Studies On The Entomological Source Authenticity Of Apis Cerana Cerana Honey And Apis Mellifera Ligustica Honey

In China,honey is mainly produced by Apis mellifera ligustica and Apis cerana cerana.The price of A.cerana cerana honey(ACH)is usually three to five times higher than that of A.mellifera ligustica honey(AMH)due to the limitation of production and a growing demand for local and traditional products,which leads to a wide adulteration and counterfeiting of ACH by AMH.In order to curb such fraud,we have studied the authenticity of the source of ACH and AMH.Based on the characteristics of honeybee origined biological differences in honeys,we have established four kinds of ACH and AMH authentication methods system,as well as developed a commercial ELISA detection kit,which could meet the industry’s need to identify the authenticity of ACH and AMH.In addition,we conducted a survey on the authenticity of ACH in the retail market.The specific results of this study are as follows:(1)We compared honeybee secretions in ACH and AMH,and found significant differences in their protein profiles,thus,we developed an SDS-PAGE based method due to the protein pattern from bee secretions.With identification of mixture honey when AMH exceeds 25 percent,our SDS-PAGE method could be used as a tool for ACH and AMH authentication,The SDS-PAGE pattern showed three species-specific bands with molecular weights between 15.0 and 29.4 kDa in ACH,and six species-specific bands in AMH with molecular weights between 13.8 and 33.1 kDa.This difference is stable and unaffected by nectar plants,concentrated processing and shelf life,but only related to the source of honey bees.Further HPLC-MS-MS results showed that the main protein in these bands were MRJPs,or rather,the fragments of MRJPs,and MRJP1,2 and 3 were the most abundant proteins among them.(2)Prokaryotic expression and purification were used to obtain the.Apis mellifera ligustica and Apis cerana.cerana MRJP2 protein.Polyclonal antibodies against the MRJP2 protein of Apis mellifera ligustica and Apis cerana cerana were prepared.Species specific antibodies were screened,the titers of specific MRJP2 antibodies of Apis mellifera ligustica and Apis cerana cerana were higher than 192,000 and 384,000,respectively.A double antibody sandwich method was used to develop a kit for the identification of ACH and AMH.The kit is highly sensitive,which can detect 1%AMH incorporation of ACH in less than 45 minutes.The kit meets the requirements of convenient and quick identification and could be used as a reliable tool for the identification of ACH and AMH.(3)We compared honeybee secretions in these two kinds of honey,and found significant differences in hydrocarbon components.The GC-MS-MS detection of the petroleum ether extracts of the two kinds of honey showed that 17-Pentatriacontene and Hentriacontane were the characteristic constituents of ACH and AMH,respectively.We further confirmed that the characteristic components of these two species are derived from their respective beeswax.We developed a sensitive GC method to detect 17-Pentatriacontene and Hentriacontane in honey,sensitivity of this detection method is very high,AMH could be detected even the proportion was as low as 1 percent.(4)We developed methods to discriminate ACH from AMH based on the MRJP2(major royal jelly protein 2)gene.Two pairs of species-specific primers were designed.The amplification products of A.cerana cerana and A.mellifera ligustica were 212 and 560 bp,respectively.As little as one percent incorporation of AMH honey in the mixture can be detected by duplex PCR.Additionally,another method based on the melt curve analysis using the same primers was also developed,allowing a rapid discrimination of real-time PCR product of different species.(5)A total of 81 commercial ACH samples,including 63 enterprise ACH samples and 18 ACH samples from beekeeper were tested.We used our identification system(SDS-PAGE method and antibody detection kit)to test the authenticity of ACH in Chinese retail market,and 17 samples with unclear SDS-PAGE bands were tested for syrup adulteration.The identification methods showed that Among 63 enterprise ACH samples,only 1 sample was genuine ACH,which suggested the rate of real ACH products in the enterprise samples was only 1.6%(1/63),and the rate of real ACH products was 61%(11/18)for samples from beekeeper.Taken together,the real ACH were only 15%(12/81)from all 81 samples tested.Also,no mixture of ACH and AMH were detected,the adulteration of syrup in the samples were no less than 13.6%(11/81),which indicated that ACH is seriously falsified in the Chinese retail market.