Floral Origin Identification and Amino Acid Profiles of Chinese Unifloral Honeys

Abstract

The aim of the current study was to investigate the properties of Chinese honeys. A total of 33 honeys were collected from various botanical and geographical sources in China. Nine unifloral honeys were identified as Acacia, Vetch, Linden, Coptidis, Red date, Astragalus, Codonopsis, Eriobotrya, and Eucalyptus according to their pollen granule characteristics. The chemical properties of free amino acids were further assessed; the total amino acid contents ranged from 240.7 mg/kg in light honey to 812.6 mg/kg in dark honey. Remarkable variations in proline and total amino acid contents were demonstrated in honeys from different botanical or geographical sources.

Keywords:

• Chinese unifloral honeys
• Floral sources
• Proline
• Free amino acids

INTRODUCTION

Honey is the natural sweet product produced by honey bees (Apis mellifera) from plant nectars, secretions (honeydew) of living parts of plants, or excretions of plant-sucking insects.[1] As a natural resource of sugars, honey has been used for infants, seniors, and invalids for a long time; it is a more easily digestible and palatable food carbohydrate than sucrose. Honey is widely appreciated as a food preservative[2,3] in the concentrated form of sugar.[4] Several researches have supported antimicrobial activity of honeys.[5–7 7

Honey is also considered to be one of the important traditional medicines since ancient times.[8] Its roles have been reported in the treatment of burns, gastrointestinal disorders, asthma, infections and skin ulcers,[9,10] cataracts, and other eye ailments.[6,9,11,12] Antioxidants in honey have been applied to treat different diseases caused by oxidative stress.[13,14] Honey has been used for effectively reducing the risk of heart diseases, cancers, immune system declination, cataracts, and different inflammatory processes.[15]

Amino acids account for around 1% (w/w) in honey. Proline, an indicator of honey quality,[16,17] is one of the predominant amino acid compositions that account for total amino acid contents. Proline content varies considerably in different honeys. Besides proline, there are 26 amino acids in honey; their relative proportions rely on the origin of honey. Proline and other amino acids in honey come from pollens, nectar, or are produced by the bees themselves.[18,19] Pollen from nectariferous plants is the major amino acid source of honey; the amino acid profile of a honey could reflect the characteristics of their botanical or geographical origin.[20]

Several honey samples from Burkina Faso were analyzed to determine their proline contents as well as their radical scavenging activities.[21] The correlation between radical scavenging activity and proline content was higher than that for total phenolic compounds in honeys. This suggested that the amino acid content of honey should be considered more frequently when determining its antioxidant activity. There is a long history of apiculture in China where it is rich in spices and has a wide distribution of honeybees. Some investigations have been done in Chinese honeybees and honey products, while profound studies on their chemical and biological properties are still limited. It is expected that the properties of various honeys are different because of different botanical and geographical sources. The major purpose of the current study was to evaluate nutritional and medicinal potentials of Chinese unifloral honeys, and to establish parameters valuable for further research. Therefore, the present work was to investigate the floral origins, and the food quality aspects in terms of physical properties (color, consistency, aroma, and taste) and chemical properties (amino acids).

MATERIALS AND METHODS

Honey Samples

A total of 33 honey samples were collected from various botanical and geographical sources in China (the original production places are listed in Table 1). All of the samples were harvested from 2009 to 2010 directly from beekeepers (without further processing) and then stored in dark conditions at a temperature between 0 and 4°C. Part of the unprocessed unifloral honeys were locally obtained from Wang's Bee Products (Changchun, China) with a guarantee of authenticity and known history.

