3.1. Proximate composition
The differential FTIR patterns from bee bread, royal jelly, and bee propolis reflect the different components in these products (Fig. 1). The bands in the 3700 − 3100 cm-1 might connect to stretching vibration of OH groups of water or aromatic compounds. The peaks in the 2900 − 2700 cm-1 may link to the stretching vibration of CH, CH2, and CH3 groups of lipid and fatty acids. The bands in 1300 − 900 cm-1 related to the stretching of C = O of amides, C = C of aromatic, N-H of amines, or carboxyl groups in proteins. The bands at 1100 − 500 cm-1 are due to vibration of polysaccharides, including symmetric stretching of C-O-C and OH groups17.
The chemical composition (moisture content, ash, carbohydrate, fat, and protein ad energy values) of bee bread, bee propolis, and royal jelly product were compared with egg and soya and summarized in Table 1. Principal component analysis showed that the sum of the first and second major components accounted for 94.4% total variance of the changes, with the first major component (PC1) accounting for 65.7% (eigenvalue = 4.60) and the second major component (PC2) for 28.7% (eigenvalue = 2.00) (Fig. 2). The first principal component (PC1) is positively correlated with energy (0.458), ash (0.444), fatty acid (0.351), and protein (0.318). Therefore, increasing energy, ash, fatty acid, and protein values increase the value of the first principal component. PC1 is negatively correlated with digestibility (-0.421) and moisture (-0.414). The second principal component (PC2) is positively correlated with sugar (0.670). Therefore, increasing the values of sugar increase the value of the PC2. PC2 is negatively correlated with digestibility (-0.215), moisture (-0.279), protein (-0.462) and fatty acid (-0.456). Royal jelly in the first quarter is not closely related to the measured parameters. The bee bread and propolis in the second quarter are the most related to sugar and energy. Egg in the 3rd quarter is most associated with digestibility and moisture. In the 4th quarter, soya is most associated with a fatty acid, protein, and ash18.
Table 1
Proximate analysis of bee propolis, bee bread and royal jelly in comparison with egg and soya.
Chemicals
|
Bee propolis
|
Bee bread
|
Royal jelly
|
Egg
|
Soya
|
Moisture content (g/100g)
|
15.45 ± 1.14b
|
11.81 ± 0.90a
|
25.50 ± 1.90c
|
72.00 ± 3.0b
|
9.80 ± 0.75a
|
Ash (g/100g)
|
2.76 ± 0.22b
|
2.11 ± 0.15b
|
1.46 ± 0.12a
|
1.10 ± 0.05a
|
3.37 ± 0.27c
|
Protein (g/100g)
|
11.00 ± 0.85a
|
18.50 ± 1.40b
|
21.70 ± 1.55b
|
13.70 ± 1.11a
|
39.75 ± 1.50c
|
Fatty acid (g/100g)
|
13.34 ± 1.16a
|
11.40 ± 0.84a
|
12.50 ± 1.00a
|
11.50 ± 0.87a
|
33.86 ± 2.22b
|
Sugar (g/100g)
|
57.15 ± 3.25d
|
55.60 ± 3.30d
|
38.35 ± 2.45c
|
1.40 ± 0.20a
|
12.66 ± 1.12b
|
Energy (kcal/100g)
|
392.66 ± 14c
|
399.00 ± 12c
|
352.70 ± 11b
|
163.90 ± 6.7a
|
505.38 ± 15d
|
Digestibility (g/100g raw material)
|
5.69 ± 0.45a
|
11.56 ± 0.85b
|
15.85 ± 1.20c
|
11.25 ± 0.75b
|
21.35 ± 1.55d
|
Digestibility (g/100g protein)
|
51.68 ± 2.84a
|
62.49 ± 3.70b
|
73.04 ± 4.30c
|
82.12 ± 4.76d
|
53.71 ± 3.60a
|
The values are expressed as means ± SD for three replicate experiments. Mean values with different letters within a row are significantly different by Tukey test at (p < 0.05).
