Discrimination of the entomological origin of honey according to the secretions of the bee (Apis cerana or Apis mellifera)

Accepted Manuscript Discrimination of the entomological origin of honey according to the secretions of the bee (Apis cerana or Apis mellifera)

Yan-Zheng Zhang, Yi-Fan Chen, Yu-Qi Wu, Juan-Juan Si, CuiPing Zhang, Huo-Qing Zheng, Fu-Liang Hu PII: DOI: Reference:

S0963-9969(18)30675-6 doi:10.1016/j.foodres.2018.08.049 FRIN 7857

To appear in:

Food Research International

Received date: Revised date: Accepted date:

13 June 2018 14 August 2018 18 August 2018

Please cite this article as: Yan-Zheng Zhang, Yi-Fan Chen, Yu-Qi Wu, Juan-Juan Si, CuiPing Zhang, Huo-Qing Zheng, Fu-Liang Hu , Discrimination of the entomological origin of honey according to the secretions of the bee (Apis cerana or Apis mellifera). Frin (2018), doi:10.1016/j.foodres.2018.08.049

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ACCEPTED MANUSCRIPT Discrimination of the entomological origin of honey according to the secretions of the bee (Apis cerana or Apis mellifera) Yan-Zheng Zhang, Yi-Fan Chen, Yu-Qi Wu, Juan-Juan Si, Cui-Ping Zhang, Huo-Qing Zheng* and Fu-Liang Hu*

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(College of Animal Science, Zhejiang University, Hangzhou 310058, China)

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AUTHOR INFORMATION

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Corresponding Author

*(Fu-Liang Hu) Telephone/Fax: +86-27-88982952. E- mail: [email protected]. Address:

Zheng)

Telephone/Fax:

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*(Huo-Qing

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No. 866, yuhangtang road, xihu district, Hangzhou, China.

+86-0571-88982840.

E-mail:

[email protected]. Address: No. 866, yuhangtang road, xihu district, Hangzhou,

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China.

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ORCID

Fu-Liang Hu: 0000-0002-2967-8777

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Huo-Qing Zheng: 0000-0001-8499-0694

ACCEPTED MANUSCRIPT ABSTRACT The eastern honeybee Apis cerana and the western honeybee Apis mellifera are the two most economically valuable honeybee species used in apiculture. In market, the price of Apis cerana honey (ACH) is usually several times higher than that of Apis

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mellifera honey (AMH) due to the production limit, resulting in wide adulteration and

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counterfeiting of ACH by AMH. In the present study, we compared honeybee

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secretions in these two kinds of honey, and found significant differences in protein profiles and hydrocarbon components. The SDS-PAGE pattern showed three

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species-specific bands with molecular weights between 15.0 and 29.4 KDa in ACH,

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and six species-specific bands in AMH with molecular weights between 13.8 and 33.1 KDa. The GC-MS-MS detection of the petroleum ether extracts of the two kinds of

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honey showed that 17-Pentatriacontene and Hentriacontane were the characteristic

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constituents of ACH and AMH, respectively. These two methods constitute a system to satisfy different needs for entomological authentication of honey samples.

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KEYWORDS: honey, Apis cerana, Apis mellifera, discrimination, honeybee secretions, MRJPs, beeswax compositions

ACCEPTED MANUSCRIPT 1. INTRODUCTION Honey is a sweet substance produced by bees that collect nectar from plants and deposit it into a honeycomb after digestion (National Honey Board, 2003). It is a widely consumed natural product, not only desirable for its taste and nutritional value,

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but also for its health benefits. The substances in honey mainly come from the plants

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and the honeybee secretions (Sak-Bosnar & Sakac, 2012; Escuredo, Miguez,

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Fernandez-Gonzalez & Carmen Seijo, 2013; Da Silva, Gauche, Gonzaga, Costa & Fett, 2016). These botanical and entomological origin have great influence on the

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composition and price of honey.

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The eastern honeybee Apis cerana and the western honeybee Apis mellifera are the two most economically valuable honeybee species used in apiculture (Soares et al.,

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2018). Since A. mellifera is much more productive and more suitable for modern

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breeding, it has been introduced into much of the habitats of A. cerana, such as China, Japan, South Korea, the Himalayas and Vietnam (Partap & Verma, 1998; Verma,

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1990). In some regions of Asia, such as China, Japan and South Korea, there are extent competition between the two species, resulting in the decline of population of A. cerana (Won, Li, Kim, & Rhee, 2009; He et al., 2013). For example, 7 million A. mellifera colonies but only 2 million A. cerana colonies are raised in China. It is estimated that the annual production of honey exceeds 400,000 tons in China and 90 per cent are produced by A. mellifera. The price of A. cerana honey (ACH) is usually three to five times higher than that of A. mellifera honey (AMH) due to the limitation of production and a growing demand for local and traditional products, which leads to

ACCEPTED MANUSCRIPT a wide adulteration and counterfeiting of ACH by AMH (Won, Li, Kim, & Rhee, 2009; Gao, & Zhao, 2015). Thus, there is an urgent need to establish methods that can determine the entomological origin of honey produced by A. cerana to protect the interests of consumers and honest beekeepers.

