Non-enzymatic browning and protein aggregation in royal jelly during

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Non-enzymatic browning and protein aggregation in royal jelly during room-temperature storage Jiangtao Qiao, Xueyu Wang, Liqiang Liu, and Hongcheng Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04955 • Publication Date (Web): 03 Feb 2018 Downloaded from http://pubs.acs.org on February 6, 2018

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Journal of Agricultural and Food Chemistry

Non-enzymatic browning and protein aggregation in royal jelly during room-temperature storage

Jiangtao Qiaoa, Xueyu Wanga, Liqiang Liuc, Hongcheng Zhanga,b*

a

Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing,

100093, China b

National Research Center of Bee Product Processing, Ministry of Agriculture, Beijing,

100093, China c

College of Life Sciences and Food Engineering, Hebei University of Engineering, Handan

056021, China;

*Corresponding author: Tel: +86 10 62590442; Fax: +86 10 62590442; Email: [email protected] (H. Zhang) Address: Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Xiangshan, Beijing, 100093, China

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ACS Paragon Plus Environment


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ABSTRACT Royal jelly possesses numerous functional properties. Improper storage usually

3

causes bioactivity loss, especially queen differentiation activity. To determine changes in

4

royal jelly, we investigated non-enzymatic browning and protein changes in royal jelly

5

during room-temperature storage from one to six months. Our results indicate that royal

6

jelly experiences non-enzymatic browning and protein aggregation. The products of

7

non-enzymatic browning dramatically increased, especially Nε-carboxymethyl lysine

8

(CML) with growth of approximately sevenfold. We speculate that CML may be

9

recognized as a freshness marker for royal jelly. Our results also demonstrate that major

10

royal jelly protein 1 (MRJP1) monomer gradually aggregated with MRJP1 oligomers into

11

new oligomers of about 440 kDa and 700 kDa. This suggests that the reduction of MRJP1

12

monomer may be attributable to aggregation. We provide the novel explanation that the

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differentiation loss of royal jelly may be due to the aggregation of MRJP1 limiting the

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honeybees' ability to digest and absorb royal jelly.

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Keyword: Queen Differentiation; CML; Freshness marker; Protein aggregation; MRJP1

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monomer;

17

Running title: Changes in royal jelly during storage

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1. INTRODUCTION

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As standards of living rise and the pursuit of functional foods grows, royal jelly has

22

drawn increasing attention during the last decades. Royal jelly represents a creamy white

23

viscous secretion from the mandibular and hypopharyngeal glands of worker bees (1).

24

Royal jelly plays a pivotal role in the growth and development of honeybees. In the

25

society of honeybees (Apis mellifera), the queen bees have shorter development time and

26

larger body size than worker bees. In addition, the queen bees are long-lived and

27

typically reach 1 to 2 years, whereas the worker bees only live 40 days (2, 3). These

28

circumstances are not a consequence of genetic difference but a diet difference. Queen

29

bees consume royal jelly throughout their lifetime, whereas worker bees only eat royal

30

jelly less than three days (2, 3). Royal jelly also presents human health benefits;

31

specifically, it enhances the body's immunity (4), decreases cardiovascular disease (5),

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and slows down the aging process (6). These functional properties may be accredited to

33

its characteristic components such as carbohydrates, lipids, minerals, vitamins, free

34

amino acids and proteins (7).

35

Since the bioactivities and the quality of royal jelly are largely labile, and principally

36

influenced by its storage conditions, suitable storage conditions are essential to guarantee

37

the quality of royal jelly. Some researchers strongly recommend storing royal jelly at –

38

20 ℃ or lower in commerce (1, 8, 9). Conversely, improper storage of royal jelly can

39

cause changes in its physical and chemical features, resulting in the loss of functional 3



40

properties (9). For instance, after reared with royal jelly stored at 40 ℃ for 7 days, larvae

41

presented longer developmental times, lower body weight and smaller ovary size,

42

compared to those with fresh royal jelly (10). After fed with royal jelly stored at 40 ℃ for

43

30 days, larvae developed into a full worker bee instead of a queen bee (10). Thus, it

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seems very important to focus on the effects of different storage conditions on the

45

compositional changes in in royal jelly.

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Royal jelly mainly consists of water (50-70%), crude proteins (9-18%), total sugars

47

(10-16%) and lipids (3-6%) (7, 11, 12). Royal jelly undergoes various changes during

48

storage, such as increase of viscosity, acidity and color; degradation of protein; and

49

chemical reduction of sugars (13, 14). These changes are perhaps related to the

50

non-enzymatic browning reaction (14). Non-enzymatic browning, the Maillard reaction,

51

is initiated by condensation between amino groups of proteins and reducing sugars

52

during food processing and storage (15). It is worth noting the Maillard reaction is

53

divided as initial, intermediate and final stages (16). Different reaction stages deliver

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different reaction products, such as glycoprotein, furosine, hydroxymethylfurfural (HMF)

55

and advanced glycosylation end products (AGEs) (17). AGEs, as a mixture with a variety

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of chemical structures, have gained much more attention in recent years, such as

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Nε-carboxymethyl lysine (CML), pentosidine, pentodilysine, crossline, pyrropyridine and

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argpyrimidine (18). The accumulation of these products in food can usually cause the

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changes in the food quality, especially like decreasing digestibility and forming toxic

60

compounds (15). Furthermore, numerous studies have revealed that AGEs are associated 4


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with the pathogenesis, for example, diabetes, uremia, atherosis, Alzheimer and caducity

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(19-21). Unfortunately, except HMF (8) and furosine (17), little attention has been paid

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to other non-enzymatic browning products or protein changes in royal jelly during

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storage.