|
3.2. Fatty acid composition
The fatty acid composition of bee bread, bee propolis, and royal jelly product were stated in Table 2. Linoleic, palmitic, oleic, α-linolenic, behenic, and myristic acids are the primary fatty acids in propolis. Linolenic, palmitic, α-linolenic, myristic oleic, and behenic acids are the primary fatty acids in bee bread. α-linolenic, 2-dodecenedioic, 10-hydroxy-2-decenoic, decanedioic, linoleic, 10-hydroxydecanoic, 3-hydroxy-decanoic, palmitic, 11-hydroxy-dodecanoic, palmitoleic, and oleic acids are primary fatty acids in the royal jelly (Table 2). The fatty acid composition of fatty acids from bee bread, bee propolis, royal jelly from other geographical regions differs. Fatty acids such as; capric, linoleic, palmitic, stearic, linolenic, oleic, behenic, arachidic, lauric, decanoic, dodecanoic, tetradecanoic, octadecenoic, tetracosanoic, eicosanoic, and hexacosanoic acids, are reported in bee products19.
Table 2
Fatty acid composition (percent of area) of fatty acid from bee bread, bee propolis, and royal jelly in comparison with egg and soya.
Common Name
|
Systematic Name
|
Lipid Numbers
|
Bee propolis
|
Bee bread
|
Royal jelly
|
Egg
|
Soya
|
Levulinic acid
|
Levulinic acid
|
C5H8O3
|
0.00 ± 0.00a
|
0.75 ± 0.03a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
Benzoic acid
|
Benzoic acid
|
C7H6O2
|
0.43 ± 0.02b
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
Hyrocinnamic acid
|
Hyrocinnamic acid
|
C9H10O2
|
0.70 ± 0.03b
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
Cinnamic acid
|
Cinnamic acid
|
C9H8O2
|
0.43 ± 0.02b
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
Anethole
|
Anethole
|
C10H12O
|
2.80 ± 0.15c
|
0.00 ± 0.00a
|
0.40 ± 0.01b
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
Octadecane
|
Octadecane
|
C18H38
|
3.65 ± 0.17c
|
0.00 ± 0.00a
|
1.30 ± 0.05b
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
Pentacosane
|
Pentacosane
|
C25H52
|
3.46 ± 0.18b
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
Heptadecane
|
Heptadecane
|
C27H56
|
3.10 ± 0.14b
|
3.19 ± 0.17b
|
0.50 ± 0.02a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
Queen bee acid
|
10-hydroxy-2-decenoic acid
|
C10:1
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
19.50 ± 1.70b
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
Sebacic acid
|
decanedioic acid
|
C10:0
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
16.60 ± 1.45b
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
10-Hydroxydecanoic acid
|
10-hydroxydecanoic acid
|
C10:0
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
8.80 ± 0.73b
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
3-hydroxy-decanoic acid
|
3-hydroxy-decanoic acid
|
C10:0
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
7.50 ± 0.65b
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
2- dodecenedioic acid
|
2- dodecenedioic acid
|
C12:1
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
19.90 ± 1.65b
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
11-hydroxy-dodecanoic acid
|
11-hydroxy-dodecanoic acid
|
C12:0
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
2.40 ± 0.13b
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
Lauric acid
|
Dodecanoic acid
|
C12:0
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
Myristic acid
|
Tetradecanoic acid
|
C14:0
|
1.89 ± 0.008c
|
13.43 ± 0d
|
0.00 ± 0.00a
|
0.47 ± 0.03b
|
