Author Contributions
The contributions made by several authors are described as follows: Formal analysis, C.K.C.-B., J.A.L.-P. and B.B.; Funding acquisition, J.-A.A. and B.B.; Investigation, C.K.C.-B., J.A.L.-P., T.T., R.T.-D., N.C.-F. and B.B.; Methodology, C.K.C.-B., J.A.L.-P., T.T., R.T.-D., N.C.-F. and B.B.; Project administration, J.-A.A. and B.B.; Supervision, J.-A.A. and B.B.; Writing – original draft, C.K.C.-B.; Writing – review & editing, C.K.C.-B., J.A.L.-P., J.-A.A. and B.B.
Figure 1.
Chemical Structures of bixin and norbixin.
Figure 1.
Chemical Structures of bixin and norbixin.
Figure 2.
Encapsulation efficiencies for pure bixin crystals (PB) or carbohydrates rich bixin (CRB) extract spray dried with different wall materials. Error bars represent the standard deviation (n = 6). (MD: Maltodextrin, GA: Gum arabic, CMC: Carboxymethylcellulose, CRB-encap: Spray dried CRB).
Figure 2.
Encapsulation efficiencies for pure bixin crystals (PB) or carbohydrates rich bixin (CRB) extract spray dried with different wall materials. Error bars represent the standard deviation (n = 6). (MD: Maltodextrin, GA: Gum arabic, CMC: Carboxymethylcellulose, CRB-encap: Spray dried CRB).
Figure 3.
SEM images of (a) CRB: Carbohydrates rich bixin extract prior spray drying; (b) CRB-encap: Microcapsules of spray dried CRB extract; (c) CRB-Sucrose: Microcapsules of spray dried CRB extract together with sucrose; (d) PB-Sucrose: Spray dried pure bixin together with sucrose.
Figure 3.
SEM images of (a) CRB: Carbohydrates rich bixin extract prior spray drying; (b) CRB-encap: Microcapsules of spray dried CRB extract; (c) CRB-Sucrose: Microcapsules of spray dried CRB extract together with sucrose; (d) PB-Sucrose: Spray dried pure bixin together with sucrose.
Figure 4.
SEM images of Spray Dried bixin microcapsules using different wall materials. Left column corresponds to PB (bixin crystals) microcapsules whereas right column shows CRB (Carbohydrate rich bixin extract) microcapsules. Non-encapsulated colorant crystals can be observed when the native carbohydrates of the annatto extract are absent. Systems contain: (a) PB-maltodextrin, (b) CRB-maltodextrin, (c) PB-maltodextrin-gum arabic, (d) CRB-maltodextrin-gum arabic, (e) PB-maltodextrin-carboxylmethylcecllulose, (f) CRB-maltodextrine-carboxylmethylcecllulose, (g) PB-maltodextrine-pectin, (h) CRB-maltodextrine-pectin, (i) PB-Whey, (j) CRB-Whey.
Figure 4.
SEM images of Spray Dried bixin microcapsules using different wall materials. Left column corresponds to PB (bixin crystals) microcapsules whereas right column shows CRB (Carbohydrate rich bixin extract) microcapsules. Non-encapsulated colorant crystals can be observed when the native carbohydrates of the annatto extract are absent. Systems contain: (a) PB-maltodextrin, (b) CRB-maltodextrin, (c) PB-maltodextrin-gum arabic, (d) CRB-maltodextrin-gum arabic, (e) PB-maltodextrin-carboxylmethylcecllulose, (f) CRB-maltodextrine-carboxylmethylcecllulose, (g) PB-maltodextrine-pectin, (h) CRB-maltodextrine-pectin, (i) PB-Whey, (j) CRB-Whey.
Figure 5.