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The objective of this study was to investigate non-enzymatic browning and protein

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changes in royal jelly during room-temperature storage. We investigated the

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non-enzymatic browning by measuring the fluorescence intensities of browning products

68

and the contents of glycoprotein and CML. The conformational changes of royal jelly

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proteins were also analyzed using fluorescence wavelength scanning and synchrotron

70

radiation circular dichroism (SRCD). Finally, we explored protein variation in royal jelly

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by SDS-PAGE, Native-PAGE, Liquid chromatography-tandem mass/mass spectrometry

72

(LC-MS/MS) and size exclusion chromatography - high performance liquid

73

chromatography (SEC-HPLC).

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2. MATERIALS AND METHODS

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2.1. Royal jelly samples

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Fresh royal jelly was acquired from queen cell cups where larvae had been grafted 72

77

h earlier at the apiary of the Institute of Apicultural Research, Chinese Academy of

78

Agricultural Sciences (Beijing, China) during the flowering season in June 2016. The

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royal jelly samples were dispensed into seven sterile air-tight glass bottles and then stored

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at room temperature for one to six months. The average room temperature was about

81

25±3 ℃. All analyses were done in duplicate. 5



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2.2 Chemicals

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Concentrated sulfuric acid, potassium sulfate, copper sulfate, trichloroacetic acid,

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Nitroblue Tetrazolium and Triton were obtained from Solarbio (Beijing, China). Pronase

85

E was from Roche (Basel, Switzerland). Standards of 1-deoxy-1-morpholino-D-fructose

86

(DMF), pentosidine, pentodilysine, crossline, pyrropyridine, argpyrimidine and L

87

(+)-Arginine were purchased from Sigma (St. Louis, MO, USA). The

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Nε-(Carboxymethyl)lysine (CML) ELISA Kit (contain CML-HSA Standard, Sample

89

Dilution Buffer, Standard Dilution Buffer, First Antibody, HRP-conjugated detection

90

antibody, antigen-coated microplate, substrate reagent and stop solution) was purchased

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from MBL (Nagoya, Japan). Standard marker proteins were obtained from Phenomenex

92

(Torrance, CA, USA). A pre-stained Protein Marker (no. 26616) was purchased from

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Thermo Scientific (Rockford, IL, USA). Novex® 4 - 12% Tris-glycine gel, 2 × native

94

sample buffer, 10 × Tris-Glycine native running buffer and NativeMark™ unstained

95

protein standards were purchased from Invitrogen (Carlsbad, CA). The TSK-GEL

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G3000SWXL column was from Tohso (Tokyo, Japan). All other reagents were from

97

Sigma (St. Louis, MO, USA).

98

2.3 Determination of glycoprotein content

99

To determine the content changes of glycoprotein, a nitroblue tetrazolium (NBT)

100

colorimetric assay was performed by modification of a previous method (22).

101

1-Deoxy-1-morpholino-D-fructose (DMF) was used as the standard substance.

102

DMF standard curve. Standards with 20, 50, 100, 150 and 200 µL of DMF standard 6


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solute ion (6.0 mM) were dispensed into five tubes with a fixed amount of saline to 1 mL.

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Next, 2 mL of NBT (0.5 mM, pH 10.8, 57 ℃) and 2 mL of Triton X-100 (0.02%, pH 10.8,

105

57 ℃) were added to 100 µL of DMF diluted solution. After reacting in a 57 ℃ water

106

bath for about 20 min, the absorbance was measured at 530 nm by UV-2550

107

spectrophotometer (Shimadzu Co., Ltd., Kyoto, Japan). The standard curve was

108

established by DMF concentration and absorbance value.

109

Royal jelly (0.5 g) was homogenized in 100 mL of ultrapure water, and then 100 µL

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royal jelly solution was mixed with 2 mL of NBT (0.5 mM, pH 10.8, 57 ℃) and 2 mL of

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Triton (0.02%, pH 10.8, 57 ℃). After reacting in a 57 ℃ water bath for about 20 min, the

112

absorbance was measured at 530 nm. The content of glycoprotein was calculated

113

according to the DMF standard curve.

114 115

M=

c × v × 249 m

Where M is the content of glycoprotein per RJ gram (mg/g); c is the content of

116

DMF (mM); v is the volume of saline to dissolve 1 g of RJ; 249 represents the molar

117

mass of DMF (g/mol); m is the protein content per gram of royal jelly.

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2.4 Determination of CML content

119

Standard curve. The CML-HAS standard solution was diluted to 5, 2.5, 1.25, 0.63, 0.31,

120

0.16 and 0.08 ng/mL (standards 1-7). A total of 60 µL of each standard solution

121

(Std1-Std7, Blank) was pipetted into the appropriate wells of a sample preparation

122

microplate. Next, 60 µL of the first antibody working solution was pipetted into each

123

well and mixed well. Then, 100 µL of the mixtures prepared above were transferred to 7



124

each well of an antigen-coated microplate and incubated at room temperature for one

125

hour with shaking at 300 rpm on an orbital microplate shaker. The plate was washed four

126

times by filling each well with wash buffer (350 µL) using a squirt bottle. Then, 100 µL

127

of substrate reagent was added, and the wells were incubated at room temperature for 20

128

minutes with shaking at 300 rpm on an orbital microplate shaker. Finally, 100 µL of stop

129

solution was added to each well in the same order as the previously added substrate

130

reagent, and the absorbance was measured in each well at dual wavelengths of 450 nm.

131

The standard curve was established from the CML-HAS concentration and the

132

absorbance value.

133

Royal jelly samples of 2 g were dissolved in ultrapure water and diluted to 10 mL.

134

Then, the samples were diluted with the sample buffer at a ratio of 1:4 (v/v). A total of 60

135

µL of the above sample solution was pipetted into the well and mixed with 60 µL of first

136

antibody working solution. The other steps are the same as those of the standard curve.

137

All assays were carried out in triplicate.

138

2.5 Fluorescence-emission of different fluorescence compounds

139

Preparation of sample: One gram of royal jelly was dissolved in ultrapure water and

140

diluted to 2.5 mL and then ultrasound dissolved for 20 minutes at 20 ℃.

