Effect of Seasonal on Strength of Bee and Volatile Compounds in Multi Flora Honey
Mohammed A. Salman*, Dhia F. Alfekaiki and Ali Zachi A. Alhilfi
College of Agriculture, University of Basrah, Basrah, Iraq
*Email: [email protected] Abstract
This study was performed in Basra - Iraq, the Aims of Identify of the seasonal effect on the strength of honeybee colonies and Determination of volatiles compounds in honey by Gas Chromatography-Mass Spectrometry (GC-MS), The results showed significant differences (p<0.05) between means , it showed an increase in the area of the Capped brood during the months March, April, May and June, then decreased in July and August, then increase activity during September and October and then decreased in November. the Determination of volatile compounds in spring and autumn honey it was appeared in both types the compounds: Diethyl Phthalate, and Acetic acid, [bis[(trimethylsilyl)oxy]phosphinyl]-, trimethylsilyl ester, and Octadecanoic acid, and Eicosane and Others in Different rates, the volatile Compounds in spring season honey appeared including: Diethyl Phthalate and Acetic acid, [bis[(trimethylsilyl)oxy]phosphinyl]-, trimethylsilyl ester and 4H-Pyran-4-one, 2,3- dihydro-3,5-dihydroxy-6-methyl- and 1,2-Benzenedicarboxylic acid, butyl 2- methylpropyl ester and Trisiloxane, respectively, while in autumn season honey appeared including: l-(+)- Ascorbic acid 2,6-dihexadecanoate, and Dodecanoic acid, and Diethyl Phthalate, and 6-Octadecenoic acid, and Hexatriacontane, respectively.
Key words: Seasonal change, bee brood, honey , GC-MS ,Volatile Compounds.
Introduction
The length of the honeybees life has been evaluated in All the seasons, it have a apparent bimodal apportionment at the temperature zones, the honeybees live for 30–
40 days in spring, while in summer 15–38 days, however in autumn 50–60 days, and for 150–200 days in winter, wherever it has been evaluated the variation of life length between seasons may come of foraging and activity of brood-rearing (Remolina &
Hughes. 2008).
The honey bee colonies strength is significantly positively correlated between the amount of brood breeding and The amount of bees in colonies prior nectar overflow correlated with the honey production is highly significantly positively (Gąbka. 2014).
Honey is a natural complex bee product from Multi floral sources may have various tastes and aromas due to several volatile compounds, The geographical and botanical
Annals of R.S.C.B., ISSN:1583-6258, Vol. 25, Issue 6, 2021, Pages. 10533 - 10541 Received 25 April 2021; Accepted 08 May 2021.
of the flora are also based on the methods of extraction for transformation (Jerkovic et al., 2010).So The botanical origin may be determined by a highest concentration of compounds in several types of honey collecting than in others(Castro- Várquez et al., 2006).
Materials and methods
All experiments was performed in Basrah –Iraq in 2019 on 9 colonies of Apis mellifera L. bees in hives All queens in hives were one-year old, produced from one reproductive queen, and were naturally intermarry, In the beginning of February until the end of November 6 colonies covering 6 combs. Check each colony included brood , adult bee pests. The Number of brood was estimated of the basis of brood surface area by Squares measuring of 2 cm 2.
Honey samples
Honey produced by two different seasons (spring and Autumn) from honey bee which was in blooming from the beginning in Basrah –Iraq. All The honey samples were producers from Apis mellifera the samples were kept in a dark place at 25 °C . Extraction of volatile organic compounds
The Clevenger SD unit has been used. The boiler was filled with 200 ml of the sample (80 g of honey /100 ml of water). The method was calibrated for 4 hours and the condenser of the unit was cooled for water at 20 °C (Eleftherios et al.,2005).
Conditions for GC-MS
The study was carried out using the Shimadzu GC-MS QP2010, The temperature of the injector was 280 °C. Inside the section processing, a solution of honey was injected with a split ratio of 1/60. Column capillary Rtx-5MS95 percent Dimethyl Polysiloxane-5 percent Diphenyl (30 m × 0.25 mm × 0.25 ìm). Career gas used helium at a steady speed of 1,00 mL/min. The oven temperature was as follows: at the first temperature of 60 °C, held for 2 min, then increased for 10 °C/min until 260 °C held for 10 minutes. The MS ionization capacity was 70 eV, the temperatures were as follows: interface 260 °C, source Ions 280 °C. Scanning the mass spectrophotometry from 40-550 (Khan et al., 2017).
Statistical analyses
Statistical analyses were performed and the significance differences between the means of groups were calculated by L.S.D test (Ali et al., 2019).
