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Alginulose is a reducing disaccharide, currently only found in natural stingless bee honey, difficult to synthesize chemically, and studies have shown that only certain microorganisms can convert sucrose through sucrose isomerase to produce isomaltulose and treginulose
.
These sucrose isomers have higher acid stability than sucrose, which in some ways can increase the competitive advantage
of the species.
In addition to causing metabolic diseases such as obesity, high blood pressure, high blood lipids, and high blood sugar, a diet high in sucrose can also cause systemic and brain inflammation, as well as increase the risk
of colorectal cancer.
Stingless bee honey contains a variety of functional ingredients, consumer demand for stingless bee honey-derived products is growing rapidly, and the identification, quality control and standards of stingless bee honey have important supporting significance
for the application of stingless bee honey.
However, the research on stingless bee products in China has just begun, and the research on its chemical composition and nutritional composition has just begun
.
Zheng Xing, Wang Kai*, Peng Wenjun* and others from the Bee Research Institute of the Chinese Academy of Agricultural Sciences took stingless bee honey in China as the research object, and analyzed the alginulose in the stingless bee honey samples in China by establishing a detection method for alginulose in stingless bee honey
.
Through the collection location, time and species of samples, the content and stability of alginulose in stingless bee honey were analyzed, aiming to provide a certain reference for the establishment of relevant food standards
.
1.
Chromatographic detection of sugar standards and honey samples
As shown in Figure 1A, the chromatograms of the six single standard solutions show that trehalose and sucrose peak times are exactly the same, but they can be significantly separated
from alginulose.
The three main high response peaks in the chromatogram of the stingless bee honey sample solution in Figure 1B were glucose (peak 1, retention time 16.
59 min), tretulose (peak 2, retention time 18.
61 min) and fructose (peak 3, retention time 23.
02 min).
Taking acacia honey, jujube nectar, wattle honey and manuka honey as the control of common honey, it can be seen from Figure 1B that the glucose and fructose content of stingless bee honey is lower than that of ordinary honey, but it contains higher tretulose
.
Due to the rich function and bioavailability of alginulose, the detection of alginulose content in stingless bee honey is of great research value
.
2.
High performance liquid chromatography-electrospray ion source quadrupole time-of-flight tandem mass spectrometry analysis of alginulose
As can be seen from Figure 2, the monomer isolated from stingless bee honey is consistent with the secondary spectrum of the alginulose standard, and the main secondary fragments of the precursor ion ([M-H]-)m/z 341.
108 9 are m/z 113, 119, 179 and 221
.
The main secondary fragments of sucrose in the precursor ion ([M-H]-)m/z 341.
108 9 are m/z 113, 119 and 179, without 221
in alginulose.
The main secondary fragments of maltose in the precursor ion ([M-H]-)m/z 341.
108 9 are m/z 113, 119, 161 and 221, which are also significantly different
from the secondary ions of alginulose.
This is consistent with the results of FLETCHER ET AL.
WITH UPLC-MS/MS analysis, tretulose and sucrose and maltose in the main secondary fragments of the precursor ion ([M-H]-)m/z 341.
108 9, and the three sugars are isomers
of each other.
Therefore, the target compound with a retention time of 18.
61 min can be further determined by high performance liquid chromatography-electrospray ion source quadrupole time-of-flight tandem mass spectrometry as alginulose
.
3.
Methodology verification
As can be seen from Figure 3, trebulose is completely separated from the other 5 sugars, and the retention time is consistent with
that of tretulose monomer.
Alginulose can be completely separated by section 1.
3.
2 chromatographic conditions, and the method has good selectivity, so this chromatographic condition can be used as a method
to detect the content of alginulose in honey.
The standard curve obtained by the detection of alginone standard solution of 0.
5, 1, 2.
5, 5, 10, 25, 50 mg/mL under the chromatographic conditions of section 1.
3.
2, the linear equation is Y=1.
56×105X+2.
21×102,R2=0.
999 6, so the linear relationship between treginlose in the range of 0.
5~50 mg/mL is good
.
This method determined alginulose with a LOD of 0.
07 mg/mL and a LOQ of 0.
22 mg/mL
.
It can be seen from Table 2 that the RSD of the peak area of six 50 mg/mL alginone standard solution was 2.
46% after injection, indicating that the reproducibility of this extraction method was good.
After the injection of the same alginulose standard, the RSD of the peak area was 0.
18%, indicating that the precision of the chromatographic method was good.
After the determination of three alginulose standards of the same mass concentration at different times, the RSD of the peak area was 2.
43%, indicating that the chromatographic method had good
stability within 5 days.
Table 3 shows that the recovery rate of different amounts of treginulose in fructose syrup is between 93.
50%~95.
71%, and the RSD is not higher than 0.
61%, indicating that this method has a good recovery rate and the results are accurate and reliable
.
4.
Determination of alginulose content in stingless bee honey samples
1~6 is yellow-streaked stingless bee honey collected in Yunnan in May 2020, and the mass fraction of alginulose is 22.
90%~30.
82%; Sample No.
7~10 was the top stingless bee honey collected in Yunnan in May 2020, and the mass fraction of alginulose was 17.
30%~27.
91%; Sample No.