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The proteins in the 2.5-mL royal jelly solution were precipitated with 2.5 mL of 24%

142

(w/v) trichloroacetic acid (TCA). Then, the solution was centrifuged at 13,000 × g for 20

143

min at room temperature. The supernatant was preserved at 4 ℃ until use.

144

Fluorescence measurement: Fluorescence measurement was carried out as described by 8


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Palomobo (23), with minor modification, using a fluorescence spectrophotometer

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(F-4600, Hitachi Ltd., Ibaragi, Japan). The scanning speed of 2400 nm/min, EX and EM

147

slit of 5 nm, EX sampling interval of 10 nm and voltage of 400 V were used for analysis.

148

Fluorescence values were measured at the following excitation and emission wavelengths

149

reported by Palomobo (23): AGEs (excitation: 347 nm, emission: 415 nm); pentosidine

150

(excitation: 335 nm, emission: 385 nm); pentodilysine (excitation: 366 nm, emission: 440

151

nm); cross-link (excitation: 379 nm, emission: 463 nm); pyrropyridine (excitation: 370

152

nm, emission: 455 nm); and argpyrimidine (excitation: 320 nm, emission: 382 nm).

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2.6 SRCD analysis

154

To investigate the secondary structure changes in royal jelly proteins, royal jelly

155

samples were analyzed by Synchrotron radiation circular dichroism (SRCD) at the

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Beijing Synchrotron Radiation Facility (BSRF) (Beijing, China). Royal jelly samples of

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1 g were dissolved in 10 mL of ultrapure water. The homogenate was centrifuged at

158

12,000 × g for 10 min at 4 ℃. The supernatant (14 µL) was loaded into a CaF2 cell with a

159

0.09-mm path length. Spectra were measured using a wavelength range from 180 nm to

160

260 nm, by a bandwidth of 1 nm and a time constant of 1 s. Each spectrum was measured

161

three times at room temperature of 25 ℃. The SRCD spectra were recorded after

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equilibrating each sample. The CD Tool software was used for data processing, and

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replicate scans were averaged, smoothed, and baselines subtracted. Protein secondary

164

structures were calculated from processed SRCD spectra on the DICHROWEB and

165

analyzed by the CONTIN LL method or CDSSTR algorithm with reference set SP175. 9



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2.7 The wavelength scanning of royal jelly protein fluorescence One gram of royal jelly was dissolved in ultrapure water and diluted to 100 mL. Next,

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10 mL was pipetted to centrifuge at 12,000 × g for 10 min at 4 ℃. The supernatant was

169

analyzed by fluorescence spectrophotometer (F-4600, Hitachi Ltd., Ibaragi, Japan). The

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start excitation wavelength was set at 290 nm, the start and end emission wavelengths

171

were set at 300 nm and 500 nm. The scanning speed of 2400 nm/min, EX and EM slit of

172

5 nm, EX sampling interval of 10 nm and voltage of 400 V were used for analysis.

173

2.8 Measurement of viscosity

174

The viscosity was measured with a Physica MCR-301 rotational rheometer (Anton

175

Paar GmbH, Graz, Austria). The RJ sample was previously equilibrated to 25 ℃. The

176

measurement was taken three times with a Cone plate diameter of 50 mm, cone angle of

177

1℃, and shear rate of 50 s-1 at room temperature of 25 ℃.

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2.9 SDS-PAGE and Native-PAGE analysis

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Royal jelly samples of 1 g were homogenized in phosphate buffer (10 mL, 50 mM,

180

pH 7.0) containing 150 mM NaCl. Then, the homogenate was centrifuged at 10,000 × g

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for 30 min at 4 ℃. The supernatant was then preserved at 4 ℃ until use.

182

SDS-PAGE was carried out as described by Laemmli (1970) with minor modification.

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Samples (15 µL) were run at 150 V at room temperature. After the bromophenol blue dye

184

had run off the gel, the proteins in the gel were stained with Coomassie brilliant blue

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R-250. Molecular weights were calibrated using a standard pre-stained protein marker

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range from 10 to 170 kDa. 10


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Seven royal jelly samples were prepared in 2 × native sample buffer and loaded onto

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a precast Novex® 4 - 12% Tris-glycine gel. Native-PAGE was run with Tris-glycine

189

native running buffer at 125 V according to the manufacturer’s protocol (Invitrogen,

190

Carlsbad, CA). NativeMark™ unstained protein standards were used as molecular weight

191

markers.

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2.10 SEC-HPLC analysis

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To understand the chromatographic behavior of royal jelly proteins during

194

room-temperature storage, highly sensitive SEC-HPLC was performed with a model

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LC-6AD high-performance liquid chromatography instrument (Shimadzu Co., Ltd.,

196

Kyoto, Japan) equipped with a TSK-GEL G3000SWXL column (30 cm × 7.8 mm ID, 5

197

µm particles, Tohso, Tokyo, Japan).

198

Royal jelly sample preparation was the same as in section 2.8. The sample injection

199

volume was 50 µL. The elution buffer was 50 mM Tris-HCl (pH 7.5) with 150 mM

200

L-arginine. The flow rate was 0.5 mL/min. Protein elution profiles were monitored at

201

280 nm.