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In order to identify honey produced by different bee species, the substance of bee

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origin is the key for authentication. Substance of bee origin in honey include DN A

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and secretions of bees. DNA-based method has been successfully used to determine the botanical and entomological origins of honey (Prosser & Hebert, 2017; Kek, Chin,

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Tan, Yusof & Chua, 2017; Kim, Lee & Choi, 2017; Soares et al., 2018). The

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secretions of bees in honey are the endogenous substance of honey. It is another suitable marker for the entomological origins identification of honey. The MRJPs

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(Major Royal Jelly Proteins) and digestive enzymes are the major proteins secreted

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into honey by worker bees. Most of the digestive enzymes are instable in honey. Their activity decreased quickly due to heating and prolonged storage (Ahmed et al., 2013;

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Li et al., 2017), so they are not suitable markers to distinguish ACH from AMH. The MRJPs is a group of homologous proteins consisting of nine members, namely MRJP1-9 (Albert & Klaudiny, 2004; Drapeau, Albert, Kucharski, Prusko & Maleszka, 2006). As the main proteins in royal jelly (Tamura et al., 2009), MRJPs also consist of the main proteins in honey with pollen proteins (Sera, Deugchan, Seukhyun, Jangwon & Haeik, 2008; Di Girolamo, D'Amato & Righetti, 2012; Chua, Lee & Chan, 2015). The MRJPs of different bee species are specific. Sera et al. (2008) found that the MRJP1 in A. cerana honey and in A. mellifera honey have different molecular

ACCEPTED MANUSCRIPT weights and surface structures, based on this difference they successfully identified the entomological origin of these two kinds of honey. SDS-PAGE is a widely used technique to separate and identify proteins. In previous studies, SDS-PAGE has been commonly used to identify the botanical and

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geographical authenticity of honey (Mohammed & Babiker, 2009; Nisbet, Guler,

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Ciftci & Yarim, 2009; Deng et al., 2012; Mohammed & Azim, 2012; Can, Ebru Çakır,

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Şirin & Kolayli, 2017), and has also been used in some studies to identify the entomological origin of honey (Deug-Chan, Sang- Young, Sang-Hoon, Yong-Soon &

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Hae-Ik, 1998; Ramónsierra, Ruizruiz & De, 2015). Ramónsierra et al. (2015) used

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SDS-PAGE to analyze the protein profiles of Melipona beecheii honey, Trigona spp honey and A. mellifera honey. SDS-PAGE patterns showed distinctive bands for each

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kind of honey, indicating that electrophoresis characterisation of protein could be used

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as a method to establish the entomological origin of stingless bee honeys. Another bee secretion that may be used to identify the entomological origin of

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honey is beeswax. Beeswax is secreted by wax glands of honeybee workers and is used to build honeycomb (Fratini, Cilia, Turchi & Felicioli, 2016). It is a complex mixture of hydrocarbons, free fatty acids, esters of fatty acids and fatty alcohol, diesters and exogenous substances (Tulloch, 1970; Tulloch, 1980). Xu et al. (1989) found that there were differences in the content and composition of hydrocarbons, monoesters and free acids between the beeswax of A. cerana cerana and A. mellifera ligustica. In honeybee colony, honey is stored in the comb, thus, tiny beeswax fragments are inevitably existing in honey due to the behavior of honeybee and the

ACCEPTED MANUSCRIPT process of harvesting honey. Zuccato et al. (2017) have analyzed the chloroform extracts spectra of Geotrigona-Trigona, Melipona, Scaptotrigona, and A. mellifera Ecuadorian honeys using 1 H NMR coupled with chemometrics. They achieved a successful entomological discrimination between these honeys, which showed some

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diagnostic entomological markers are derived from beeswax.

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In this study, we hypothesized that differences between A. cerana and A. mellifera

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secretions may be used to identify their respective honey. We compared protein profiles and hydrocarbon compositions in ACH and AMH. Our goal is to establish a

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method system for the discrimination of honey produced by A. cerana and A.