0.30 ± 0.01b
|
Myristoleic acid
|
9-tetradecenoic acid
|
C14:1n-5
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.15 ± 0.02b
|
0.12 ± 0.01b
|
Pentadecanoic acid
|
Pentadecanoic acid
|
C15:0
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.15 ± 0.02b
|
0.00 ± 0.00a
|
Palmitic acid
|
Hexadecanoic acid
|
C16:0
|
33.50 ± 2.00d
|
20.31 ± 1.30c
|
5.00 ± 0.23a
|
31.37 ± 2.35d
|
11.36 ± 1.00b
|
Palmitoleic acid
|
9-hexadecenoic acid
|
C16:1n-7
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
1.60 ± 0.07c
|
3.55 ± 0.25d
|
0.23 ± 0.02b
|
Hexadecadienoic acid
|
7,10-hexadecadienoic acid
|
C16:2n-6
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
Hexadecatrienoic acid
|
7,10,13-hexadecatrienoic acid
|
C16:3n-3
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
Margaric acid
|
heptadecanoic acid
|
C17:0
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.14 ± 0.03b
|
0.37 ± 0.06c
|
Heptadecanoic acid
|
10-heptadecenoic acid
|
C17:1n-7
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.16 ± 0.02b
|
0.08 ± 0.001a
|
Stearic acid
|
octadecanoic acid
|
C18:0
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
11.00 ± 1.13c
|
3.77 ± 0.77b
|
Oleic acid
|
9-Octadecenoic acid
|
C18:1n-9
|
27.27 ± 1.450d
|
10.89 ± 0.94b
|
1.55 ± 0.03a
|
38.10 ± 1.75e
|
20.88 ± 1.56c
|
Linoleic acid
|
9,12-octadecadienoic acid
|
C18:2n-6
|
11.04 ± 0.97b
|
24.04 ± 1.40c
|
4.45 ± 0.56a
|
9.53 ± 0.65b
|
52.70 ± 2.35d
|
α-Linolenic acid
|
9,12,15-octadecatrienoic acid
|
C18:3n-3
|
8.03 ± 0.54b
|
24.40 ± 1.30c
|
8.55 ± 0.55b
|
0.03 ± 0.001a
|
8.22 ± 0.37b
|
γ-Linolenic acid
|
6,9,12-octadecatrienoic acid
|
C18:3n-6
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
1.23 ± 0.05b
|
0.03 ± 0.01a
|
0.04 ± 0.01a
|
Arachidic acid
|
Eicosanoic acid
|
C20:0
|
0.00 ± 0.00a
|
0.02 ± 0.01a
|
0.00 ± 0.00a
|
0.07 ± 0.02a
|
0.04 ± 0.02a
|
Paullinic acid
|
13-Eicosenoic acid
|
C20:1n-7
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.47 ± 0.03a
|
0.00 ± 0.00a
|
Gondoic acid
|
11-eicosenoic acid
|
C20:1n-9
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.03 ± 0.01a
|
0.00 ± 0.00a
|
Eicosatrienoic acid
|
11,14,17-eicosatrienoic acid
|
C20:3n-3
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.07 ± 0.01a
|
0.02 ± 0.01a
|
Dihomo-γ-linolenic acid
|
8,11,14-Eicosatrienoic acid
|
C20:3n-6
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.07 ± 0.01a
|
0.05 ± 0.01a
|
Arachidonic acid
|
5,8,11,14-eicosatetraenoic acid
|
C20:4n-6
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
1.10 ± 0.04c
|
0.26 ± 0.02b
|
Eicosapentaenoic acid
|
5,8,11,14,17-Eicosapentaenoic acid
|
C20:5n-3
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.28 ± 0.02b
|
0.32 ± 0.02b
|
Behenic acid
|
Docosanoic acid
|
C22:0
|
3.09 ± 0.17b
|
2.18 ± 0.11b
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
Dosapentaenoic acid
|
4,7,10,13,16-docosapentaenoic acid
|
C22:5n-6
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.27 ± 0.012b
|
0.39 ± 0.02b
|
Cervonic acid
|
4,7,10,13,16,19-docosahexaenoic acid
|
C22:6n-3
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
2.13 ± 0.13b
|
0.00 ± 0.00a
|
Total FA
|
Total FA
|
Total FA
|
99.39 ± 2.80a
|
99.21 ± 3.0a
|
99.28 ± 2.65a
|
99.20 ± 2.0a
|
99.25 ± 2.70a
|
The values are expressed as means (standard deviation) of three replicates. Mean values with different letters within a row are significantly different by Tukey test at (p < 0.05).