11~13 was the stingless bee honey collected in Yunnan in May and November 2020, with a mass fraction of 19.
72%~27.
74% in alginulose.
Sample No.
14 was a mixture of yellow streak stingless bee, top stingless bee and Goth's stingless bee collected in the same bee farm in Yunnan in November 2020, with a treginone mass fraction of 24.
03%; Samples No.
15 and 16 collected in the same apiary in Hainan in November 2020 and November 2019 had similar mass fractions of alginulose of 18.
16% and 18.
52%, respectively.
Sample No.
17~21 was Heterotrigona itama stingless bee honey collected in Malaysia in 2019, with a mass fraction of 10.
36%~20.
92%; Sample No.
22~31 was a yellow streak stingless bee honey collected in Yunnan in January 2021, with a mass fraction of alginone sugar of 6.
97%~20.
61%.
Conclusion
The content of alginone in honey was determined by high performance liquid chromatography-differential detector, the honey sample was dissolved with pure water, the ion ligand exchange column Hi-Plex Pb (300 mm× 7.7 mm, 8 μm) was used as the separation column, pure water was used as the mobile phase, the flow rate was 0.
5 mL/min, the injection volume was 10 μL, the column temperature was 50 °C, the temperature of the differential detection cell was 50 °C, and the mass concentration of alginone sugar in the range of 0.
5~50 mg/mL was linear, R2 is 0.
999 6
.
By high performance liquid chromatography-electrospray ion source quadrupole time-of-flight tandem mass spectrometry, it can be seen that the target compound with a retention time of 18.
61 min in the stinging-free bee sample is alginulose
.
The methodological test confirmed that treginone sugar was separated from other carbohydrates at baseline, and the recovery rate of this method was high and reproducible, with LOD of 0.
07 mg/mL, LOQ of 0.
22 mg/mL, and recovery rate of 93.
50%~95.
71% (RSD≤0.
61%)
.
This method is simple, fast and quantitative, which can realize the accurate quantitative analysis of alginulose in stingless bee honey samples, which provides a scientific basis for the quality evaluation of stingless bee honey in China, and the detection method established in this study can also provide reference
for the development of relevant food standards.
01 Correspondence author profile
Peng Wenjun, male, born in 1970, second-level researcher, doctor, doctoral supervisor
.
Category C leading talent of agricultural science of Chinese Academy of Agricultural Sciences, director of the Bee Research Institute of Chinese Academy of Agricultural Sciences, chief scientist of the innovation team of resource insect product processing and function evaluation team, post expert of national bee industry technology system, mainly engaged in the research of bee product processing technology and equipment, extraction and identification of active ingredients of bee products, and nutritional function evaluation
of bee products 。 In recent years, he has won 1 second prize of National Technological Invention Award, 6 provincial and ministerial awards, 4 Academy Awards, published more than 100 papers in academic journals such as Nature Communications, Journal of Agricultural and Food Chemistry, published more than 10 academic works, and trained more than 10 doctoral and master's
students.
Wang Kai, male, born in 1988, Ph.
D.
, associate researcher, master supervisor
.
Since July 2016, he has been working at the Bee Research Institute of the Chinese Academy of Agricultural Sciences, and is now the scientific backbone of the Bee Product Quality and Risk Assessment Innovation Team of the Bee Research Institute of the Chinese Academy of Agricultural Sciences, with the main research direction of bees and bee products
.
He has presided over the National Natural Science Foundation of China, the Youth Fund, the Youth Innovation Special Project of the Chinese Academy of Agricultural Sciences, and the Special Project of Basic Scientific Research Business Funds of Central Public Welfare Scientific Research
Institutes.
In the past five years, he has published more than 60 papers in important journals at home and abroad, including 20 SCI papers published as the first/corresponding author (the highest impact factor of 14.
224), 1 ESI highly cited paper, and an H-index of 30
。 He is also the Associate Editor of Frontiers in Nutrition (2021 IF=6.
59, JCR Q1), BMC Complementary Medicine and Therapies (2021 IF= 2.
838), and Editorial Board Member
of Oxidative Medicine and Cellular Longevity (2021 IF= 7.
31).
02 First author profile
Zheng Xing, female, born in 1996, has been a doctoral student
at the Bee Research Institute, Chinese Academy of Agricultural Sciences since September 2019.
Mainly engaged in the extraction and identification of bioactive components of bee products, nutritional function and evaluation of bee products
.
During his studies at the Chinese Academy of Agricultural Sciences, he won the First Class Outstanding Student Scholarship, Outstanding Student Cadre, Outstanding Student Party Member and Outstanding Award for Postgraduate Mid-term Assessment
.
He has published one article each in the journals Nutrients, Food Science, and Food Industry Science and Technology, and two articles
in the journal China Bee Industry.
This article "Detection of Alginulose Content in Stingless Bee Honey by High Performance Liquid Chromatography - Differential Refractive Detection Method" is from Food Science, Vol.
43, No.
16, 2022, pages 217-225, authors: Zheng Xing, Wang Kai, Xue Xiaofeng, Wang Zhengwei, Pan Peng, Wu Liming, Zhao Yazhou, Peng Wenjun
.
DOI:10.
7506/spkx1002-6630-20210520-251
。 Click to view information about
the article.