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2.11 LC-MS/MS analysis

203

To identify protein sequence, the two new bands from Native-PAGE were separated

204

for in-gel digestion. Then, the samples were disulfide reduced with 25 mM dithiothreitol

205

and alkylated with 55 mM iodoacetamide. Pepsin (Promega, Madison, WI, USA) was

206

used for in-gel digestion in 25% formic acid at 37°C overnight. The peptides were

207

extracted twice with 1% trifluoroacetic acid in 50% acetonitrile aqueous solution for 30 11



208 209

min. The peptide extracts were then centrifuged in a SpeedVac to reduce the volume. Peptide analyses were performed by LC–MS/MS using an Easy-nLC 1000 system

210

coupled to a Thermo Orbitrap Fusion mass spectrometer (Thermo-Fisher Scientific, San

211

Jose, CA, USA). The digestion products were loaded onto a Thermo Scientific Acclaim

212

PepMap C18 column (100 µm × 2 cm, 3 µm particle size) and eluted with a 60-min

213

gradient at a flow rate of 300 nL/min. Mobile phase A consisted of 0.1% formic acid in

214

water, and mobile phase B consisted of 0.1% formic acid in acetonitrile. The Orbitrap

215

Fusion mass spectrometer was operated in the data-dependent acquisition mode using

216

Xcalibur3.0 software, and there was a single full-scan mass spectrum in the Orbitrap

217

(350-1550 m/z, 120,000 resolutions) followed by top-speed MS/MS scans in the Ion-trap.

218

The MS/MS spectra from each LC-MS/MS run were searched against the Honeybee.fasta

219

from the UniProt/Swiss-Prot database using an in-house Proteome Discoverer (Version

220

PD1.4, Thermo-Fisher Scientific, USA).

221 222

3. RESULTS

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3.1. Non-enzymatic browning

224

Glycoprotein and AGEs are respectively the initial and final products of

225

non-enzymatic browning. Glycoprotein is formed by covalent crosslinking between

226

proteins and reducing sugars. Nitroblue Tetrazolium (NBT) Colorimetric Method is a

227

quantitative analysis for determining the content of glycoprotein. CML, one of

228

characterized AGEs markers, is usually quantified by enzyme-linked immunosorbent 12


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assay (ELISA). Fig. 1 A, B illustrates the content changes of glycoprotein (A) and CML

230

(B) in royal jelly during room-temperature storage. As can be seen, both glycoprotein and

231

CML tended to increase dramatically within six months. It is worthwhile to note that the

232

content of CML increased approximately six-fold and that the level of glycoprotein

233

improved nearly to two-fold.

234

In order to further study the non-enzymatic browning of royal jelly, the fluorescence

235

intensity changes of reaction products were measured at 347/415 nm (24). As can be seen

236

in Fig. 1C, the fluorescence intensity of different fluorescent products increased

237

obviously with the storage time. It is worth noting that the fluorescence intensity of all

238

fluorescent products increased more than one-fold.

239

3.2. Conformational changes in RJ proteins

240

Synchrotron radiation circular dichroism (SRCD) spectroscopy has many advantages

241

over circular dichroism (CD) spectroscopy. SRCD can provide high-intensity ultraviolet

242

and vacuum ultraviolet light, and has an especial ability to measure lower-wavelength

243

data. This somewhat helps to detect subtle changes in proteins and offer the secondary

244

structural information of proteins. We analyzed the secondary structure changes in royal

245

jelly proteins during room-temperature storage using SRCD. As can be seen, the royal

246

jelly proteins varied obviously in structure composition (Table 1). Fresh royal jelly

247

proteins consisted of β-sheets and β-turns up to 50.4%, while α-helixes only accounted

248

for 28%. The α-helixes and β-turns of the royal jelly proteins slowly decreased during

249

room-temperature storage, while the β-sheets and P2 structures gradually increased. It is 13



250

worth noting that α-helixes were reduced by 3%, while β-sheets and P2 increased by 1.3%

251

and 4.2%, respectively.

252

Fig. 2 presents the fluorescence wavelength scanning spectra of seven royal jelly

253

samples. As can be seen, the fluorescence peak positions and fluorescence intensities of

254

the samples were noticeably different. The fluorescence peak positions had a tendency to

255

red-shift when the storage time was prolonged. After storage for six months, the

256

fluorescence intensities of the samples decreased by nearly 70%, compared with fresh

257

royal jelly.

258

3.3. Protein aggregation

259

Viscosity refers to the internal friction of fluid. Supplementary Fig. 1 demonstrates the

260

viscosity changes in royal jelly analyzed by cone-plate viscometer. As can be seen, the

261

viscosities of royal jelly tended to increase rapidly within six months. The viscosity of

262

the sample during six-month storage was four-fold higher than that of fresh royal jelly.

263

Furthermore, a remarkable change was that the slope of the viscosity curve climbed

264

sharply during room temperature storage.

265

To investigate the changes in royal jelly proteins during room-temperature storage,

266

the samples were analyzed by SDS-PAGE and Native-PAGE. As can be seen on

267

SDS-PAGE (Fig. 3A), the bands of major royal jelly protein 1 (MRJP1) and MRJP2 did

268

not significantly change, while MRJP3 gradually weakened and MRJP5 disappeared

269

within 6 months. Native-PAGE showed that bands with molecular weights of 70 kDa,

270

290 kDa and 670 kDa slowly weakened during storage (Fig. 3B). It is worth noting the 14


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emergence of two new bands with molecular weights of 440 kDa and 700 kDa (Fig. 3B).

272

In previous studies, we analyzed royal jelly proteins by size exclusion chromatography

273

- high performance liquid chromatography (SEC - HPLC), and nine representative peaks

274

were obtained (25). Peaks 1-4 represented MRJP1 oligomer 2 (639 kDa), MRJP1

275

oligomer 1 (228 kDa), MRJP3s and MRJP2 with a small amount of MRJP 1 monomer in

276

sequence, while peaks 5-9 were small peptides and free amino acids (25). To detect

277

protein changes, the seven royal jelly samples were analyzed by SEC-HPLC. The

278

chromatographic profiles of all royal jelly samples looked very different. Most notable is

279

that a new peak (peak N) occurred between peak 1 and peak 2 after storage for three

280

months. The area of peak N after six-months of storage was approximately twice as

281

many as that after three-months storage. Another notable finding is that peak 6 gradually

282

disappears during storage.