|
Principal component analysis showed that the sum of the first and second major components accounted for 73.3% total variance of the changes, with the first major component (PC1) accounting for 45.7% (eigenvalue = 16.43) and the second major component (PC2) for 27.6% (eigenvalue = 9.93) (Fig. 3). The first principal component (PC1) is positively correlated with C14:1n5 (0.239), C15:0 (0.210), C16:1n7 (0.174), C17:0 (0.153), C17:1n7 (0.245), C18:0 (0.241), C18:1n9 (0.179), C20:0 (0.242), C20:1n7 (0.210), C20:1n9 (0.210), C20:3n3 (0.238), C20:3n6 (0.242), C20:4n6 (0.234), C20:5n3 (0.220), C22:5n6 (0.203), and C22:6n3 (0.210). PC1 is negatively correlated with benzoic acid (-0.105), hyrocinnamic acid (-0.105), cinnamic acid (-0.105), anethole (-0.123), octadecane (-0.146), pentacosane (0.105), heptadecane (-0.165), 10-hydroxy-2-decenoic acid (-0.103), decanedioic acid (-0.103), 10-hydroxydecanoic acid (-0.103), 3-hydroxy-decanoic acid (-0.103), 2-dodecenedioic acid (-0.103), 11-hydroxy-dodecanoic acid (-0.103), α-Linolenic acid (-0.146), and docosanoic acid (-0.146). The second principal component (PC2) is positively correlated with 10-hydroxy-2-decenoic acid (0.269), decanedioic acid (0.269), 10-hydroxydecanoic acid (0.269), 3-hydroxy-decanoic acid (0.269), 2-dodecenedioic acid (0.269), and 2-dodecenedioic acid (0.269). PC2 is negatively correlated with benzoic acid (-0.223), hyrocinnamic acid (-0.223), cinnamic acid (-0.223), anethole (-0.190), octadecane (-0.131), pentacosane (-0.223), heptadecane (-0.201), palmitic acid (-0.262), oleic acid (-0.194), and docosanoic acid (-0.246). Royal jelly in the first quarter is closely related to 2-dodecenedioic acid, 10-hydroxy-2-decenoic acid, decanedioic acid, 10-hydroxydecanoic acid, 3-hydroxy-decanoic acid, and 6,9,12-octadecatrienoic acid. The soya in the second quarter is the most related to linoleic acid, oleic acid and palmitoleic acid. Bee bread and bee propolis in the 3rd quarter are extremely associated with 9,12,15-octadecatrienoic acid, tetradecanoic acid, heptadecane, levulinic acid, hexadecanoic acid, octadecane, pentacosane, heptadecane, and docosanoic acid. In the 4th quarter, the whole egg is most associated with 9-octadecenoic acid, hexadecanoic acid, octadecanoic acid, 9,12-octadecadienoic acid, 9-hexadecenoic acid, 4,7,10,13,16,19-docosahexaenoic acid, and 5,8,11,14-eicosatetraenoic acid. Hydroxylated acids (like 10-hydroxy-2-decenoic, 10-hydroxydecanoic, and 3-hydroxydecanoic acids) and dicarboxylic fatty acids (like decanedioic and 2-dodecenedioic acids) are only found in royal jelly17. Myristic acid is only found in bee bread. Stearic acid finds in eggs and soya but not in bee products. Linoleic, palmitic, linolenic, and oleic acids are found in bee bread, bee propolis, royal jelly, egg, and soya but in different quantities18.