Light microscope images of pure bixin and CRB microcapsules imbibed in MCT oil. (a) PB-Sucrose, non-polarized light ×100; (b) CRB-Sucrose, non-polarized (left) and polarized light (Right) ×50; (c) Bi-MD-GA, polarized light, ×50; (d) CRB-MD-GA, non-polarized light, ×100; (e) PB-Whey, non-polarized, ×50; (f) CRB-Whey, non-polarized (left) and polarized light (Right), ×100; (g) PB-MD dispersed in water, non-polarized light, ×20; (h) PB-MD dispersed in water, ×50; (g,h) are representative images of all the water dispersions of spray dried systems containing PB and CRB respectively.
Figure 5.
Light microscope images of pure bixin and CRB microcapsules imbibed in MCT oil. (a) PB-Sucrose, non-polarized light ×100; (b) CRB-Sucrose, non-polarized (left) and polarized light (Right) ×50; (c) Bi-MD-GA, polarized light, ×50; (d) CRB-MD-GA, non-polarized light, ×100; (e) PB-Whey, non-polarized, ×50; (f) CRB-Whey, non-polarized (left) and polarized light (Right), ×100; (g) PB-MD dispersed in water, non-polarized light, ×20; (h) PB-MD dispersed in water, ×50; (g,h) are representative images of all the water dispersions of spray dried systems containing PB and CRB respectively.
Figure 6.
Aqueous dispersions of CRB (a) and PB (b) microcapsules at 0.3 mg/mL and 1.5 mg/mL respectively at alkaline pH. Aqueous dispersions of CRB and CRB-encap at 0.048 mg/mL. From left to right I: MD, II: MD-GA, III: MD-CMC, IV: MD-Pectin, V: Whey, VI: Sucrose, VII: (a) CRB-encap, VII: (b) CRB.
Figure 6.
Aqueous dispersions of CRB (a) and PB (b) microcapsules at 0.3 mg/mL and 1.5 mg/mL respectively at alkaline pH. Aqueous dispersions of CRB and CRB-encap at 0.048 mg/mL. From left to right I: MD, II: MD-GA, III: MD-CMC, IV: MD-Pectin, V: Whey, VI: Sucrose, VII: (a) CRB-encap, VII: (b) CRB.
Figure 7.
Visible spectra of pure bixin microcapsules aqueous solution/dispersions before and after physical stability tests. At 0 h, concentrations of CRB and CRB-encap were 0.1 mg/mL and 0.048 mg/mL respectively. Visible spectra after physical stability tests were taken of the supernatant of samples prepared at 0.3 mg/mL for CRB and CRB-encap. For all other systems, 1.5 mg/mL was employed. The solid lines show the visible spectra at time 0 h, whereas the dashed line shows the lack of absorbance (characteristic bands) after the colorant particles have settled.
Figure 7.
Visible spectra of pure bixin microcapsules aqueous solution/dispersions before and after physical stability tests. At 0 h, concentrations of CRB and CRB-encap were 0.1 mg/mL and 0.048 mg/mL respectively. Visible spectra after physical stability tests were taken of the supernatant of samples prepared at 0.3 mg/mL for CRB and CRB-encap. For all other systems, 1.5 mg/mL was employed. The solid lines show the visible spectra at time 0 h, whereas the dashed line shows the lack of absorbance (characteristic bands) after the colorant particles have settled.
Figure 8.
Visible spectra of pure CRB microcapsules aqueous solution/dispersions before and after physical stability tests. Stability tests conducted at 0.3 mg/mL for all systems. The solid lines show the characteristic bands for the CRB systems at 0 h, whereas the dashed lines show the decrease in the absorbance after 40 days. Notice the lack of absorbance for CRB-Whey (dashed line) after 40 days.
Figure 8.
Visible spectra of pure CRB microcapsules aqueous solution/dispersions before and after physical stability tests. Stability tests conducted at 0.3 mg/mL for all systems. The solid lines show the characteristic bands for the CRB systems at 0 h, whereas the dashed lines show the decrease in the absorbance after 40 days. Notice the lack of absorbance for CRB-Whey (dashed line) after 40 days.