283

The two new bands on Native-PAGE and the new peak (peak N) on SEC-HPLC were

284

then analyzed by LC-MS/MS to identify protein sequences. The data shown in Table 2

285

demonstrate that the 440 kDa protein was identified as MRJP1 of Apis mellifera L. with

286

179 matched peptides and 89.36% sequence coverage. The 700 kDa protein was also

287

identified as MRJP1 of Apis mellifera L. with 283 matched peptides and 91.64%

288

sequence coverage. Additionally, peak N was similarly identified as MRJP1 of Apis

289

mellifera L. with 221 matched peptides and 88.35% sequence coverage.

290

4. DISCUSSION

291

Prior work has documented that changes in royal jelly compounds have a significant 15



292

impact on its functional properties; Kamakura, for example, reported that royal jelly

293

during room-temperature storage gradually loses a caste differentiation property (10). In

294

this study, we analyzed changes in non-enzymatic browning products and proteins of

295

royal jelly during room-temperature storage.

296

Our results indicate that non-enzymatic browning occurs in royal jelly during

297

room-temperature storage. The experiments showed that the content of glycoprotein

298

improved nearly to two-fold (Figure 1A). Glycoprotein, the initial product of the

299

non-enzymatic browning reaction, is formed by covalent binding of proteins and

300

reducing sugars. Royal jelly contains 8.5% to 16.0% carbohydrates and 12% to 14%

301

protein (7, 11, 12); therefore, those compounds may be responsible for glycoprotein

302

formation. In addition, we find that the contents of AGEs, such as CML, pentosidine,

303

pentodilysine, crossline, pyrropyridine and argpyrimidine, increased along with storage

304

time; in particular, CML increased approximately 6-fold (Figure 1B). CML, as a final

305

product of non-enzymatic browning, has been used as an indicator of non-enzymatic

306

browning in foods (26). For instance, Birlouez-Aragona and colleagues determined the

307

non-enzymatic browning levels of milk by measuring the contents of CML (27) . Our

308

study seems to provide compelling evidence that royal jelly undergoes non-enzymatic

309

browning during room-temperature storage. It is the first study to our knowledge to

310

investigate glycoprotein and CML changes in royal jelly during storage.

311 312

Since royal jelly presents a large variety of bioactivities, its freshness is important for royal jelly quality. Studies have proposed a considerable number of markers for 16


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evaluating royal jelly quality. These markers mainly involve apalbumin 1 (a 57 kDa

314

proteins) (9, 10, 28), free amino acid (29), sugar (14), furosine (30), glucose-oxidase (30)

315

and 10-hydroxy-2-decenoic acid (32). However, these markers probably suffer from

316

some disadvantages; for example, the analytical method is not completely validated (8),

317

and the markers require uncommon instrumentation to be detected. In addition, they are

318

unable to clarify all the phases during the entire shelf life of royal jelly (8).

319

10-hydroxy-2-decenoic acid, a stable unsaturated fatty acid specific to RJ, also lacks

320

suitability as a freshness marker (8). Moreover, Shen et al. reported a very important

321

solution to detect royal jelly freshness using ELISA to quantity apalbumin 1 with a

322

highly specific anti-body (28). This solution seems to lack available ELISA kits in

323

commercial application. In this study, our results indicate that the non-enzymatic

324

browning of royal jelly gradually intensified along with storage time. CML, as a

325

non-enzymatic browning indicator, increased from 50.09 ng/g to 198.02 ng/g in royal

326

jelly after one month of storage (Figure 1B), suggesting that the content of CML may be

327

associated with the freshness of royal jelly. This finding seems to concur with

328

Birlouez-Aragon’s research that the CML level increased significantly in milk after heat

329

treatment (27). Furthermore, CML determination usually uses a commercial ELISA kit

330

with many advantages, such as simple operation and high sensitivity and specificity.

331

Thus, we recommend that CML can be recognized as a suitable freshness marker for

332

royal jelly.

333

Royal jelly proteins account for about 50% of royal jelly dry weight and 90% of them 17



334

belong to a family identified as the major royal jelly proteins (MRJPs) (33). The major

335

royal jelly protein (MRJP) family is a group of highly homologous proteins in honeybees.

336

So far, nine major royal jelly proteins (MRJP1-9) have been identified in the cDNA

337

sequences of a honeybee head cDNA library (34). MRJP1 is the most abundant

338

component among royal jelly proteins, making up about 31% of the total royal jelly

339

proteins. Our previous research demonstrates that MRJP1 can be found as a 57 kDa

340

monomer and three oligomers: a 228 kDa MRJP 1 oligomer 1, a 408 kDa MRJP 1

341

oligomer 2 and a 639 kDa MRJP 1 oligomer 3 (25). Kamakura reported in Nature (2011)

342

that royalactin (MRJP1 monomer) is considerably responsible for inducing queen

343

differentiation. In addition, improper storage leads to royal jelly losing queen

344

differentiation, and this loss may be contributed to the reduction of royalactin (10).

345

Kamakura supposed that this reduction can arise from royalactin degradation (10). In

346

addition, many studies have also showed that the reduction of royalactin was due to

347

protein degradation (1, 8, 9). However, our results provide reliable evidence that the

348

decrease of MRJP1 monomer on Native-PAGE is caused by aggregation rather than

349

degradation. As shown in Figure 3A, MRJP1 did not decrease on SDS-PAGE, indicating

350

no degradation. Along with the decrease of MRJP1 monomer, two new bands occurred

351

with molecular weight of 440 kDa and 700 kDa on Native-PAGE (Figure 3B). This

352

finding is consistent with Kamakura’s Native-PAGE in Supplementary Figure 3 (10).