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mellifera. 2. MATERIALS AND METHODS

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2.1. Chemicals

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Nonacosane (analytical standard, PubChem CID:11903), Hentriacontane (analytical standard, PubChem CID: 12410), 1-Triacontanol (analytical standard, PubChem

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CID:62194) and 17-Pentatriacontene (analytical standard, PubChem CID:5365022 ) were purchased from Sigma-Aldrich (Shanghai, China). Petroleum ether 60-90℃ (chromatographically purity) was purchased from National Pharmaceutical Chemical Reagent Co., Ltd. 2.2. Honey and beeswax samples ACH (A. cerana honey) samples were harvested directly by beekeepers from China (n=34), Vietnam (n=3). AMH (A. mellifera honey) samples were harvested directly by beekeepers from China (n=37), Australia (n=3), Brazil (n=3), South Africa (n=2). All

ACCEPTED MANUSCRIPT honey samples were extracted from the colonies that have made their own waxes. 12 ACW (A. cerana beeswax) samples and 18 AMW (A. mellifera beeswax) samples were collected from the the same colony that the honey samples been collected. 8 ACW (A. cerana beeswax) samples and 8 AMW (A. mellifera beeswax) samples were

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collected from the experimental apiary of Zhejiang University. To avoid the

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contamination from comb foundation, we only cut a small piece of wax from the

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surface of a comb without touching the foundation. The collection time of all the samples were in 2016-2018. All honey and beeswax samples were stored in glass

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bottles at 4℃ until analyzed.

2.3.1. Honey protein extraction

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2.3. Honey protein analysis

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Honey protein was extracted by cool ethanol. Pollen protein is the main source of

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protein in honey, but it can not be used to determine the entomological origin of honey. In order to better analyze proteins from bees in honey, we removed pollen. A

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diluted honey solution (10 g in 10 mL distilled water) was centrifuged at 8000×g for 10 min at 4℃ in order to remove pollen and other impurities. 20 mL ethanol which was pre-cooled at -20℃ was added into the purified honey solution. The mixed solution was stirred tenderly in ice water for 15 min. After that, the mixed solution was centrifuged at 15000×g for 10 min at 4℃. The supernatant was discarded and the sediment was collected. 1.5 mL distilled water was added to dissolve the sediment honey protein. The protein concentration was determined by the Bradford assay (Bradford, M., 1976), with bovine serum albumin serving as a standard.

ACCEPTED MANUSCRIPT 2.3.2. Determination of protein profile and molecular weight Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Bizani et al. (2015) with appropriate modifications. A 15 per cent sodium dodecyl sulfate polyacrylamide gel (15 cm long, 12 cm wide, 1.5 mm

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thick) was prepared and used for honey major protein separation. The samples were

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mixed with loading buffer (5×) and then incubated for 5 min at 95℃ in order to favour

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the denaturation of proteins. The loading quantity of protein was 200 μg each well.

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Gel was run at 100 V until the dye reached the bottom of the gel. A similar process was applied to the protein marker that loaded on the first and last well o f the gel as a

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standard for the determination of the molecular weight of the proteins, then the gel was carefully washed several times with distilled water and immobilized with fixative

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solution consisted of ethanol, acetic acid and distilled water in a ratio of 2:1:5 for an

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hour. After immobilization, the gel was stained with Coomassie brilliant blue for 3 hours. The gel was held overnight with destaining solution consisted of ethanol, acetic

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acid and distilled water in a ratio of 3:1:10 to wash away the remaining Coomassie brilliant blue. The relative molecular weight of the bands of honey samples was determined with assistance of the software Quantity One v4.6.2. 2.3.3. In-gel digestion and HPLC-MS-MS The visible specific protein bands were excised into s mall pieces and digested according to Katayama, Nagasu & Oda (2001).

ACCEPTED MANUSCRIPT After digestion, the samples were re-suspended with Nano-RPLC buffer A (0.1 per cent methanoic acid, 2 per cent methanoic acid, 98 per cent distilled water). The online Nano-RPLC was employed on the Eksigent nanoLC-Ultra™ 2D System (AB SCIEX, USA). The samples were loaded on C18 nanoLC trap column (100 µm×3 cm,

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C18, 3 µm, 150Å) and washed by Nano-RPLC buffer A at 2 μL/min for 10 min. An

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elution gradient of 5-35 per cent acetonitrile (0.1 per cent methanoic acid) in 90 min

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gradient was used on an analytical ChromXP C18 column (75 μm x 15 cm, C18, 3 μm 120 Å) with spray tip. Data acquisition was performed with a Triple TOF 5600

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System (AB SCIEX, USA) fitted with a Nanospray III source (AB SCI EX, USA) and

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a pulled quartz tip as the emitter (New Objectives, USA). Based on combined MS and MS/MS spectra, proteins were successfully identified based on 95 per cent or higher

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confidence interval of their scores in the MASCOT V2.3 search engine (Matrix

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Science Ltd., London, U.K.). The database of Uniprot and protein sequence repository of A. cerana and A. mellifera from the NCBI website were used for matching

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purposes.