Figure 9.
Delta transmittance profile of Bi-MD-Pectin microcapsules dispersed in water obtained over 40 days of storage in sample flasks. Sedimentation is shown in the bottom of the flasks (heights between 1 mm and 3 mm) and flocculation in the middle section of the flasks (heights between 3 mm and 37 mm). A blue line shows proximity to day 0. A red line shows proximity to day 40. (a) is an expansion of (b) between 1 mm and 3 mm.
Figure 9.
Delta transmittance profile of Bi-MD-Pectin microcapsules dispersed in water obtained over 40 days of storage in sample flasks. Sedimentation is shown in the bottom of the flasks (heights between 1 mm and 3 mm) and flocculation in the middle section of the flasks (heights between 3 mm and 37 mm). A blue line shows proximity to day 0. A red line shows proximity to day 40. (a) is an expansion of (b) between 1 mm and 3 mm.
Figure 10.
CRB and PB microcapsules dissolved/dispersed in water after 40 days of the physical stability study (0.3 mg/mL). From left to right: I: CRB-MD, II: CRB-Sucrose, III: CRB-Whey, IV: CRB-encap, V: CRB, VI: CRB-MD-CMC, VII: CRB-MD-GA, VIII: CRB-MD-Pectin, IX: PB-MD, X: PB-Whey, XI: PB-Sucrose, XII: PB-MD-GA, XIII: PB-MD-CMC.
Figure 10.
CRB and PB microcapsules dissolved/dispersed in water after 40 days of the physical stability study (0.3 mg/mL). From left to right: I: CRB-MD, II: CRB-Sucrose, III: CRB-Whey, IV: CRB-encap, V: CRB, VI: CRB-MD-CMC, VII: CRB-MD-GA, VIII: CRB-MD-Pectin, IX: PB-MD, X: PB-Whey, XI: PB-Sucrose, XII: PB-MD-GA, XIII: PB-MD-CMC.
Figure 11.
Monosaccharide composition of the hydrolyzed native carbohydrates determined by HPAEC-PAD. The chromatograms show presence of arabinose, galactose and glucose. The standards include arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl) and manose (Man). The samples were analyzed in triplicate (a, b, c).
Figure 11.
Monosaccharide composition of the hydrolyzed native carbohydrates determined by HPAEC-PAD. The chromatograms show presence of arabinose, galactose and glucose. The standards include arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl) and manose (Man). The samples were analyzed in triplicate (a, b, c).
Figure 12.
Linear tendency of increasing encapsulation efficiency as a result of decreasing average volume diameter of colorant particles. Each of the encapsulated system replicas was graph against their encapsulation efficiency. Two parallel tendencies are observed: ♦ CRB microcapsules and ■ PB microcapsules.
Figure 12.
Linear tendency of increasing encapsulation efficiency as a result of decreasing average volume diameter of colorant particles. Each of the encapsulated system replicas was graph against their encapsulation efficiency. Two parallel tendencies are observed: ♦ CRB microcapsules and ■ PB microcapsules.
Table 1.
Initial compositions of the total solids for each prepared system prior spray drying.
Table 1.
Initial compositions of the total solids for each prepared system prior spray drying.