353

However, Kamakura ignored these emerging bands and misinterpreted the MRJP1

354

monomer degradation. We identified the two new bands and the emerging peak N in 18


Page 18 of 32

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355

SEC-HPLC as MRJP1 of Apis mellifera L using LC-MS/MS (Table 2). This suggests that

356

MRJP1 monomer can undergo further aggregation with oligomers during improper

357

storage and then produce new oligomers with larger molecular weights. This finding

358

seems to explain that protein aggregates form clumps and precipitate, leading to increase

359

the viscosity (Supplementary Fig. 1). Chen also reported that water-soluble protein

360

aggregation may increase the viscosity of royal jelly (14). In addition, we find that

361

MRJP3 gradually weakened and MRJP5 disappeared within 6 months on SDS-PAGE

362

(Fig. 3A). We speculate that the disappearance of MRJP3 and MRJP5 may also be

363

attributed to protein aggregation. This finding is in accordance with Li’s reports that

364

MRJP3 could be polymerized during storage by proteomics analysis (1). This is probably

365

because the two proteins hold exceptional structures with a variable number of tandem

366

repeats (VNTR) located at the C-terminal part of the coding region. Further research is

367

necessary to investigate the aggregations of MRJP3 and MRJP5.

368

Protein aggregations are usually associated with conformational changes (35). Our

369

results suggest that royal jelly proteins undergo conformational unfolding during storage.

370

A partial unfolding reaction occurred in the secondary structure of royal jelly proteins,

371

leading to a decrease in α-helixes and an increase in β-turns (Table 1). Meanwhile, the

372

unfolding reaction also took place in the tertiary structure of royal jelly proteins, leading

373

to a red shift of the emission spectra and decrease of fluorescence intensity (Figure 2).

374

This suggests that fluorescent groups of proteins transfer from the internal hydrophobic

375

environment to the external hydrophilic environment. This unfolding structure can 19



376

promote royal jelly protein aggregation. This finding is consistent with Karoui’s report

377

that the proteins of egg yolks undergo conformational changes and aggregation during

378

room-temperature storage (36). On the other hand, protein aggregation may also be

379

associated with non-enzymatic browning. Miller reported that non-enzymatic browning

380

products may promote the formation of lysine-lysine cross-links, thus resulting in protein

381

aggregation (37). In addition, our previous research illustrated that the β-sheet levels of

382

MRJP1 monomer, MRJP 1 oligomer 1 and MRJP 1 oligomer 2 ranged from 60% to 33%

383

(25). This suggests that the high levels of β-sheet tend to aggregation (38) between

384

MRJP1 monomer and oligomers. Taking these results together, we think that the

385

aggregation of MRJP1 monomer with oligomers derives from secondary structure

386

characteristics, protein unfolding, and non-enzymatic browning.

387

Protein aggregation and non-enzymatic browning have significant impacts on the

388

nutritional value of foods. Protein aggregation usually changes biological activity and

389

protein digestibility (15). Furthermore, the products of non-enzymatic browning may also

390

decrease food digestibility (15). Thus, we provide a novel explanation that the

391

differentiation loss of royal jelly may be because the aggregation of MRJP1 monomer

392

with oligomers limits the honeybees' ability to digest and absorb royal jelly.

393

In conclusion, our results reveal that royal jelly experiences non-enzymatic browning

394

and protein aggregation during room-temperature storage. CML, as the non-enzymatic

395

browning product, can be recognized as an eligible freshness marker for royal jelly.

396

Protein aggregation may lead to the differentiation loss of royal jelly. Our further study 20


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Page 21 of 32


397

will focus on physiological and pharmacological functions of MRJP1 monomer and

398

oligomers, MRJP3 and MRJP5.

399

CONFLICTS OF INTEREST

400 401

The authors declare that there are no conflicts of interest.

ACKNOWLEDGEMENTS

402

This research was supported by the Modern Agro-industry Technology Research

403

System (CARS-44-KXJ19) and the Agricultural Science and Technology Innovation

404

Program (CAAS-ASTIP-2015-IAR) from the Ministry of Agriculture of P.R. China.

405 406

References:

407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427

1. Li, J.; Feng, M.; Zhang, L.; Zhang, Z.; Pan, Y., Proteomics Analysis of Major Royal Jelly Protein Changes under Different Storage Conditions. Journal of Proteome Research 2008, 7, (8), 3339. 2. Jr, P. R.; Peng, C. Y., Aging and development in social insects with emphasis on the honey bee, Apis mellifera L. Experimental Gerontology 2001, 36, (4–6), 695-711. 3. Detienne, G.; Haes, W. D.; Ernst, U. R.; Schoofs, L.; Temmerman, L., Royalactin extends lifespan of Caenorhabditis elegans

through epidermal growth factor signaling. Experimental Gerontology 2014, 60,

129-35. 4. Sver, L.; Orsolić, N.; Tadić, Z.; Njari, B.; Valpotić, I.; Basić, I., A royal jelly as a new potential immunomodulator in rats and mice. Comparative Immunology Microbiology & Infectious Diseases 1996, 19, (1), 31. 5. Matsui, T.; Yukiyoshi, A.; Doi, S.; Sugimoto, H.; Yamada, H.; Matsumoto, K., Gastrointestinal enzyme production of bioactive peptides from royal jelly protein and their antihypertensive ability in SHR. Journal of Nutritional Biochemistry 2002, 13, (2), 80-86. 6. Guo, H.; Kouzuma, Y.; Yonekura, M., Structures and properties of antioxidative peptides derived from royal jelly protein. Food Chemistry 2009, 113, (1), 238-245. 7. Bogdanov, S., Royal jelly, bee brood: composition, health, medicine: a review. Lipids 2011,. 8. Ciulu, M.; Floris, I.; Nurchi, V. M.; Panzanelli, A.; Pilo, M. I.; Spano, N.; Sanna, G., A possible freshness marker for royal jelly: the formation of 5-hydroxymethyl-2-furaldehyde as a function of storage temperature and time. Journal of Agricultural & Food Chemistry 2015, 63, (16), 4190. 9. Kamakura, M.; Fukuda, T.; Fukushima, M.; Yonekura, M., Storage-dependent degradation of 57-kDa 21