2.4. Analysis of beeswax compositions in honey samples 2.4.1. Sample preparation A diluted honey solution (40 g in 100 mL distilled water) was treated through vacuum filtering with slow filter paper. The filter paper was soaked in 15 mL Petroleum Ether 60-90℃ and the filter residue was dissolved by ultrasonic for 5 min. Then the filter paper was discarded. The solution was concentrated to 1.5 mL using

ACCEPTED MANUSCRIPT Termovap Sample Concentrator. The beeswax produced by A. cerana and A. mellifera were treated by dissolving about 0.5 g in 10 mL of Petroleum Ether 60-90℃ and dissolved by ultrasonic for 5 min. The Petroleum Ether extracts of honey and wax were filtered through a 0.22 μm membrane filter before use. Standard substances were

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dissolved in a suitable concentration with Petroleum Ether 60-90℃.

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2.4.2. GC detection

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The gas chromatographic instrument used for GC detection was Shimadzu

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GC-2010, and the chromatographic column was InertCap-5 capillary column (30

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m×0.25 mm i.d., and 0.25 μm film thickness). Carrier gas (He, purity 99.999 per cent) flow was kept constant at 0.8 mL/ min. The split ratio was 10:1. During the whole

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analysis, the injection port temperature was 280 ℃. The oven temperature was initially set at 50℃ (1 min hold), followed by a temperature ramp of 20℃/min to 180℃, then

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increased to 280℃ at a rate of 10℃/min (20 min hold), for a total run time of 37.5 min.

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2.4.3. GC-MS-MS Identification The analysis was performed using a 7890B gas chromatograph coupled to a mass spectrometer model 7000C (Agilent Technologies, Palo Alto, CA, USA). The separation was achieved using a HP-5ms capillary column (30 m×0.25 mm i.d., and 0.25 μm film thickness). Carrier gas (He, purity 99.999 per cent) flow was kept constant at 0.8 mL/min. Split ratio was 10:1. During the whole analysis, the injection port temperature was 280℃. The oven temperature was initially set at 50℃ (1 min

ACCEPTED MANUSCRIPT hold), followed by a temperature ramp of 20℃/min to 180℃, then increased to 280℃ at a rate of 10℃/min (20 min hold), for a total run time of 37.5 min. The mass scan ranging from 30 to 550 amu. Nonacosane, Hentriacontane, 1-Triacontanol and 17-Pentatriacontene petroleum

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ether solutions with 1 mg/mL concentration were prepared respectively, and the

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detection method was the same as used for honey and beeswax samples.

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Agilent Mass Hunter (Qualitative Analysis B.06.00) was used as the search engine.

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Individual peak identification was achieved by fragmentation pattern comparison with

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the NIST 11 (National Institute of Standards and Technology) mass spectra database. The results of GC-MS-MS identification were confirmed with the reference standard

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substance.

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2.4.4. Validation

The approach described here was verified according to Codex guideline (1993),

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including requisite values for linearity, accuracy, precision, and limits of detection (LODs). The linearity was assessed from matrix- fortified calibration measured at six concentration levels. The concentration of Hentriacontane was at six concentration levels: 0.625, 1.25, 2.5, 5, 10 and 20 μg/mL, and the concentration of 17-Pentatriacontene was 10, 20, 40, 80, 160 and 320 μg/mL. Repeatability (intraday precision) were assessed (n=5) in 1 day. For assessment of inter-day precision (reproducibility), the same samples were tested over three consecutive days. The LODs was calculated at three times the signal-to-noise (S/N) ratio.

ACCEPTED MANUSCRIPT 3. RESULTS AND DISCUSSION 3.1. Honey protein profile We performed SDS-PAGE analysis on 82 honey samples. We found that the protein profiles were identical within the same bee species, and there were stable and

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consistent differences between A. cerana honey and A. mellifera honey. Fig. 1 shows

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a representative result of honey protein SDS-PAGE analysis of A. cerana honey (lane

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1-4) and A. mellifera honey (lane 5-8). The botany origin of the honey samples of lane

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1-8 was Brassica napus L. There are obvious differences in the composition of protein between these two kinds of honey. Three distinctive bands for ACH and six for AMH

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were detected in the separating gel. The molecular weight of these distinctive proteins was found to be between 13.8 and 33.1 KDa. ACH was characterized by the presence

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of three bands with approximate molecular weights of 15, 20 and 29 KDa. AMH was

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characterized by the presence of six bands with approximate molecular weights of 14, 17, 18, 19, 21 and 33 KDa (Table 1). These species-specific protein bands could be

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clearly detected in all analyzed samples, which demonstrated that these proteins are secretions from honeybees and are stably expressed and exist in honey. Thus, these species-specific bands could be used to distinguish ACH from AMH. In order to test the detection sensitivity of SDS-PAGE method on the adulteration of AMH, we mixed ACH with AMH in different proportions and analyzed their protein. When the percentage of AMH in the mixture exceeds 25 percent, one of the species-specific bands (about 16.5 KDa) of AMH appeared and the color of this band

ACCEPTED MANUSCRIPT deepens with the increase of AMH content (Fig. 2). Hence, when the protein profile of an ACH sample shows a band with molecular weight of 16.5 KDa, it can be reasonably doubted that this ACH was adulterated with AMH. Therefore, SDS-PAGE

of entomological origin of the two kinds of honey.