System | Bixin | Native Carbohydrates | Sucrose | MD | Whey | CMC | Pectin | GA | Na2CO3 |
---|
PB-Sucrose | 9.7% | | 87.6% | | | | | | 2.6% |
PB-MD | 9.7% | | | 87.6% | | | | | 2.6% |
PB-MD-GA | 9.7% | | | 73.0% | | | | 14.6% | 2.6% |
PB-MD-CMC | 9.7% | | | 86.7% | | 1.0% | | | 2.6% |
PB-MD-Pectin | 9.7% | | | 82.8% | | | 4.9% | | 2.6% |
PB-Whey | 9.7% | | | | 87.6% | | | | 2.6% |
CRB-Sucrose | 11.9% | 11.9% | 71.3% | | | | | | 4.9% |
CRB-MD | 11.9% | 11.9% | | 71.3% | | | | | 4.9% |
CRB-MD-GA | 11.9% | 11.9% | | 57.0% | | | | 14.3% | 4.9% |
CRB-MD-CMC | 11.9% | 11.9% | | 70.3% | | 1.0% | | | 4.9% |
CRB-MD-Pectin | 11.9% | 11.9% | | 66.5% | | | 4.8% | | 4.9% |
CRB-Whey | 11.9% | 11.9% | | | 71.2% | | | | 5.1% |
CRB | 50% | 50% | | | | | | | |
Table 2.
Total bixin (TB) contents and water activity (Wa) of spray dried powders, volume weighted average diameter of the microcapsules dissolved/dispersed in water after spray drying , and the one of their supernatants after 40 days of the physical stability test (). (--: not measured, nd: not detected, (1) & (2): Replicates 1 and 2).
Table 2.
Total bixin (TB) contents and water activity (Wa) of spray dried powders, volume weighted average diameter of the microcapsules dissolved/dispersed in water after spray drying , and the one of their supernatants after 40 days of the physical stability test (). (--: not measured, nd: not detected, (1) & (2): Replicates 1 and 2).
Sample | TB (%) | Wa | | | Sample | TB (%) | Wa | | |
---|
CRB * | 36.0 ± 1.60 | -- | (1) | 74.5 | nd | CRB-encap | 49.3 ± 2.14 | 0.41 | (1) | 8.86 | 0.19 |
(2) | 42.4 | -- | (2) | 6.57 | -- |
PB-Sucrose | 6.2 ± 0.41 | 0.37 | (1) | 7.44 | nd | CRB-Sucrose | 8.46 ± 0.11 | 0.30 | (1) | 7.62 | 0.23 |
(2) | 6.45 | -- | (2) | 17.54 | -- |
PB-MD | 8.8 ± 1.31 | 0.39 | (1) | 10.25 | nd | CRB-MD | 9.62 ± 0.58 | 0.27 | (1) | 4.54 | 0.31 |
(2) | 4.23 | -- | (2) | 4.72 | -- |
PB-MD-GA | 8.4 ± 0.62 | 0.38 | (1) | 15.69 | nd | CRB-MD-GA | 9.63 ± 0.23 | 0.29 | (1) | 4.78 | 0.25 |
(2) | 4.23 | -- | (2) | 5.09 | -- |
PB-MD-CMC | 6.5 ± 1.56 | 0.41 | (1) | 11.46 | nd | CRB-MD-CMC | 7.34 ± 0.12 | 0.28 | (1) | 7.94 | 0.24 |
(2) | 10.85 | -- | (2) | -- | -- |
PB-MD-Pectin | 7.4 ± 0.29 | 0.38 | (1) | 16.34 | nd | CRB-MD-Pectin | 7.79 ± 0.25 | 0.33 | (1) | 7.24 | 0.85 |
(2) | 11.90 | -- | (2) | 7.63 | -- |
PB-Whey | 7.5 ± 0.79 | 0.34 | (1) | 9.84 | nd | CRB-Whey | 6.62 ± 0.16 | 0.30 | (1) | 7.85 | nd |
(2) | 12.71 | -- | (2) | 11.46 | -- |
Table 3.
CIE Lab parameters a, b, L, and turbidity (t) measurements of water dispersions containing 1.5 mg/mL of PB microcapsules, 0.3 mg/mL of CRB microcapsules, or 0.048 mg/mL of CRB and CRB-encap.
Table 3.
CIE Lab parameters a, b, L, and turbidity (t) measurements of water dispersions containing 1.5 mg/mL of PB microcapsules, 0.3 mg/mL of CRB microcapsules, or 0.048 mg/mL of CRB and CRB-encap.