Results and discussion
The Seasonal change in the levels of capped bee brood:
The Fig 1 showed the change of Capped brood area of honey bees, which was based on various months of the year. the statistical analysis showed significant differences
between the means (P˂ 0.05) The mean of bee brood area in February Values was 4676.5 cm 2 , It increased during March, April, May and June to Values its highest level 9674.4 cm 2, 13245 cm 2 , 13957 cm 2, 13654.6 cm 2 respectively. Then it decreased in July and August, which is the lowest level of the brood Values 5047.3 cm
2 and 5679 cm 2 respectively. then The honeybees reactivated in September and October to 12467.4 cm2 and 10233 cm 2 respectively. In November the brood area was 4373 cm 2.
*L.S.D: 1525 significant differences (P <0.05) between means
Fig. 1: Seasonal change in the levels of capped bee brood.
The amount of brood is usually positively correlated with bee colony growth, the exact correlation differing on various sources, In the spring the strong colonies rearward more brood, however the differences are not forever statistically significant, and The observed seasonal variance in dry weight with the highest during April and May partly reflect colony evolution, with higher protein Enabled for larvae breeding in spring (Requier et al., 2015).
The strength of bee colony was proportional with the brood rearing, the strong colonies constantly reared more broods. A larger number of bee workers can care and feed to a larger area of brood (Bhusal et al.,2011), So The strength of colony depends on many factors in the spring, but firstly, on climatic conditions, so colony strength in wintering and measures taken during the spring (Jevtić et al.,2013).
Gąbka, J.(2014) found the positive correlation between the area of bee brood, honey and the colony strength at April to May, it caused by the reality that a more amuont of worker bees were obtainable to feeding and warming the brood.
Annals of R.S.C.B., ISSN:1583-6258, Vol. 25, Issue 6, 2021, Pages. 10533 - 10541 Received 25 April 2021; Accepted 08 May 2021.
Volatile Compounds in Honey:
The results in Fig 2 and Table 1 show the volatile compounds in honey in the spring season, the diagnosis of volatile compounds in honey by Gas Chromatography Mass Spectrometry (GC-MS) technique, From the figure shown, 50 peaks of volatile compounds are observed which are represented by the peak 14 of Diethyl Phthalate with a similarity of 89% , Followed by the peak 5 of Acetic acid, [bis[(trimethylsilyl)oxy]phosphinyl]-, trimethylsilyl ester with a similarity of 87%, Then the peak 1 was 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- with 96%
similarity, Then the peak 31 of the compound 1,2-Benzenedicarboxylic acid, butyl 2- methylpropyl ester with a similarity of 96%, Then the 9th peak, Trisiloxane, 1,1,1,5,5,5-hexamethyl-3,3-bis[(trimethylsilyl)oxy]- with the similarity 94%, Then the 40th peak of Cholest-5-en-3-ol, (3.alpha.)-, and the peaks of compounds Benzeneethanamine, N-[(pentafluorophenyl) methylene]-.beta., 3,4 - tris [(trimethylsilyl),17-(1,5-Dimethylhexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11, 12,13,14, 15, 16,17-tetradeca, and The compounds of Octadec-9-enoic acid, and other compounds have appeared, including [Dimethyl-(3-trimethylsilanyloxy-propyl)- silanyl]-benzene -, then the compound Octadecanoic acid, and Cyclopropanenonanoic acid, 2-[(2-butylcyclopropyl) methyl]-, methyl ester and other compounds within different Rates.
Fig. 2: GC-MS Chromatogram of Spring Season Honey.
Received 25 April 2021; Accepted 08 May 2021.