Table 1  Pollen identification and sensory characteristics of Chinese unifloral honeys.^a

Categories	Sample ID	Floral origin (frequency)	Place of production	Color	Consistency	Aroma and taste
Acacia	H1	Robinia pseudoacacia L. (61.7%)	Western Gansu Province	Colorless	Crystallized, creamy	Light fragrant, mellow
	H2	Robinia pseudoacacia L. (62.1%)	Northern Shannxi Provence	White	Crystallized, creamy	Light fragrant, mellow
	H3	Robinia pseudoacacia L. (66.7%)	Kunming, Yunnan Province	Colorless	Clear, viscous	Sophora aroma, mellow
Vetch	H4	Vicia cracca L. (71.6%)	Yunnan Province	Ivory	Crystallized, creamy	Light fragrant, mellow
	H5	Vicia cracca L. (71.7%)	Changbai Mountains, Northeast of China	Ivory	Crystallized, creamy	Light fragrant, mellow
	H6	Vicia cracca L. (60.9%)	Jiangsu Province	Pale amber	Crystallized, creamy	Light fragrant
	H7	Vicia cracca L. (67.7%)	Sichuan Province	Pale amber	Noncrystalline	Light fragrant, mellow
Linden	H8	Tilia spp. (69.1%)	Tonghua, Jilin Province	Amber	Crystallized, creamy	Strong fragrant, mellow
	H9	Tilia mandschurica (76.8%)	Xing'an Forestry, Heilongjiang Province	Less amber	Crystallized	Mellow
	H10	Tilia mandschurica (69.1%)	Huadian County, Jilin Province	Pale amber	Noncrystalline	Fragrant
	H11	Tilia mandschurica (65.1%)	Antu County, Jilin Province	Pale amber	Crystallized, creamy	Strong fragrant
	H12	Tilia mandschurica (69.0%)	Xing'an Forestry, Heilongjiang Province	Pale amber	Noncrystalline	Strong fragrant
Coptidis	H13	Coptis chinensis (67.8%)	Changbai Mountains, Northeast of China	Pale yellow	Noncrystalline, transparent	Sweet, light bitter
	H14	Coptis chinensis (62.4%)	Sichuan Province	Yellow	Noncrystalline, transparent	Sweet, light bitter, agreeable sweetness
	H15	Coptis chinensis (62.4%)	Longyao, Hebei Province	Yellow	Noncrystalline, transparent	Sweet, light bitter, agreeable sweetness
	H16	Coptis chinensis (60.1%)	Beijing	Yellow	Noncrystalline	Sweet, light bitter
Red date	H17	Ziziphus jujuba Mill. (66.4%)	Dali County, Shannxi Province	Dark amber	Noncrystalline, transparent	Syrupy
	H18	Ziziphus jujuba Mill. (61.0%)	Zhongning, Ningxia	Dark amber, reddish	Noncrystalline	Syrupy, heavy aftertaste
	H19	Ziziphus jujuba Mill. (65.7%)	Xinjiang Autonomous Region	Amber	Noncrystalline	Syrupy
	H20	Ziziphus jujuba Mill. (67.7%)	Shandong Province	Less amber	Noncrystalline	Syrupy, heavy aftertaste
Astragalus	H21	Astragalus microcephalus Willd. (72.4%)	Northern Zhejiang Province	White	Noncrystalline	Fragrant, mellow
	H22	Astragalus microcephalus Willd. (74.8%)	Ganshu Province	Less amber	Crystallized, creamy	Fragrant, mellow
	H23	Astragalus microcephalus Willd. (60.4%)	Southern Zhejiang Province	Less amber	Noncrystalline	Fragrant
Codonopsis	H24	Codonopsis pilosula (Franch.) Nannf. (71.8%)	Eastern Ganshu Province	Less amber	Noncrystalline viscous	Fragrant, sweet
	H25	Codonopsis pilosula (Franch.) Nannf. (68.6%)	Northern Ganshu Province	Less amber	Noncrystalline	Fragrant, sweet
	H26	Codonopsis pilosula (Franch.) Nannf. (61.3%)	Ganshu Province	Less amber	Noncrystalline	Fragrant, sweet
Eriobotrya	H27	Eriobotrya japonica (Thunb.) Lindl. (62.7%)	Fujian Province	Light amber	Crystallized, granulated	Fragrant, mellow
	H28	Eriobotrya japonica (Thunb.) Lindl. (68.0%)	Zhejiang Province	Amber	Crystallized	Fragrant, mellow
	H29	Eriobotrya japonica (Thunb.) Lindl. (61.2%)	Zhejiang Province	Light amber	Noncrystalline	Fragrant
Eucalyptus	H30	Eucalyptus robusta Smith. (70.5%)	Guangdong Province	Dark amber	Crystallized, granulated	Eudesmol odor, sweet, light sour
	H31	Eucalyptus robusta Smith. (65.4%)	Fujian Province	Brown	Crystallized, granulated	Eudesmol odor, sweet, light sour
	H32	Eucalyptus exserta F. Muell. (66.2%)	Guangxi Province	Brown, reddish	Noncrystalline	Eudesmol odor, sweet, light sour
	H33	Eucalyptus citriodora Hook. F (63.9%)	Guangxi Province	Dark amber	Noncrystalline	Lemon odor, sweet, light astringent
^aGB 18796-2005 China Mandatory National Standards were utilized.