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3.2. HPLC-MS-MS identification of species-specific bands

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analysis could be considered to be an analytical method to determine the authenticity

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In order to clarify the composition of these species-specific bands, we made a further HPLC-MS-MS identification. The composition of all these species-specific

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bands are shown in Table 1. Our data revealed that all these bands were composed of

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mixed proteins. The main protein in these bands were MRJPs, or rather, the fragments of MRJPs, and MRJP1, 2 and 3 were the most abundant proteins in them. In addition,

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digestive enzymes and unknown protein LOC408608 of A. mellifera were also

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detected in the protein bands of AMH with molecular weight of 13.8 and 16.5 KDa. This finding was in line with previous studies which reported the detection of MRJPs

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in honey ( Š imú th, Bí liková , Kováčová , Kuzmová & Schroder, 2004; Sera, Deugchan, Seukhyun, Jangwon & Haeik, 2008; Di Girolamo, D'Amato & Righetti, 2012; Chua, Lee & Chan, 2015). MRJPs are intrinsic ingredients of honey and has been confirmed in many previous research. The differences between A. cerana and A. mellifera on MRJPs are stable, their molecular weight and protein amino acid sequences can be found in the NCBI database. This difference can be reflected by SDS-PAGE analysis. Therefore, ACH and AMH have different molecular weight protein bands and these species-specific

ACCEPTED MANUSCRIPT bands could be used to determine the authenticity of their entomological o rigin. From Table 1, we know that all the specific bands contain multiple MRJPs and their molecular weight were all below 35 KDa. The molecular weight of the whole MRJPs was about 45-87 KDa (Ramanathan, Nair & Sugunan, 2018), so the MRJPs in specific

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bands were the degraded products. The members of MRJP family are homologous

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proteins with sequence consistency over 60 per cent (Drapeau, Albert, Kucharski,

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Prusko & Maleszka, 2006; Shen, Xing, Yang & Gao, 2007). We speculate that different MRJPs were degraded in a similar pattern so one protein band contains a

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variety of MRJPs.

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3.3. Detection of beeswax composition in honey

In order to increase the accuracy and sensitivity of our identification method, we

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further explored the beeswax compositions in honey, whic h is another kind of

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honeybee secretion found in honey and may be used to determine the authenticity of entomological origin of different types of honey.

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We tested 37 ACH samples and 45 AMH samples by GC method. The GC chromatography of the honey samples that from the same kind of bee was similar. The typical GC chromatography of the two kinds of honey was shown in Fig. 3. There were significant difference in the composition of the petroleum ether extracts between ACH and AMH (Fig. 3). The petroleum ether extract of AMH and ACH have a characteristic component peak with a retention time of 22.95 and 32.77 min, respectively. The peak with a retention time of 32.77 min was detected in all honey samples produced by A. cerana but not in any of those by A. mellifera , while the

ACCEPTED MANUSCRIPT peak with a retention time of 22.95 min was detected in all honey samples produced by A. mellifera but not in any of those by A. cerana. In order to verify the source of these differences, we compared the composition o f ACH and AMH with their corresponding beeswax. We tested all the 46 beeswax samples. The GC

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chromatography of the beeswax that from same bee species was consistent. As shown

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in Fig.3, the peaks of honey were almost all coincide with beeswax of the same

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honeybee species. This was consistent with previous report demonstrating that some of entomological markers for stingless bee honey come from their waxes (Zuccato,

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Finotello, Menegazzo, Peccolo & Schievano, 2017). These findings allow us to

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confirm that the characteristic peaks in honey come from beeswax, and the chemical components of these peaks could be used as entomological markers for these two

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kinds of honey. Despite the difference between honeybee species was highly

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consistent in our samples, it still must be noted that, colony management and apicultural practice during beekeeping may introduce exogenous wax into colonies

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and cause inaccuracy of the detection method. For example, comb foundation widely used in modern beekeeping is such a factor. The activity using A. mellifera combs for honey production in A. cerana colonies may have even greater impact. The GC-MS-MS analysis results showed that the peaks with the same retention time contain the same chemical substance. The chemical components of these peaks were mainly hydrocarbons (Table 2). The characteristic peak of ACH with a retention time of 32.77 min was identified as 17-Pentatriacontene, and the characteristic peak of AMH with retention time of 22.95 min was Hentriacontane. 17-Pentatriacontene

ACCEPTED MANUSCRIPT and Hentriacontane were the markers of ACH and AMH, respectively. The peaks with the retention time of 20.15 and 26.52 min were identified as Nonacosane and 1-Triacontanol, respectively. The ratio of hydrocarbons with a predominant chain length of C27-C33, mainly heptacosane, nonacosane, hentriacontane, pentacosane and

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tricosane is 12-16 per cent in beeswax (Fratini, Cilia, Turchi & Felicioli, 2016).

