Study On The Differences Of The Proteins In Different Honey

In this thesis, we use acacia honey as a model to build the SDS-PAGE systems of the proteins in the honey, and study the similarities and differences of the proteins in12samples of six kinds of honey from different botanical sources, which include Eurya, loquat, rape, litchi, longan and acacia. And we further researched on the change of protein species and quantities about the incorporation of fructose syrup, doping rape honey to the other five kinds of honey and doping the other five types of honey to Eurya honey. We also use2-DE to study the differences of the proteins in the12samples of six kinds of honey. The results are as follows:1. The results of the total protein content in different honey (12samples:Eurya honey1, Eurya honey2, loquat honey1, loquat honey2, rape honey1, rape honey2, litchi honey1, litchi honey2, longan honey1, longan honey2, acacia honey1and acacia honey2) by Kjeldahl method and precipitation lyophilization show that:(1)The total protein content of samples1-12by Kjeldahl method as follows:0.97%,0.96%,2.20%,2.20%,0.86%,0.86%,2.17%,2.23%,3.50%,3.60%,1.07%and1.13%4The difference was not significant between the same kinds of honey; among different kinds of honey, there are no significant differences between loquat honey and litchi honey, but there are significant differences among other kinds of honey.(2) The total protein content of samples1-12by precipitation lyophilization method as follows:0.19%,0.18%,0.38%,0.29%,0.16%,0.17%,0.29%,0.40%,0.55%,0.i53%,0.18%and0.18%. Between the same kinds of honey, the difference between the two samples of loquat honey and litchi honey is significant, but the differences are no significant between the two samples of the four kinds of honey (Eurya honey, rape honey, longan honey and locust honey); among different kinds of honey, the differences between Eurya honey and acacia honey are no significant, but the differences are significant among other kinds of honey.2. In order to establish the SDS-PAGE system of proteins in the honey, we optimize the SDS-PAGE conditions, including the honey protein extraction methods, sample buffer, ultrasonic time of dissolution and the concentration separation glue. The results showed that the optimum experimental method is the use of sulfuric acid-sodium Tung state precipitation method to extract protein in2g honey, and add200μL sample buffer (100mmol/L,1.0M Tris-HCl(pH6.8),4%SDS,0.2%bromophenol blue,20%glycerol,200mmol/L β-mercaptoethanol), then to ultrasound (ultrasonic power200w) dissolved for10min, the selection of the concentration of12%separating gel, appropriate sample5μL.3. The results of12samples of six kinds of honey by SDS-PAGE gel electrophoresis show that:(1) The number of protein bands between the two samples of the same kinds of honey is basically the same, but slightly different in the protein content. These indicate the good reproducibility between the samples of the same kind of honey. The results also show that the species and contents of the proteins in six kinds of honey are different.(2) The molecular mass of12samples of six kinds of honey is mainly ranged between16kD-90kD. The number of the protein bands is approximately11-17. They contain four kinds of proteins, whose molecular weights are64kD,30kD,25kD,16.5kD, respectively.(3)6kinds of honey have their own main proteins. The number of the main protein species in Eurya honey, loquat honey, rape honey, litchi honey, longan honey and acacia honey is7,6,6,7,8and7, respectively.(4) Compared with the other five kinds of honey, Eurya honey has two specific proteins, whose molecular weight is49kD and32kD. Eurya honey does not contain two main proteins of the other five kinds of honey whose molecular weight is52kD (or53kD) and47kD (or48kD).(5) The content of two proteins (35kD and30kD) in Loquat honey was significantly higher than the content of other five kinds of honey.(6) The content of proteins in rape honey is less than the other five kinds of honey.(7) The molecular weight of proteins in litchi honey is between16.5kD-64kD. Litchi honey does not contain two main proteins in Eurya honey, loquat honey and acacia honey, which molecular weights are85kD and72kD. Compared with the other five kinds of honey, litchi honey has a64kD protein, which content was significantly higher than other honey. This indicates that the64kD protein is a specific protein in litchi honey.(8) Longan honey and litchi honey do not contain the57.5kD protein, and longan honey contains the38kD protein, without the39.5kD protein of litchi honey; longan honey does not contain the21kD protein of other four kinds of honey (loquat honey, rape honey, litchi honey and acacia honey). Thus the21kD protein can be used as a specific protein in the detection of longan honey.(9) The content of21kD protein in sample11, acacia honey, is significantly higher than the other11samples.4. Studies of the different proportions of honey mixed with fructose syrup, different proportions of the rape honey mixed into the other five kinds of honey and the other five kinds of honey in different proportions incorporation into Eurya honey by SDS-PAGE system, indicated that:with the increase of fructose syrup or honey proportion, some protein bands of the honey will be pale or even disappear, while it will show the protein bands of the added honey.5. The studies of proteins in12samples of six honey (Eurya honey, loquat honey, rape honey, litchi honey, longan honey and acacia honey) by2-DE show that:(1) The suitable sample volume is75μg for7cm pH3-10strips, silver staining;(2) The proteins’molecular weights of these honeys distribute in16kD-90kD, and the isoelectric point (pI) are mainly distributed in the3.5-8.5. But the most abundant proteins distribute in the region of40kD-80kD and pI3.5-7.5, which is the main distribution area of the honey proteins;(3) the distribution and abundance of the proteins are differences among the six kinds of honey, and all kinds of honey have their unique protein distribution;(4) Between the two samples of the same kind of honey, the proteins distribution is similar, especially for Eurya honey and longan honey. Therefore; research on the honey proteins by2-DE technology can provide effective information for the establishment of the honey protein database, and also provided technical supports for the identification of honey types and honey adulteration.