Chemicals and Reagents

The 17 amino acid standards, including alanine (Ala), arginine (Arg), aspartic acid (Asp), cysteine (Cys), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ileu), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tyrosine (Tyr), and valine (Val), as well as three internal standards, including norleucine (Nle), phenylisothiocyanate (PITC), and triethylamine (TEA), were obtained from Agela Technologies Inc. (Newark, DE, USA). Sodium acetate, acetonitrile, and methanol (high performance liquid chromatography [HPLC] grade) were obtained from Fisher Chemical Co. (Hampton, NH, USA). All other chemicals were of analytical grade.

Sensory Test of Chinese Unifloral Honeys

A series of organoleptic tests were performed to certify the validity of the floral origins of the collected honey samples. The floral origins of the samples were specified regarding hive locations, seasons, and available floral sources, and further confirmed by means of sensory analyses, such as aroma, taste, consistency, and color characteristics, according to previous reports.[10,16,22]

Pollen Analysis

All of the samples were subjected to pollen analysis according to the method of Lutier and Vassière.[23] For identification of the floral source, 5 g of diluted honey sample was centrifuged at 10,000 rpm for 15 min, to separate the pollens. The separated pollen granules were spread with the help of a brush on a slide containing a drop of lactophenol. The slide was examined microscopically at 400×, using an optical SZ61 bright-field microscope (Olympus, Tokyo, Japan). The slides of pollen granules from flowers of nectariferous plants (Acacia, Vetch, Linden, Coptodis, Red date, Astragalus, Codonopsis, Eriobotrya, and Eucalyptus) of Chinese honeys were also made for comparison. The aim of the analysis was to confirm that the analyzed samples could be declared as unifloral honeys.

Amino Acid Analysis of Honeys by HPLC

The total amino acid extracts were isolated from 33 honey samples according to the method described by Bouseta et al.,[24] starting from 12.5 g of honey treated with 375 μl of norleucine (an internal standard) solution (1 mg/ml in 0.1 N HCl). After extraction, the final volume was 10 ml, the pH was adjusted to 3.2, and the sample was refrigerated prior to derivatization. The extracts (300 μl) were dried under reduced pressure, at t ≤ 38°C, and allowed to react for 10 min with 18 μl of solution of water, triethylamine, and absolute ethanol (1:1:7 (v/v/v)), and 2 μl of phenylisothiocyanate. After filtration through a nylon membrane of 0.45 μm, direct injections of the derivatizated samples were subjected to a High Performance Liquid Chromatograph (Shimadzu, Tokyo, Japan), equipped with a Venusil-AA amino acid analysis column (5 μm, 250 × 4.6 mm, Angela Technologies Inc.). The CTO-10Avp thermostatted column compartment was set at 40°C during the HPLC analyses. Detection was done at 254 nm with an SPD-10Avp detector.

Chromatographic condition for separation was carried out according to Agela Chemical Co. with a gradient of two mobile phases. Phase A: sodium acetate buffer (25 mM, pH 6.5)–acetonitrile; Phase B: acetonitrile–water (v/v = 4:1). Linear gradient elution was programmed as 0.0 min, 0% B; 4 min, 3% B; 16 min, 11% B; 17 min, 21% B; 32 min, 34% B; 34 min, 100% B; 38 min, 0% B. The flow rate was set at 1.0 ml/min. Amino acid identification was done by means of comparing the retention times of HPLC peaks obtained from standard compounds and honey samples. Quantification was achieved using calibration curves obtained from amino acids and ammonium solutions of known concentrations containing the same amount of internal standard.