3.3. Lipid nutritional quality
The lipid nutritional quality of bee products including UFA, PUFA, omega-3 PUFA, SFA, omega-6 PUFA, MUFA, omega-7, omega-9, H/H, PUFA/SFA, AI, TI, NVI, and PI are stated in Table 3. Principal component analysis showed that the sum of the first and second major components accounted for 80.9% total variance of the changes, with the first major component (PC1) accounting for 50.0% (eigenvalue = 8.00) and the second major component (PC2) for 30.9% (eigenvalue = 4.94) (Fig. 4). The first principal component (PC1) is positively correlated with UFA (0.340), PUFA (0.325), Omega-6 (0.349), Omega-5 (0.135), Omega-6/Omega-3 (0.244), PUFA/SFA (0.352), H/H (0.325), PI (0.293), and NVI (0.241). PC1 is negatively correlated with SFA (-0.326), MUFA (-0.188), Omega-7 (-0.140), AI (-0.109), and TI (-0.168). The second principal component (PC2) is positively correlated with PUFA (0.138), Omega-3 (0.398), AI (0.216), PI (0.127). PC2 is negatively correlated with MUFA (-0.292), Omega-5 (-0.392), Omega-7 (-0.350), Omega-9 (-0.334), Omega-6/Omega-3 (-0.318), TI (-0.283), NVI (-0.309). Propolis and royal jelly in the first quarter is closely related to the AI. The bee bread in the second quarter is the most related to omega-3, PI, omega-6 and PUFA/SFA. Egg in the 3rd quarter is most associated with TI, MUFA, omega-9 omega-7. In the 4th quarter, soya is most associated with omega-5, NVI, omega-6/omega-3, UFA, H/H and PUFA/SFA17.
Table 3
Lipid nutritional quality of fatty acid from bee bread, bee propolis, and royal jelly in comparison with egg and soya.
Common Name
|
Bee propolis
|
Bee bread
|
Royal jelly
|
Egg
|
Soya
|
Saturated fatty acid (SFA)
|
38.48 ± 2.25d
|
35.94 ± 1.10cd
|
32.80 ± 1.85b
|
43.20 ± 2.50e
|
15.921 ± 0.90a
|
Unsaturated fatty acid (UFA)
|
46.34 ± 2.70a
|
59.33 ± 3.40b
|
56.78 ± 3.17b
|
55.99 ± 3.05b
|
83.33 ± 4.25c
|
Monounsaturated fatty acid (MUFA)
|
27.27 ± 1.70c
|
10.89 ± 0.75a
|
42.55 ± 2.57d
|
42.47 ± 2.40d
|
21.32 ± 1.24b
|
Polyunsaturated fatty acid (PUFA)
|
19.07 ± 1.20
|
48.44 ± 2.70
|
14.23 ± 0.85
|
13.52 ± 0.75
|
62.00 ± 3.50
|
Omrge-3 PUFA
|
8.03 ± 0.55b
|
24.40 ± 1.35c
|
8.55 ± 0.54b
|
2.51 ± 0.16a
|
8.56 ± 0.53b
|
Omega-6 PUFA
|
11.04 ± 0.77b
|
24.04 ± 1.40c
|
5.68 ± 0.45a
|
11.00 ± 0.70b
|
53.44 ± 3.00d
|
Omega-5
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
0.15 ± 0.01b
|
0.12 ± 0.01b
|
Omega-7
|
0.00 ± 0.00a
|
0.00 ± 0.00a
|
1.60 ± 0.07c
|
4.19 ± 0.27d
|
0.32 ± 0.02b
|
Omega-9
|
27.27 ± 1.75d
|
10.89 ± 0.76b
|
1.55 ± 0.08a
|
38.13 ± 2.25e
|
20.88 ± 1.35c
|
Omega-6/Omega-3
|
1.37 ± 0.08b
|
0.95 ± 0.06a
|
0.66 ± 0.05a
|
4.37 ± 0.26c
|
6.24 ± 0.40d
|
PUFA/SFA
|
0.49 ± 0.03a
|
1.35 ± 0.07b
|
0.43 ± 0.03a
|
0.31 ± 0.02a
|
3.89 ± 0.20c
|
Atherogenicity index (AI)
|
0.88 ± 0.04a
|
1.25 ± 0.05b
|
0.09 ± 0.01a
|
0.59 ± 0.03a
|
0.15 ± 0.01a
|
Thrombogenicity index (TI)
|
0.80 ± 0.05b
|
0.37 ± 0.02a
|
0.10 ± 0.01a
|
1.24 ± 0.07b
|
0.24 ± 0.01a
|
Hypocholesterolemic/hypercholesterolemic fatty acids (H/H)
|
1.30 ± 0.08a
|
1.76 ± 0.10a
|
2.91 ± 0.16a
|
1.49 ± 0.08a
|
7.02 ± 0.35b
|
Peroxidizability index (PI)
|
27.80 ± 1.70a
|
73.12 ± 4.0c
|
25.10 ± 1.50a
|
35.80 ± 2.00b
|
75.20 ± 4.40c
|
Nutritive value index (NVI)
|
0.81 ± 0.05b
|
0.54 ± 0.03a
|
0.31 ± 0.02a
|
1.56 ± 0.09b
|
2.17 ± 0.14c
|
The values are expressed as means (standard deviation) of three replicates. Mean values with different letters within a row are significantly different by Tukey test at (p < 0.05).