428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468

protein in royal jelly: a possible marker for freshness. Bioscience Biotechnology & Biochemistry 2001, 65, (2), 277-84. 10. Kamakura, M., Royalactin induces queen differentiation in honeybees. Nature 2011, 473, (7348), 478-483. 11. Viuda-Martos, M.; Ruiz-Navajas, Y.; Fernández-López, J.; Pérez-Alvarez, J. A., Functional properties of honey, propolis, and royal jelly. Journal of Food Science 2008, 73, (9), R117. 12. Sabatini, A. G.; Marcazzan, G. L.; Caboni, M. F.; Bogdanov, S.; Almeida-Muradian, L. B. D., Quality and standardisation of Royal Jelly. Journal of Apiproduct & Apimedical Science 2009, 1, (1), 1-6. 13. Jian-Ke, L. I.; Hua-Wei, L. I.; Zhang, L., Analysis of the Proteome of the Larvae of the High Royal Jelly Producing Worker Bees (Apis mellifera L.). Scientia Agricultura Sinica 2008, 41, (3), 880-889. 14. Chen, C.; Chen, S. Y., Changes in protein components and storage stability of Royal Jelly under various conditions. Food Chemistry 1995, 54, (2), 195-200. 15. Cécile, R.; Delphine, L.; Emilie, R.; Carole, P.; Thierry, S., Mitigation strategies of acrylamide, furans, heterocyclic amines and browning during the Maillard reaction in foods. Food Research International 2016, 90, 154-176. 16. Hodge, J. E., Hodge J E. Dehydrated foods: chemistry of browning reactions in model systems. J. Agr. Food Chem. 1:928-43, 1953. 1978,. 17. Radamendoza, M.; Sanz, M. L.; Olano, A.; Villamiel, M., Formation of hydroxymethylfurfural and furosine during the storage of jams and fruit-based infant foods. Food Chemistry 2004, 85, (4), 605-609. 18. Obayashi, H.; Nakano, K.; Shigeta, H.; Yamaguchi, M.; Yoshimori, K.; Fukui, M.; Fujii, M.; Kitagawa, Y.; Nakamura, N.; Nakamura, K., Formation of crossline as a fluorescent advanced glycation end product in vitro and in vivo. Biochemical & Biophysical Research Communications 1996, 226, (1), 37-41. 19. Srikanth, V.; Maczurek, A.; Phan, T.; Steele, M.; Westcott, B.; Juskiw, D.; Münch, G., Advanced glycation endproducts and their receptor RAGE in Alzheimer's disease. Neurobiology of Aging 2011, 32, (5), 763. 20. Kume, S.; Takeya, M.; Mori, T.; Araki, N.; Suzuki, H.; Horiuchi, S.; Kodama, T.; Miyauchi, Y.; Takahashi, K., Immunohistochemical and ultrastructural detection of advanced glycation end products in atherosclerotic lesions of human aorta with a novel specific monoclonal antibody. American Journal of Pathology 1995, 147, (3), 654. 21. Ehrlich, H.; Hanke, T.; Frolov, A.; Langrock, T.; Hoffmann, R.; Fischer, C.; Schwarzenbolz, U.; Henle, T.; Born, R.; Worch, H., Modification of collagen in vitro with respect to formation of Nepsilon-carboxymethyllysine. International Journal of Biological Macromolecules 2009, 44, (1), 51. 22. Chung, H. F.; Lees, H.; Gutman, S. I., Effect of nitroblue tetrazolium concentration on the fructosamine assay for quantifying glycated protein. Clinical Chemistry 1988, 34, (10), 2106-11. 23. Palombo, R.; Gertler, A.; Saguy, I., A Simplified Method for Determination of Browning in Dairy Powders. Journal of Food Science 2010, 49, (6), 1609-1609. 24. Morales, F. J.; Majsvan, B., A study on advanced Maillard reaction in heated casein/sugar solutions: colour formation. International Dairy Journal 1997, 7, (11), 675-683. 25.

Wang, X., Dong, J., Qiao, J., Zhang, G., Zhang, H. (2017). Purification and Characteristics of Major

Royal Jelly Protein 1-3 and Identification of Two Novel MRJP 1 Oligomers. Journal of Apicultural 22


Page 22 of 32

Page 23 of 32

469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503


Research. Under Review. 26. Erbersdobler, H. F.; Somoza, V., Forty years of furosine – Forty years of using Maillard reaction products as indicators of the nutritional quality of foods. Molecular Nutrition & Food Research 2007, 51, (4), 423. 27. Birlouez-Aragon, I.; Pischetsrieder, M.; Leclère, J.; Morales, F. J.; Hasenkopf, K.; Kientsch-Engel, R.; Ducauze, C. J.; Rutledge, D., Assessment of protein glycation markers in infant formulas. Food Chemistry 2004, 87, (2), 253-259. 28.

Shen, L. R.; Wang, Y. R.; Zhai, L.; Zhou, W. X.; Tan, L. L.; Li, M. L.; Liu, D. D.; Xiao, F.,