Statistical Analysis

All analyses were carried out in triplicate and the data were expressed as means ± standard deviations. Statistical analyses between experimental results were based on the Student's t-test. Significant differences were statistically considered at the level of p < 0.05.

RESULTS AND DISCUSSION

Sensory Properties

A sensory test of a honey sample was carefully conducted according to the international rules of the International Honey Committee. The harmonized methods of previous literature[10,16,22] and the European Honey Commission[25] were utilized. The honey samples were characterized according to several selected sensory characteristics, such as aroma, taste, color, and consistency. The main sensory characteristics are characterized in Table 1. The colors of Chinese honeys defined by visual observation were noticeably different and varied from almost colorless to dark brown. The brightest were Acacia, Eriobotrya, and Linden honeys, almost colorless to pale yellow and white to cream or ivory, with a yellow or dark brown tinge, respectively. Current results showed that honeys from different botanical and geographical sources possessed diverse sensory properties in aroma, color, and consistency, which verified sensory diversity of Mexico honeys.[26]

Identification of Floral Source

Pollen in natural honey comes from respective nectariferous plants; therefore, the pollen composition can be extensively applied to identify the botanical and geographical origin of honey, to discriminate honey types, and to examine honey quality as well. The microscopic observation on pollen morphologic characteristics and composition is one of the prevailing ways of honey examination. Generally, a honey is considered mainly from one plant (unifloral) if the pollen frequency of that plant is higher than 45%. Pollen granules from taxa are under- or over-presented in relation to their flowers yield of respective nectariferous plant. For unifloral honeys with under-represented pollen, the minimum percentage of the taxon that gives the honey name is 10–20% or 20–30%; for unifloral honeys with over-represented pollen, the minimum percentage of the taxon that gives the honey is 70–90%.[27] Pollen analysis was used to specifically identify the floral origins of 33 honeys and the results are shown in Table 1. The micro-structures of Acacia pollen, Vetch pollen, Linden pollen, Coptidis pollen, and Red date pollen were clearly presented on the microscopic slides.

The morphological characteristics of Acacia pollen were near triangle and three apertures, with a side length of 0.079 μm and height of 0.059 μm. While the morphological characteristics of pollen from Acacia honey were also triangle and three apertures, similar to the appearance and structure of Acacia pollen (Fig. 1a). The morphological characteristics of Vetch pollen were oval and one aperture, with a long diameter of 0.13 μm, and a short diameter of 0.086 μm. The morphological characteristics of pollen from Vetch honey were also oval and one aperture, similar to the appearance and structure of Vetch pollen (Fig. 1b). The morphological characteristics of Linden pollen were near triangle and three apertures, with a side length of 0.092 μm and height of 0.066 μm, while the morphological characteristics of pollen from Linden honey were also triangle and three apertures, similar to the appearance and structure of Linden pollen (Fig. 1c). The morphological characteristics of Ziziphus jujuba pollen were near triangle and three apertures, with a side length of 0.076 μm and height of 0.042 μm. The morphological characteristics of pollen from Red date honey were also triangle and three apertures, similar to the appearance and structure of Ziziphus jujuba pollen (Fig. 1d). The morphological characteristics of Coptidis pollen were near triangle and three apertures, with a long diameter of 0.064 μm, and a short diameter of 0.056 μm. The morphological characteristics of pollen from Coptidis honey were oval and three apertures, similar to the appearance and structure of Coptidis pollen (Fig. 1e).

Figure 1 Micro-structure of pollen granules from several unifloral honeys. (a) Acacia honey; (b) Vetch honey; (c) Linden honey; (d) Red date honey; (e) Coptidis honey. (Color figure available online.)