|
The high omega-3 diet causes a decrease in triglycerides, increases mitochondrial biogenesis, prevents inflammation, and restores insulin sensitivity. Besides, an omega-3 rich diet reduces chronic diseases such as obesity, diabetes, heart disease, increases blood vessels' elasticity, and prevents lipid plaques in the arteries20. Prostaglandin E3, thromboxane A3, prostacyclin I3, and leukotriene B5 generated from the omega-3 fatty acids can suppress thrombosis and inflammation. On the other hand, prostaglandin E2, prostacyclin I2, thromboxane A2, and leukotriene B4 are produced from omega-6 fatty acids, leading to pro-inflammatory and prothrombotic with the enhancement in blood viscosity21. The proportion of omega-6 / omega-3 is vital for lowering the risk of chronic diseases and preventing and managing obesity disorders. The ratio of omega-6/omega-3 lower than 3.0 is significant for healthy food. Bee products are important as sources of functional fatty acids with a well-proportioned omega-6 to omega-3, which is a priority for dietary supplements.
The H/H values describe the impacts of specific fatty acids on cholesterol level and metabolism. The higher proportion of H/H directly relates to the higher content of PUFA, which is beneficial for human healthiness. The P/S ratio is an index that expresses the lipid's nutritional quality in a specific diet. The high ratios of P/S consider undesirable for foodstuffs due to their potentials in inducing high blood cholesterol levels. The favorable ratio of PUFA/SFA that reflects the lipid nutritional quality of a specific diet is the range of 1–2 that reduce blood cholesterol and the risk of cardiovascular diseases15.
Low AI values indicate the higher protective of foodstuffs potentials in preventing heart and coronary diseases and general and abdominal obesities and AI values. Moreover, there is a direct relationship between gestational diabetes mellitus and AI in pregnant women14. Myristic acid and palmitic acid consider as the most atherogenic agents. Stearic acid is a thrombogenic fatty acid22. Foodstuffs with low TI values have a high potential to protect against coronary and heart diseases, reduce general and abdominal obesities, and prevent gestational diabetes mellitus in pregnant women. The AI and TI values of bee products are somewhat lower or similar to the AI values of egg and soya8.
The highest NVI values belong to soya, followed by egg and then bee propolis, bee bread, and royal jelly (Table 3). The NVI values of bee products range between 0.310 to 0.814, which is lower than an egg (1.565) and soya (2.172) (Table 3). The higher NVI values in the soya and egg attribute to the higher proportion of stearic and oleic acids and the lower levels of palmitic acid8. The lowest PI value belongs to royal jelly, followed by bee propolis, egg, and bee bread, then soya oil. Royall jelly and bee propolis had a PI value similar to egg, while the PI value of bee bread was identical to soya (Table 3). The higher PI value of soya and bee bread can be related to higher PUFA and MUFA in the soya and bee bread oil (Table 3). The PI represents the susceptibility of fatty acid to oxidation, and this index signifies the stability of PUFA against oxidation processes. The favorable ratio of PI that reflects the lipid nutritional quality of a specific diet is the range of 70–90 that reduces blood cholesterol and the risk of cardiovascular diseases. All bee products in this work have PI values lower than 90. Big PI values lead to a higher level of fatty acids oxidation. However, great PI values owing to the high content of omega-3 and omega-6 lead to more protective effects against oxidation and inflammation23.
