phenolic compounds,proteins,enzymes and other compounds that are beneficial to health.The production process of honey is a very complicated biological conversion process.The mechanism of converting nectar into honey is not only the starting point of honey production,but also a way to directly reflect its authenticity.Therefore,it is necessary to clarify the substance changes during honey ripening.In this study,high performance liquid chromatography with evaporative light-scattering detection(HPLC-ELSD)was used to study the sugar changes of 6 kinds of honey during the maturation and detect the composition of sugars in rape nectar at different periods of fullbloom stage.And qRT-PCR was used to detect the expression of key genes related to nectar formation in different periods of nectar maturation and fullbloom stage,so as to understand the molecular mechanism of the differences of carbohydrate composition in different periods of fullbloom stage.The main research contents and results of this article are as follows:1.A method was established for analyzing carbohydrates in honey by HPLC-ELSD combining with standard products.The method presents very good repeatability(RSD 0.04-0.144%),precision(RSD 0.013-0.078%)and stability(RSD 0.081-0.159%);The standard recovery rates of high,medium and low concentrations in the six carbohydrates were 105.89-115.37%,96.02-106.09%and 92.14-98.84%.2.Using the established HPLC-ELSD method,the nectar,honey sac,immature honey and honey of Brassica napus and acacia were used as experimental materials to study the changes of sugar during the maturation process.The fructose,glucose,sucrose,maltose,trehalose and unknown peaks(retention time was 24.35 min)were measured by HPLC-ELSD.The total contents of these sugars showed an upward trend during the maturation process.The content of sucrose in acacia nectar was 52.14 g/100g.With the addition of digestive enzymes such as a-glucosidase secreted by bees,sucrose was continuously converted into fructose and glucose,so the sucrose content gradually decreased from 52.14 g/100 g to 1.39 g/100 g,the content of fructose and glucose gradually increased from 18.33 g/100 g to 72.79 g/100 g.The variation trends of Brassica napus honey and acacia honey were consistent.During the honey ripening process,the sugar content is becoming more and more rich.The difference is that the content of fructose and glucose in rape nectar is higher(11.68 g/100g and 11.16 g/100g,respectively),while the content of sucrose is lower(1.2 g/100g).This is due to the biological evolution of plants to adapt to the foraging preferences of the visitors.While the ratio of monosaccharide:sucrose still indicates that sucrose is continuously hydrolyzed into fructose and glucose.The melibiose was also detected during the ripening process of Tilia amurensis,Citrus reticulata,navel orange and Camellia oleifera honey,in addition to the fructose,glucose,sucrose,maltose and trehalose,and its change trend was consistent with that of the acacia honey during ripening process.The above results show that the sugar substances in mature honey are very complex and diverse,and mature honey is of higher quality.3.The diurnal variation of sugar was clarified for the first time in rapeseed nectar by HPLC-ELSD.With the extension of time,the water content of nectar decreased significantly from 92.15%to 37.84%;only fructose and glucose were detected before 14:00,the contents were 6.81 g/100g,15.23 g/100g,16.2 g/100g and 17.45g/100g respectively,and sucrose(1.18±0.15g/100g)was detected in the nectar from 16:00.4.The expression of genes was detected by qRT-PCR which related to the nectar secreted by the nectaries at different flowering periods of Brassica napus.The relative expression levels of BrSBE2-1,BrMYB305 and BrAMY3 genes showed an upward trend and then a downward trend.The peak reached at 16:00,so more raw materials for sucrose synthesis were produced than other time periods.Under the action of enzymes such as BrSUS5 and BrSPS2,starch degradation products were resynthesized into sucrose;And then sucrose was then transported to the cell gap near the opening of the nectary by its carrier BrSWEET9,while the expression level of BrCWINV4 was relatively stable throughout the day.At 16:00,sucrose was transported to the cell gap in large quantities.And BrCWINV4 is unable to hydrolyze a large amount of sucrose into fructose and glucose in time.Then sucrose is secreted to the outside of the nectary through the open pores on the surface of the nectary along with fructose,glucose and water.thereby forming nectar.Therefore,a small amount of sucrose could be detected from the nectar of Brassica napus at 16:00,while it was not detected in other periods.In summary,the main changes of honey are hydrolysis of sucrose to fructose and glucose during ripening.With the continuous processing of bees,the composition of honey is becoming more and more complex.The diurnal variation rule of sugar in rapeseed nectar was clarified for the first time by HPLC-ELSD.Using qRT-PCR technology to analyze the expression of related genes of sucrose secreted by Brassica napus nectaries,so as to preliminarily clarify the inherent molecular mechanism of sucrose detected from Brassica napus nectar around 16:00.This study provides an important reference value for understanding the rules of sugar changes during honey maturation and for improving the molecular level regulation of nectar synthesis and secretion. |
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