As shown in Table 1, the samples of honey H1, H2, and H3 were classified as Acacia (Robinia pseudoacacia L.). The samples of honey H4, H5, H6, and H7 were classified as Vetch (Vicia cracca L.). The samples of honey H8, H9, H10, H11, and H12 were classified as Linden (Tilia spp.). The samples of honey H13, H14, H15, and H16 were classified as Coptidis (Coptis chinensis). The samples of honey H17, H18, H19, and H20 were classified as Red date (Ziziphus jujuba Mill.). The samples of honey H21, H22, and H23 were classified as Astragalus (Astragalus microcephalus Willd.). The samples of honey H24, H25, and H26 were classified as Codonopsis (Codonopsis pilosula (Franch.) Nannf.). The samples of honey H27, H28, and H29 were classified as Eriobotrya (Eriobotrya japonica (Thunb.) Lindl.). The samples of honey H30, H31, H32, and H33 were classified as Eucalyptus (Eucalyptus robusta Smith.).

Free Amino Acid Contents in Chinese Unifloral Honeys

The quantitative determination of free amino acids in honeys were performed by following a RP-HPLC method in which the extraction and derivatization of analyses with phenylisothiocyanate (PITC) were developed and validated by Bouseta et al.[24] HPLC was able to separate and quantify 17 amino acids in honeys. The chromatogram of 17 amino acid standards is shown in Fig. 2a and a typical chromatogram of free amino acids from a Linden honey sample is shown in Fig. 2b. The proline contents varied from 139.2 to 631.5 mg/kg (Table 2). The highest proline content was observed in a Eucalyptus honey (sample H30, a dark-colored honey) and the lowest proline content was observed in a Vetch honey (sample H7, a light-colored honey). There were no significantly (p < 0.05) different proline contents within several species (Vetch, Red date, Astragalus, and Eriobotrya) of unifloral honeys from different geographical sources. Part of the samples from unifloral honeys (Acacia, Linden, Coptidus, Codonopsis, and Eucalyptus) with different geographical sources exhibited significant (p < 0.05) differences in proline contents, while part of samples from these sources did not exhibit significant (p < 0.05) differences in proline content within the same species of unifloral honey. Among all unifloral honey species, the Eucalyptus honeys exhibited the highest proline content (average 574.7 mg/kg), followed by Red date honeys (average 529.1 mg/kg), Coptidis honeys (average 423.1 mg/kg), Linden honeys (average 421.7 mg/kg), Codonopsis honeys (average 346.8 mg/kg), Eriobotrya honeys (average 285.4 mg/kg), Astragalus honeys (average 272.4 mg/kg), Acacia honeys (average 247.9 mg/kg), and Vetch honeys (average 144.9 mg/kg) in a descending order.

Figure 2 Chromatogram of amino acids. (a) Chromatogram of 17 amino acid standards; (b) typical chromatogram of a sample (Linden honey). 1. Asp; 2. Glu; 3. Ser; 4. Gly; 5. His; 6. Arg; 7. Thr; 8. Ala; 9. Pro; 10. Tyr; 11. Val; 12. Met; 13. Cys; 14. Ile; 15. Leu; 16. Nle (internal standard); 17. Phe; 18. Lys. (Color figure available online.)