The Fig 3 and Table 2 show the volatile compounds in honey in the autumn season the diagnosis it through by the (GC-MS) technique, From the figure shown, 58 peaks of volatile compounds are observed which are represented by the peak 36 of l-(+)-
Table 1. Volatile compounds of spring season honey identified by GC-MS
Peak R.TimeArea% Name
1 7.251 5.87 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- 2 10.579 0.38 Benzeneacetic acid
3 10.730 0.08 Nonanoic acid
4 11.135 0.09 Methoxy-4-vinylphenol
5 11.606 7.07 Acetic acid, [bis[(trimethylsilyl)oxy]phosphinyl]-, trimethylsilyl ester 6 11.779 0.25 Phenol,2-etyl-4methyl-
7 12.039 0.13 Tetradecane, 4-methyl-
8 12.309 0.28 1H-Indene-1,2-diol, 2,3-dihydro-1-methyl-, cis-
9 15.468 4.81 Trisiloxane, 1,1,1,5,5,5-hexamethyl-3,3-bis[(trimethylsilyl)oxy]- 10 15.560 0.20 Dodecane, 2,6,11-trimethyl-
11 15.729 0.15 Heptadecane, 2,6,10,15-tetramethyl- 12 15.914 0.04 1-Octadecanesulphonyl chloride
13 16.120 0.16 1,3,5,7,9-Pentaethyl-1,9-dibutoxypentasiloxane 14 16.658 37.01 Diethyl Phthalate
15 17.002 0.21 Benzophenone
16 17.136 4.58 Benzeneethanamine, N-[(pentafluorophenyl)methylene]-.beta.,3,4-tris[(trimethylsilyl) 17 17.360 1.06 Cyclopropanenonanoic acid, 2-[(2-butylcyclopropyl)methyl]-, methyl ester
18 17.586 0.33 Tetracosane 19 17.684 0.13 Tetradecanal
20 17.935 0.20 2-Propenoic acid, (1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)] ester 21 18.034 0.43 Tetradecanoic acid
22 18.220 0.35 Eicosane 23 18.357 0.17 Octadecanal
24 18.465 0.08 2,6-Octadiene, 1-(1-ethoxyethoxy)-3,7-dimethyl- 25 18.505 0.10 1,2-Epoxynonane
26 18.624 0.70 Phthalic acid, decyl isobutyl ester
27 18.805 0.46 1-(4-Hydroxy-3,5-di-tert.-butylphenyl)-2-methyl-3-morpholinopropan-1-one 28 18.892 0.43 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione
29 18.955 0.23 Octadecanoic acid, 3-hydroxy-2-tetradecyl-, methyl ester, (2R,3R)- 30 19.078 0.47 Cyclopentadecanone, 2-hydroxy-
31 19.160 5.03 1,2-Benzenedicarboxylic acid, butyl 2-methylpropyl ester 32 19.331 0.76 Hexadecanoic acid, ethyl ester
33 19.453 0.39 .beta.-D-Glucopyranoside, methyl-4-O-decyl- 34 19.586 0.27 17-Pentatriacontene
35 19.724 3.15 [Dimethyl-(3-trimethylsilanyloxy-propyl)-silanyl]-benzene 36 19.865 0.87 Hexatriacontane
37 20.072 3.79 Octadec-9-enoic acid 38 20.185 3.14 Octadecanoic acid
39 20.276 4.28 17-(1,5-Dimethylhexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradeca 40 20.365 4.70 Cholest-5-en-3-ol, (3.alpha.)-
41 20.645 0.30 Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester 42 20.775 0.35 9-Tricosane,(Z)-
43 20.937 0.62 Tetratetracontane 44 21.145 0.62 Oxirane, hexadecyl- 45 21.350 0.99 13-Docosenamide, (Z)- 46 21.645 0.21 Tetratriacontane
47 22.033 3.73 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-, (all-E)- 48 22.534 0.50 Tetrapentacontane, 1,54-dibromo-
49 22.878 0.72 1,2-Benzenedicarboxylic acid, diisooctyl ester 50 23.409 0.17 Cholest-5-en-3-ol (3.beta.)-, nonanoate
Annals of R.S.C.B., ISSN:1583-6258, Vol. 25, Issue 6, 2021, Pages. 10533 - 10541 Received 25 April 2021; Accepted 08 May 2021.
Ascorbic acid 2,6-dihexadecanoate, with a similarity of 89% , Then the peak 17 was Dodecanoic acid with the similarity 96% , and the 18th peak, which was Diethyl Phthalate with similarity 94% ,then the 41th peak, which was 6-Octadecenoic acid, (Z)- with similarity 94%, and the peak 40 was Hexatriacontane with the similarity 96% ,and the peak 52 was 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23- hexamethyl-, (all-E)- with similarity 94% ,then the 47th peak which was compounds Tetratetracontane, then 2,5-Furandicarboxaldehyde, and Tetradecanoic acid,and Tetrapentacontane, 1,54-dibromo-, and Octadecanoic acid, and Cholest-5-en-3-ol, (3.alpha.)-Respectively and other compounds within a different rates.
Fig. 3: GC-MS chromatography of the autumn season of honey.