3.4. Inhibition of the α-amylase activity
Bee propolis, bee bread, and royal jelly inhibit amylase with a lower level than acarbose. Kinetic parameters of amylase in the presence of acarbose and bee products measure through kinetic analysis. Figure 5a demonstrates plots of amylase activity at varying concentrations of acarbose. According to the Lineweaver-Burk plot, acarbose inhibits with a competitive inhibition trend. Since Vmax of amylase remains constant, in contrast, the Km/Vmax and Km increase (Table 4). Acarbose is a competitive inhibitor of amylase activity, and as the concentration of acarbose increases, the Vmax values do not change, but the Km values increase (Fig. 5A). Fatty acid from bee bread (Fig. 5B), royal jelly (Fig. 5C) and, bee propolis (Fig. 5D), on the other hand, are mixed un-competitive or non-competitive inhibitors. The Km/Vmax increases while Km and Vmax value decrease. By binding fatty acid to the allosteric site of the enzyme, this rises in the Km/Vmax ratio links to the reduction in the Vmax and functional enzyme24. The active compounds in fatty acid attach to allosteric sites, avoiding starch degradation. The enzyme conformation varies when inhibitors bind to the enzyme, and the affinity of the enzyme active site for starch reduces25.
Table 4
The values of kinetic parameters (Km /Vmax, Km, Vmax) of -amylase in response to acarbose bee bread, bee propolis and royal jelly fatty acid extract.
α
|
Samples
|
Parameters
|
0.00 mg/ml
|
0.30 mg/ml
|
0.60 mg/ml
|
0.90 mg/ml
|
Acarbose
|
Km/Vmax
|
4.78 ± 0.25a
|
9.07 ± 0.52b
|
10.04 ± 0.57b
|
13.12 ± 0.70c
|
Acarbose
|
Vmax
|
0.73 ± 0.04a
|
0.74 ± 0.05a
|
0.70 ± 0.04a
|
0.68 ± 0.03a
|
Acarbose
|
Km
|
3.47 ± 0.20a
|
6.74 ± 0.35b
|
7.06 ± 0.40b
|
8.96 ± 0.45c
|
Bee bread
|
Km/Vmax
|
5.05 ± 0.25a
|
7.52 ± 0.40b
|
8.39 ± 0.44c
|
9.40 ± 0.50d
|
Bee bread
|
Vmax
|
0.73 ± 0.04c
|
0.47 ± 0.03b
|
0.39 ± 0.02a
|
0.31 ± 0.02a
|
Bee bread
|
Km
|
3.84 ± 0.21c
|
3.60 ± 0.20bc
|
3.29 ± 0.18ab
|
2.96 ± 0.15a
|
Bee propolis
|
km/Vmax
|
4.69 ± 0.25a
|
5.79 ± 0.32b
|
6.78 ± 0.35c
|
7.68 ± 0.42d
|
Bee propolis
|
Vmax
|
0.93 ± 0.05d
|
0.70 ± 0.04c
|
0.48 ± 0,03b
|
0.36 ± 0.02a
|
Bee propolis
|
Km
|
4.38 ± 0.25b
|
4.05 ± 0.22b
|
3.28 ± 0.17a
|
2.78 ± 0.15a
|
Royal jelly
|
Km/Vmax
|
4.21 ± 0.23a
|
6.71 ± 0.35b
|
7.97 ± 0.45c
|
9.36 ± 0.50d
|
Royal jelly
|
Vmax
|
0.81 ± 0.04c
|
0.48 ± 0,03b
|
0.36 ± 0.02ab
|
0.28 ± 0,02a
|
Royal jelly
|
Km
|
3.43 ± 0.20c
|
3.23 ± 0.17bc
|
2.91 ± 0.15ab
|
2.63 ± 0.14a
|
The values are expressed as means (standard deviation) of three replicates. Mean values with different letters within a row are significantly different by Tukey test at (p < 0.05).