Table 2 Amino acid contents of 33 Chinese unifloral honeys.^a

Categories of honey	Sample ID	Proline content (mg/kg honey)	Total amino acid content (17 free amino acids) (mg/kg honey)
Acacia	H1	191.1 ± 16.5 ^a B	341.4 ± 9.62 ^a C
	H2	268.6 ± 16.9 ^b B	351.3 ± 9.46 ^ab C
	H3	284.0 ± 18.8 ^b B	370.9 ± 5.83 ^b E
Vetch	H4	151.7 ± 9.35 ^a A	268.4 ± 7.90 ^b B
	H5	147.1 ± 10.9 ^a A	280.0 ± 8.26 ^b B
	H6	141.5 ± 9.18 ^a A	240.4 ± 9.60 ^a A
	H7	139.2 ± 8.62 ^a A	263.1 ± 6.98 ^b B
Linden	H8	368.4 ± 10.1 ^a EF	694.4 ± 11.1 ^a KL
	H9	411.5 ± 6.26 ^b G	780.4 ± 6.40 ^c O
	H10	451.0 ± 11.5 ^c IJ	750.9 ± 4.27 ^b N
	H11	438.1 ± 10.7 ^c GHI	684.8 ± 9.94 ^a K
	H12	439.3 ± 10.8 ^c HI	810.5 ± 1.10 ^d P
Coptidis	H13	451.5 ± 13.9 ^b IJ	800.5 ± 11.2 ^d P
	H14	467.3 ± 6.99 ^b J	710.7 ± 3.30 ^c LM
	H15	392.3 ± 8.70 ^a GH	625.6 ± 3.92 ^a I
	H16	381.1 ± 12.1 ^a F	610.2 ± 6.19 ^a IJ
Red date	H17	541.7 ± 12.9 ^a LM	810.9 ± 8.16 ^c P
	H18	521.2 ± 9.93 ^a KL	729.9 ± 5.29 ^b M
	H19	511.6 ± 8.08 ^a K	710.9 ± 0.88 ^a LM
	H20	541.8 ± 18.3 ^a LM	717.7 ± 7.53 ^ab M
Astragalus	H21	271.1 ± 8.12 ^a C	401.5 ± 3.68 ^a EF
	H22	267.9 ± 16.4 ^a C	379.5 ± 15.1 ^a E
	H23	278.3 ± 12.9 ^a C	388.9 ± 11.7 ^a DE
Codonopsis	H24	351.8 ± 8.27 ^b DE	546.4 ± 4.69 ^b H
	H25	327.1 ± 13.5 ^a D	518.6 ± 2.97 ^a G
	H26	361.4 ± 5.49 ^b EF	596.4 ± 7.73 ^c I
Eriobotrya	H27	285.2 ± 16.0 ^a C	410.9 ± 4.32 ^a F
	H28	279.8 ± 8.65 ^a C	398.4 ± 4.90 ^a EF
	H29	291.2 ± 8.40 ^a C	411.4 ± 10.4 ^a F
Eucalyptus	H30	631.5 ± 8.71 ^b N	768.7 ± 7.01 ^b NO
	H31	551.1 ± 8.74 ^a M	710.8 ± 21.6 ^a LM
	H32	568.2 ± 14.6 ^a M	754.4 ± 13.9 ^b N
	H33	547.8 ± 15.4 ^a LM	812.3 ± 11.1 ^c P
^aData are expressed as mean ± standard deviation (n = 3). Values marked by the same small case letter within the same floral source in each column are not significantly different (p < 0.05). Values marked by the same capital letter within each column are not significantly different (p < 0.05).

The total amino acid contents (TAACs) varied from 240.7 to 812.6 mg/kg (Table 2). The highest TAAC was also observed in Eucalyptus honey (sample H30) and the lowest TAAC was observed in a Vetch honey (sample H7). Significant (p < 0.05) different TAACs were found between unifloral honeys and between samples within the same species of unifloral honey except for Astragalus and Eriobotrya honeys. Among all unifloral honey species, the Eucalyptus honeys exhibited the highest TAAC (average 761.6 mg/kg), followed by Linden honeys (average 744.2 mg/kg), Red date honeys (average 742.4 mg/kg), Coptidis honeys (average 686.8 mg/kg), Codonopsis honeys (average 553.8 mg/kg), Eriobotrya honeys (average 406.9 mg/kg), Astragalus honeys (average 389.9 mg/kg), Acacia honeys (average 354.5 mg/kg), and Vetch honeys (average 262.9 mg/kg) in a descending order. A previous study revealed that various honeys contained 11–21 free amino acids with proline predominating.[28] Proline was the most abundant amino acid in honey, and was used as a standard to quantify amino acid content in honey.

CONCLUSIONS

A total of 33 honey samples from nine floral sources were collected from various geographical areas in China. Honey contains an extensive number of amino acids. Different honey has different compositions and contents in amino acids because of the diversity of geographical areas. The concentration and type of amino acid depends on the floral origin of honey and individual geographical areas in China. Amino acids contents found in Linden honeys were all higher than those in the light colored pure honeys (Acacia and Vetch honey), closer to those in dark colored honeys (Eucalyptus, Coptidis, and Red Date honey). The Linden honey samples appeared to deserve further investigation into its individual biological active components, which may be an attractive source of nutraceuticals and medicinal ingredients.

ACKNOWLEDGMENTS

This project was sponsored by Wang's Bee Garden. The authors would like to extend thanks to Wang's Bee Garden for kindly providing honey samples.