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Hydrocarbons are one of the main components of beeswax. Our study show the

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difference in hydrocarbons between A. cerana beeswax and A. mellifera beeswax. This difference can be used to identify the entomological origins of honey produced

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by A. cerana and A. mellifera.

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In order to test the detection sensitivity of this GC method on the adulteration of ACH, we mixed ACH with AMH in different proportions and tested these samples.

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Fig. 4 is the GC chromatography of the mixture honey samples. From bottom to top,

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the proportion of AMH increased from 1 per cent to 80 per cent. With the increase of the proportion of AMH, the peak area of Hentriacontane constantly increased, while

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the peak area of 17-Pentatriacontene, the characteristic component of ACH, decreased. When the proportion of AMH was as low as 1 per cent, the peak of Hentriacontane, the characteristic component of AMH, appeared. This result demonstrated that the sensitivity of this detection method is very high, and can be used together with the SDS-PAGE method to ensure the accuracy and sensitivity of the discrimination of AMH and ACH. 3.4. Validation of beeswax composition detection method

ACCEPTED MANUSCRIPT Specificity of the characteristic components in their respective honey and beeswax samples was confirmed by comparing honey and beeswax samples to blank samples. The peak of 17-Pentatriacontene was detected in all honey and beeswax samples produced by A. cerana but not in any of those by A. mellifera, while the peak of

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Hentriacontane was detected in all honey and beeswax produced by A. mellifera but

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not in any of those by A. cerana. Neither of the two components was detected in any

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of the blank samples. These results confirmed that 17-Pentatriacontene and Hentriacontane were the characteristic components of honey and beeswax produced

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by A. cerana and A. mellifera, respectively.

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The precision and accuracy of this method were evaluated by intra and interday RSDs (relative standard deviations). The intraday RSDs were performed by five

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parallel experiments in 1 day, and triplicate measurements over three consecutive

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days gave the interday RSDs. The RSDs of the intraday precision of 17-Pentatriacontene in ACH and Hentriacontane in AMH were 1.5 per cent and 1.1

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per cent respectively, and the inter-day precision were 2.7 per cent and 2.3 per cent respectively. The results showed that high precisions were obtained with the RSD in the range of 1.1-2.7 per cent, indicating good precision and accuracy of the beeswax composition detection method. To assess the linearity of the method, standards with the concentration ranging from 10 to 320 μg/mL for 17-pentatriacontene, from 0.625 to 20 μg/mL for Hentriacontane were prepared. Good linearity was achieved with correlation coefficients (R) all

ACCEPTED MANUSCRIPT greater than 0.9999. The LODs was calculated at three times the signal- to-noise (S/N) ratio. The LODs were 0.5731 and 0.1287 μg/mL for 17-pentatriacontene and Hentriacontane, respectively, indicating a high detection sensitivity of the method. 17-pentatriacontene and Hentriacontane, as another secretions of honeybees besides

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of proteins found in honey, could be used as descrimination indexes of honey

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produced by A. cerana and A. mellifera, respectively.

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Our research have showed that A. cerana honey and A. mellifera honey could be

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identified through differences in bee secretions. Though our results indicated that these differences were only related to bee species, further testing is merited to use a

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production and extraction date.

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statistically significant number of honeys from a single botanical origin, place of

4. Conclusions

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This study confirmed the differences between ACH and AMH in the compositions of bee secretions, which can be used to accurately distinguish the entomological

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origin of them. The SDS-PAGE protein pattern from bee secretions can be used as a tool for ACH and AMH authentication. 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. Based on these discoveries, we successfully established two methods to discriminate the honey produced by A. cerana and A. mellifera. The SDS-PAGE based method is convenient to operate, while the GC based method is sensitive. These two methods

ACCEPTED MANUSCRIPT constitute a system to satisfy different needs for entomological authentication of honey samples. Acknowledgments The research was supported by grants from the National Natural Science

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Foundation of China (No. 31272512) and the earmarked fund for Modern

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Agro-Industry Technology Research System from the Ministry of Agriculture of

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China (CARS-44). Notes

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The authors declare no competing financial interest.