Table 2. Volatile Compounds in Autumn Season Honey Identified by GC-MS Peak R.Time Area% Volatile compound
1 4.934 0.42 Benzeneacetaldehyde
2 5.738 3.93 2,5-Furandicarboxaldehyde 3 6.946 0.10 Hexanoic acid, 2-ethyl-
4 7.249 0.37 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-
5 8.793 0.06 Decanal
6 9.582 1.68 2-Furancarboxaldehyde, 5-(hydroxymethyl)- 7 10.099 0.12 Benzaldehyde, 4-methoxy-
8 10.785 0.18 Nonanoic acid
9 10.864 1.37 Benzene, 1-methoxy-4-(1-propenyl)-
10 11.601 0.12 Acetic acid, [bis[(trimethylsilyl)oxy]phosphinyl]-, trimethylsilyl ester 11 13.253 0.23 n-Decanoic acid
12 14.098 0.04 Tetradecanal
13 15.464 0.12 Trisiloxane, 1,1,1,5,5,5-hexamethyl-3,3-bis[(trimethylsilyl)oxy]- 14 15.556 0.01 Dodecane, 2,6,11-trimethyl-
15 15.724 0.15 Heptadecane, 2,6,10,15-tetramethyl-
16 16.115 0.11 1,3,5,7,9-Pentaethyl-1,9-dibutoxypentasiloxane 17 16.516 13.57 Dodecanoic acid
18 16.661 6.90 Diethyl Phthalate 19 16.859 0.74 Epicedrol
20 16.973 0.70 Phenol, 2,6-bis(1,1-dimethylethyl)-4-(1-methylpropyl)- 21 17.102 0.44 2,6-Difluoro-3-methylbenzoic acid, dodecyl ester 22 17.186 1.50 Propylamine, N-[9-borabicyclo[3.3.1]non-9-yl]- 23 17.358 1.76 1,2-Benzenedicarboxylic acid, ditridecyl ester 24 17.592 2.18 Heptadecane
25 17.700 0.50 Tridecanal
26 17.790 0.43 Acetic acid, 3,7,11,15-tetramethyl-hexadecyl ester 27 17.882 0.56 Tetradecanal
28 18.064 2.96 Tetradecanoic acid
29 18.222 1.50 Eicosane 30 18.370 0.35 15-Octadecenal
31 18.464 0.30 2,6-Octadiene, 1-(1-ethoxyethoxy)-3,7-dimethyl- 32 18.500 0.30 2-Decanone, 5,9-dimethyl-
33 18.640 0.95 Pentadecanoic acid
34 18.890 2.07 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione 35 18.975 0.43 Hexadecanoic acid, methyl ester
36 19.255 15.83 l-(+)-Ascorbic acid 2,6-dihexadecanoate 37 19.336 1.58 Hexadecanoic acid, ethyl ester
38 19.485 0.58 i-Propyl 14-methyl-pentadecanoate 39 19.570 0.48 Sulfurous acid, octadecyl 2-propyl ester 40 19.840 5.45 Hexatriacontane 41 20.097 6.69 6-Octadecenoic acid, (Z)- 42 20.197 2.49 Octadecanoic acid
43 20.361 2.38 Cholest-5-en-3-ol, (3.alpha.)- 44 20.520 0.34 Oxirane, hexadecyl-
45 20.610 0.28 Tributyl acetylcitrate 46 20.776 0.57 9-Tricosene, (Z)-
47 20.940 4.08 Tetratetracontane 48 21.162 0.32 Pentadecane, 8-hexyl-
49 21.302 0.76 Hexasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11-dodecamethyl- 50 21.639 1.22 Tetratriacontane
51 21.837 0.35 Tetrapentacontane, 1,54-dibromo-
52 22.043 5.29 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-, (all-E)-
Annals of R.S.C.B., ISSN:1583-6258, Vol. 25, Issue 6, 2021, Pages. 10533 - 10541 Received 25 April 2021; Accepted 08 May 2021.
The main factors responsible for aroma are volatile organic compounds with many other factors like the flavor contribute, taste and physical, The volatile organic compounds can be produced from the nectar origin, from the conversion of flora compounds by the digestion process of the bees gut, from the honey handling, processing, during store from environmental, and microbial contamination (Jerkovic et al., 2009).
The variations between types of honey was dependent on differences on geographical origins, plant varieties and beekeeping practices. then, the botanical origin of honey must be determined Depending on plants metabolites including:
terpenes, benzene, norisoprenoids, and its derivatives (Castro- Várquez et al., 2006).
The chemical groups in the volatile compounds of honey belong included: aldehyde, hydrocarbon, alcohol, ketone, ester, furan, acid, pyran and benzene, ,terpenes and its compounds, norisoprenoids and cyclic compounds and sulfuric compounds (Barra et al.,2010).
Conclusions
The strength of honey bees varies according to the seasons of the year, as well as the honey components of volatile compounds. GC-MS is a possible, reliable method in screening of multi flora honeys for identification volatile compounds, This method can be diagnose the identity of honey quality by different volatile compounds.
Acknowledgments
The authors are thankful to the Basra Society for beekeeping and honey production to provide, hives and honey samples to complete this research.
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