Determination of royal jelly freshness by ELISA with a highly specific anti-apalbumin 1, major royal jelly protein 1 antibody. J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2015, 16, (2), 155. 29. Boselli, E.; Caboni, M. F.; Sabatini, A. G.; Marcazzan, G. L.; Lercker, G., Determination and changes of free amino acids in royal jelly during storage. Apidologie 2003, 34, (34), 129-137. 30. Marconi, E.; Caboni, M. F.; And, M. C. M.; Panfili, G., Furosine: a Suitable Marker for Assessing the Freshness of Royal Jelly. Journal of Agricultural & Food Chemistry 2002, 50, (10), 2825-9. 31. Baggio, N., Royal jelly quality during storage [bee products]. Industrie Alimentari 1998, 37, (375), 1290-1294+1297. 32. Antinelli, J. F.; Zeggane, S.; Davico, R.; Rognone, C.; Faucon, J. P.; Lizzani, L., Evaluation of ()-10-hydroxydec-2-enoic acid as a freshness parameter for royal jelly. Food Chemistry 2003, 80, (1), 85-89. 33. Beye, M.; Neumann, P.; Schmitzova, J.; Klaudiny, J.; Albert, S.; Simuth, J.; Felder, M.; Moritz, R. F. A., A simple, non-radioactive DNA fingerprint method for identifying patrilines in honeybee colonies. Apidologie 1998, 24, (34), 255-263. 34. Albert, S.; Klaudiny, J., MRJP9, an ancient protein of the honeybee MRJP family with non-nutritional function. Journal of Apicultural Research 2007, 46, (2), 99-104. 35. Patra, P.; Somasundaran, P., Evidence of conformational changes in oil molecules with protein aggregation and conformational changes at oil-'protein solution' interface. Colloids & Surfaces B Biointerfaces 2014, 120, 132-141. 36. Karoui, R.; Kemps, B.; Bamelis, F.; Ketelaere, B. D.; Merten, K.; Schoonheydt, R.; Decuypere, E.; Baerdemaeker, J. D., Development of a rapid method based on front-face fluorescence spectroscopy for the monitoring of egg freshness: 2—evolution of egg yolk. European Food Research & Technology 2006, 223, (2), 180-188. 37. Miller, A. G.; Gerrard, J., The Maillard reaction and food protein crosslinking. 2005; Vol. 1, p 69-86 38.

Esposito, L.; Pedone, C.; Vitagliano, L., Molecular dynamics analyses of cross-β-spine steric zipper

models: β-Sheet twisting and aggregation. Proceedings of the National Academy of Sciences of the United States of America 2006, 103, (31), 11533.

504

23



Figure Captions Figure. 1. The contents of glycoprotein (A) and Nε-carboxymethyl lysine (CML) (B) and the fluorescence intensities of different fluorescence compounds(C) in royal jelly during room-temperature storage Figure. 2. The fluorescence wavelength scanning spectra of royal jelly during room-temperature storage Note: F, RT/1 to RT/6 represents fresh and stored one to six months royal jelly samples, respectively. Figure. 3. SDS-PAGE (A) and Native-PAGE (B) of royal jelly protein during room-temperature storage Note: lane Mr, standard; lane F, fresh royal jelly; lane RT/1 to RT/6, royal jelly proteins stored at room temperature one to six month. Figure. 4. Size exclusion chromatography of royal jelly during room-temperature storage Note: RT/0 represents fresh royal jelly; RT/1 to RT/6, royal jelly proteins stored at room temperature one to six month. Supplementary figure. 1. The viscosity changes of royal jelly during room-temperature storage Note: 1 The abscissa indicates the shear time and the ordinate indicates the viscosity of royal jelly sample. 2 RT/1 to RT/6 represents fresh and stored one to six months royal jelly sample. Supplement figure. 2. The level ratio of stored t fresh royal jelly protein on SDS-PAGE 24


Page 24 of 32

Page 25 of 32


(A) and Native-PAGE (B). Note: lane F, fresh royal jelly; lane RT/1 to RT/6, royal jelly stored at room temperature one to six month.

25



Page 26 of 32

Table 1. The secondary structure changes of royal jelly protein during room temperature storage

α-Helix

β-Sheet

β-Turns

P2

Unordered

F

0.228

0.267

0.237

0.101

0.167

RT/1

0.219

0.266

0.229

0.123

0.164

RT/2

0.213

0.265

0.220

0133

0168

RT/3

0.212

0.268

0.216

0.139

0.165

RT/4

0.211

0.266

0.221

0.135

0.167

RT/5

0.210

0.269

0.217

0.135

0.170

RT/6

0.198

0.280

0.212

0.143

0.167

F, fresh royal jelly sample; RT/1 to RT/6 represent stored 1-6 months royal jelly samples.


Page 27 of 32


Table 2. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of the two new

bands and peak N. Fractions

Accession NO.

Coverage (%)*

Peptides*

Mw/pI*

Protein Name

440 kDa

O18330

89.36

179

48.9/5.34

MRJP 1_Apis mellifera L.

Q8ISL8

40.22

15

7.9/4.9

Apisimin_Apis mellifera L.

O18330

91.62

283

48.9/5.34


Q8ISL8

56.81

9

7.9/4.9

Apisimin_Apis mellifera L.

O18330

88.35

221

48.9/5.34


Q8ISL8

41.72

6

7.9/4.9

Apisimin_Apis mellifera L

700 kDa

Peak N



Figure. 1. The contents of glycoprotein (A) and Nε-carboxymethyl lysine (CML) (B) and the fluorescence intensities of different fluorescence compounds(C) in royal jelly during room-temperature storage 174x282mm (300 x 300 DPI)


Page 28 of 32

Page 29 of 32


Figure. 2. The fluorescence wavelength scanning spectra of royal jelly during room-temperature storage Note: F, RT/1 to RT/6 represents fresh and stored one to six months royal jelly samples, respectively.

89x58mm (600 x 600 DPI)



Figure. 3. SDS-PAGE (A) and Native-PAGE (B) of royal jelly protein during room-temperature storage Note: lane Mr, standard; lane F, fresh royal jelly; lane RT/1 to RT/6, royal jelly proteins stored at room temperature one to six month.

115x202mm (600 x 600 DPI)


Page 30 of 32

Page 31 of 32


Figure. 4. Size exclusion chromatography of royal jelly during room-temperature storage Note: RT/0 represents fresh royal jelly; RT/1 to RT/6, royal jelly proteins stored at room temperature one to six month.

234x426mm (600 x 600 DPI)



TOC Graphic 84x47mm (300 x 300 DPI)


Page 32 of 32

Non-enzymatic browning and protein aggregation in royal jelly during

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