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Ram ó nsierra, J. M., Ruizruiz, J. C., & De, L. (2015). Electrophoresis characterisation of protein as a method to establish the entomological origin of stingless bee honeys. Food Chemistry, 183, 43-48. Sak-Bosnar, M., & Sakac, N. (2012). Direct potentiometric determination of diastase activity in honey. Food Chemistry, 135 (2), 827-831. Sera, W., Deugchan, L., Seukhyun, K., Jangwon, K., & Haeik, R. (2008). Honey major protein characterization and its application to adulteration detection. Food Research International, 41, 952-956.

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Entomological authentication of stingless bee honey by 1 H NMR-based metabolomics

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ACCEPTED MANUSCRIPT TABLES AND ARTWORK Table 1. The HPLC-MS-MS identification results of species-specific bands of A. cerana honey and A. mellifera honey. Table 2. Composition of Petroleum Ether extracts of honey and beeswax produced by

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A. mellifera and A. cerana.

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Fig. 1. SDS-PAGE patterns of honey proteins of different entomological origin. Lane

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1-4, A. cerana. Lane 5-8, A. mellifera.

Fig. 2. The protein pattern of A. cerana honey mixed with A. mellifera honey. From

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left to right, the proportion of A. mellifera honey was 5, 10, 15, 25, 40, 50, 60, 70, 85

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per cent.

Fig. 3. Comparison of GC-MS-MS chromatography of the petroleum ether extracts

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between a) A. mellifera honey(AMH) and A. cerana honey (ACH) , b) A. mellifera

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honey(AMH) and A. mellifera beeswax(AMW) and c) A. cerana honey (ACH) and A. cerana beeswax (ACW).

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Fig. 4. GC chromatography of A. cerana honey mixed with A. mellifera honey. From bottom to top , the proportion of A. mellifera honey is 1, 5, 10, 20, 40, 80 per cent.

ACCEPTED MANUSCRIPT Table 1 molecul Ban

ar

d

weight

Scor Accession e

number

Protein

Specie s

Amino acid sequence fragment

(KDa)

185

1 gi|5642203 7

K.YIDYDFGSEERR.Q, K.IVNNDFNFNDVNFR.I

jelly protein 2 cerana major royal

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156

K.YIDYDFGSEER.R,

Apis

R.LTSNTFDYDPR.Y,

Apis

jelly protein 3 cerana

K.IINNDFNFNDVNFR.I, R.GEALIIYQNSDDSFHR.L

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29.4

major royal

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C1

gi|4021830

did not show 17matched peptide sequences.

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major royal gi|3335839 Apis 786 jelly protein 1 4 cerana precursor

R.EYILVLSNR.M, K.EALPHVPIFDR.Y,

76

96

C3

15

203

111

M1

1 gi|5642203 7

33.1

major royal

Apis

jelly protein 2 cerana major royal

Apis

jelly protein 3 cerana

gi|3335839 major royal Apis 4 jelly protein 1 cerana

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77

gi|4021830

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20.2

Apis

jelly protein 1 cerana

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C2

4

major royal

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510

gi|3335839

gi|4021830 1

major royal

Apis

jelly protein 2 cerana

gi|5642203 major royal Apis 7 jelly protein 3 cerana

K.IKEALPHVPIFDR.Y,R.IMDANVNDLI

LNTR.C, K.MANNDYNFNDVNFR.I, R.TSNYEQNAVHYEGVQNILDTQSSAK. V K.YIDYDFGSEER.R, K.YIDYDFGSEERR.Q

K.NYPFDVDR.W, K.HIDYDFGSVER.R

R.EYILVLSNR.M, K.EALPHVPIFDR.Y R.ENLELVAQNEK.T, K.IVNNDFNFNDVNFR.I, K.YIDYDFGSEER.R

R.QNDNNQNNNQNDNNR.N

Apis 692 gi|5858509 major royal mellife 6 8 jelly protein 1 ra

did not show 30matched peptide sequences.

204 gi|2411770

did not show

7

43

major royal

Apis

jelly protein 2 mellife

27matched peptide

sequences.

ACCEPTED MANUSCRIPT ra Apis 236 gi|2013895 major royal mellife 0 3 jelly protein 3 ra

did not show 24matched peptide sequences.

Apis gi|2013895 major royal 472 mellife 4 jelly protein 4 ra

did not show 15matched peptide sequences.

131 gi|4094963

38

107

503

gi|1892123

major royal

75

jelly protein 8

gi|6291092

major royal

5

jelly protein 9

gi|8988557 alpha-glucosid

M2

4

M3

gi|5858509 8

21.3 107

19.1

478

ase

gi|5858514 alpha-amylase

422 gi|6448461

567

major royal jelly protein 7

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576

gi|5858510 8

gi|5858509 8

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sequences.

did not show

mellife ra

18matched peptide

sequences.

ra Apis

16matched peptide

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precursor

7

9

Apis

jelly protein 6 mellife

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0

major royal

did not show

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8

mellife jelly protein 5 ra

did not show

18matched peptide

sequences.