|
3.5. Ultraviolet and fluorescence spectroscopic analysis
Ultraviolet-visible absorption is one of the most effective methods for monitoring enzyme conformation changes during inhibitor binding26. This study examines the absorption spectra of amylase in the presence of acarbose and fatty acids from bee products (Fig. 6). The ultraviolet absorption peak around 210–220 nm relates to the carbonyl group ππ transition of the peptide bond. The ultraviolet absorption peak around 255–280 nm attributes to the ππ shift of aromatic groups in the protein sequence like tryptophan, tyrosine, and phenylalanine16. Fatty acid and acarbose at these wavelengths did not have ultraviolet absorption. By increasing the concentration of acarbose and fatty acid, the ultraviolet absorption of amylase regularly increases. Accordingly, ligands such as fatty acid and acarbose can establish complexes with amylase, alter their conformation and expose the aromatic group to ultraviolet light in this manner, increasing the ππ transition of aromatic groups27.
Another effective technique to monitoring the interactions between amylase and inhibitors such as acarbose and fatty acid is fluorescence quenching analysis (Fig. 7). The fluorescence emission spectra of the enzyme contribute to aromatic amino acids such as tyrosine, tryptophan, and phenylalanine28. Acarbose and fatty acid did not have a fluorescence emission at this condition. The amylase exhibits a fluorescence emission peak at 360 nm after excitation at 280 nm. By increasing the concentrations of acarbose and fatty acid, the fluorescence emission of amylase declines.
Furthermore, the fluorescence intensity slightly shifts toward the blue region (Fig. 7). The fluorescence quenching and shifting of amylase directly confirm the interaction between the enzyme and its inhibitors that reflect the changes in the structural architecture, conformation, surrounding environment, and polarity of the enzyme26. The molecular interactions between fatty acid or acarbose with amylase generate a non-fluorescent amylase-inhibitor complex and change the enzyme architecture and microenvironment. These effects increase the collision between fluorescent groups in the enzymes (aromatic amino acid) and quenching agents (inhibitors), leading to a decrease in the intrinsic fluorescence intensity28.
3.6. Molecular docking simulation of amylase
Molecular docking is a technique for predicting the binding direction of small molecules (ligands) to their protein targets, as well as their affinity. Acarbose is the competitive inhibitor of amylase, and the results of docking reveal that the interaction of acarbose with amylase occur mainly through Van der Waals interactions (with Ala106, Ala198, Arg195, Asn105, Asp197, Gly104, His101, His299, Ile235, Ile51, Leu162, Leu165, Trp58, Trp59, Tyr62, and Val107) and then hydrogen binding (with Gln63, Glu233, and Thr163 of amylase) (Table S1, Figure S1-S4 in supplemental file). The related affinity of amylase for acarbose was − 8.2 kcal/mol (Table S1, Figure S1-S4 in supplemental file). Similarly, molecular docking results revealed that the interactions of fatty with amylase occurred mainly through Van der Waals interactions (Ala198, Arg10, Arg195, Arg252, Arg303, Arg398, Asn298, Asp197, Asp300, Asp356, Gln63, Gln7, Gln8, Glu233, Gly403, Gly9, His101, His201, His299, His305, Ile235, Leu162, Leu163, Leu165, Lys200, Phe335, Pro332, Pro4, Thr11, Thr163, Thr6, Trp357, Trp58, Trp59, Tyr151, Tyr62) and then hydrogen binding (with Arg421, Asp197, Asp300, Asp356, Asp402, Gln63, Glu233, Gly334, His201, Thr163, Trp59) (Table S1, Figure S1-S4 in supplemental file). But the binding pocket for fatty acid differs from the binding pocket of acarbose. The related affinity of fatty acids for acarbose ranges from − 5.8 to 4.8 kcal/mol, lower than the affinity of amylase for acarbose (Table S1, Figure S1-S4 in supplemental file). The highest binding affinity related to the γ-linolenic acid and then 3-Hydroxy-decanoic acid. The fatty acid interacts with a binding pocket other than the active site or binding pocket near the active site. It inhibits amylase activity via an un-competitive or non-competitive inhibition strategy.