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gi|5858518

Apis

Apis

mellife did not show 4matched peptide sequences. ra

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577

2

major royal

ED

777

gi|2013890

precursor glucose oxidase

Apis

mellife did not show 8matched peptide sequences. ra Apis mellife ra Apis mellife ra Apis mellife ra

did not show

22matched peptide

sequences. did not show

19matched peptide

sequences. did not show

13matched peptide

sequences.

major royal Apis jelly protein 1 mellife did not show 8matched peptide sequences. precursor

ra

major royal Apis jelly protein 2 mellife precursor

ra

major royal

Apis

jelly protein 1 mellife

K.NYPFDVDQWR.D, K.IVNDDFNFDDVNFR.I, K.NYPFDVDQWR.D + Dioxidation (W)

did not show 8matched peptide sequences.

ACCEPTED MANUSCRIPT precursor

ra

major royal Apis gi|5858510 K.IVNDDFNFDDVNFR.I, 160 jelly protein 2 mellife 8 K.SQFGENNVQYQGSEDILNTQSLAK.A precursor ra major royal Apis gi|5858514 158 jelly protein 3 mellife 2 precursor ra

K.IINNDFNFNDVNFR.I

Apis

R.NEYLLALSDR.N,

jelly protein 4 mellife R.NQNVLNNDLNLEHVNFQILGANVND precursor ra LIR.N

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0

major royal

R.QNDNQNNQNDNNR.N,

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114

gi|5858517

K.NYPFDVDR.W,

M4

gi|5858509 8

17.9

Apis

jelly protein 1 mellife precursor

ra

K.IKEALPHVPIFDR.Y,

R.IMNANVNELILNTR.C,

K.MVNNDFNFDDVNFR.I + Oxidation (M),

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374

major royal

SC

K.EALPHVPIFDR.Y, K.FFDYDFGSDER.R，

8

major royal

V

Apis

jelly protein 2 mellife precursor ra

K.IVNDDFNFDDVNFR.I

ED

70

gi|5858510

MA

R.TSDYQQNDIHYEGVQNILDTQSSAK.

16.5

major royal Apis gi|5858510 143 jelly protein 2 mellife 8 precursor ra

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M5

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major royal Apis gi|5858509 461 jelly protein 1 mellife did not show 9matched peptide sequences. 8 precursor ra

339

440 M6

gi|4809457 3

gi|5858509 8

13.8 100

gi|5858510 8

uncharacterize Apis

K.NYPFDVDQWR.D, K.YFDYDFGSEER.R, K.IVNDDFNFDDVNFR.I R.ASTPIENLIEIFDTIK.D,

R.EQVANALLEGSSDLIGALK.D, d protein mellife R.EQVANALLEGSSDLIGALKDAVELAI LOC408608 ra R.A

major royal

Apis

jelly protein 1 mellife precursor ra major royal

10matched peptide

sequences.

Apis

jelly protein 2 mellife precursor

did not show

ra

K.IVNDDFNFDDVNFR.I

ACCEPTED MANUSCRIPT

249

3

uncharacterize Apis

d protein mellife R.EQVANALLEGSSDLIGALKDAVELAI LOC408608 ra R.A

gi|5858516 alpha-glucosid 4

R.EQVANALLEGSSDLIGALK.D,

ase precursor

Apis mellife

R.TPFQWDDSVSAGFSSSSNTWLR.V

ra

AC C

EP T

ED

MA

NU

SC

RI

PT

105

gi|4809457

ACCEPTED MANUSCRIPT Table 2 Samples

Name

Formula

Retention time (min) 11.48

Pentacosane

C25H52

16.65

Heptacosane

C27H56

18.21

Nonacosane

C29H60

Hentriacontane

C31H64

22.95

1-Triacontanol

C30H62O

26.52

C16H30O2

11.48

Pentacosane

C27H56

18.21

Nonacosane

C29H60

20.15

1-Triacontanol

C30H62O

26.52

C35H70

32.77

Heptacosane

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17-Pentatriacontene

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16.65

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by A. cerana

20.15

C25H52

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Honey and beeswax produced

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Palmitoleic acid

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C16H30O2

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Honey and beeswax produced by A. mellifera

Palmitoleic acid

ACCEPTED MANUSCRIPT

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

ACCEPTED MANUSCRIPT Highlights



17-Pentatriacontene and Hentriacontane are the characteristic constituents of Apis cerana honey and Apis mellifera honey, respectively.



A sensitive GC method to detect 17-Pentatriacontene and Hentriacontane in Apis cerana honey and Apis mellifera honey, respectively, was developed.

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3. A simple SDS-PAGE protein pattern from bee secretions as a tool for Apis

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cerana honey and Apis mellifera honey authentication.

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