Sample | Colorimetry | t at 600 nm |
---|
a | b | L |
---|
CRB | 54.29 | 10.50 | 50.34 | -- |
CRB-encap | 52.77 | 13.19 | 66.24 | 0.22 |
PB-Sucrose | 45.01 | 12.07 | 46.13 | 0.41 |
CRB-Sucrose | 41.90 | 20.31 | 56.47 | 0.38 |
PB-MD | 48.06 | 10.67 | 53.70 | 0.52 |
CRB-MD | 44.00 | 19.38 | 72.62 | 0.23 |
PB-MD-GA | 48.98 | 8.15 | 40.29 | 0.41 |
CRB-MD-GA | 43.40 | 18.50 | 67.00 | 0.23 |
PB-MD-CMC | 5.63 | 9.30 | 58.81 | 0.34 |
CRB-MD-CMC | 53.20 | 11.10 | 44.68 | 0.17 |
PB-MD-Pectin | 53.63 | 6.28 | 40.46 | 0.28 |
CRB-MD-Pectin | 47.69 | 14.21 | 59.06 | 0.19 |
PB-Whey | 48.62 | 8.05 | 39.09 | 0.39 |
CRB-Whey | 48.21 | 13.89 | 39.72 | 0.24 |
Table 4.
Stability Indexes of microcapsule water dispersions at different periods of time. (--: not measured, (1) & (2): Replicates 1 and 2). Replicates (1) and (2) had a concentration of 0.3 mg/mL and 1.5 mg/mL of microcapsule powder respectively.
Table 4.
Stability Indexes of microcapsule water dispersions at different periods of time. (--: not measured, (1) & (2): Replicates 1 and 2). Replicates (1) and (2) had a concentration of 0.3 mg/mL and 1.5 mg/mL of microcapsule powder respectively.
Sample | | TSI |
---|
R | 20 min | 2 days | 8 days | 23 days | 40 days |
---|
CRB | (1) | 3.7 | 55.7 | 63.0 | 66.6 | 67.8 |
(2) | 7.8 | 38.4 | 42.4 | 55.5 | 72.0 |
CRB-encap | (1) | 2.2 | 22.8 | 28.9 | 32.4 | 33.5 |
PB-Sucrose | (1) | 1.1 | 15.7 | 22.2 | 25.1 | -- |
CRB-Sucrose | (1) | 0.7 | 17.5 | 25.7 | 34.2 | 39.3 |
PB-MD | (1) | 0.9 | 23.0 | 31.1 | 34.7 | -- |
(2) | 1.0 | 39.4 | 56.0 | 67.2 | -- |
CRB-MD | (1) | 1.3 | 17.2 | 22.2 | 27.2 | 30.3 |
PB-MD-GA | (1) | 1.2 | 22.5 | 27.9 | 29.8 | -- |
(2) | 2.0 | 22.8 | 48.8 | 65.3 | -- |
CRB-MD-GA | (1) | 1.0 | 15.5 | 20.5 | 24.8 | 28.4 |
PB-MD-CMC | (1) | 1.9 | 24.3 | 27.1 | 29.1 | -- |
CRB-MD-CMC | (1) | 1.6 | 18.1 | 22.9 | 26.6 | 28.7 |
PB-MD-Pectin | (1) | 1.9 | 21.0 | 23.6 | -- | -- |
CRB-MD-Pectin | (1) | 1.8 | 20.1 | 24.9 | 29.3 | 32.6 |
PB-Whey | (1) | 1.9 | 25.0 | 28.6 | 31.1 | -- |
CRB-Whey | (1) | 0.8 | 48.0 | 48.7 | 49.7 | 49.9 |
Table 5.
Enzymatic activities on the native carbohydrate fraction (1%
w/
v) after 14 h of incubation, determined in absorbance units by DNS assay [
31]. (nd = no detected activity).
Table 5.
Enzymatic activities on the native carbohydrate fraction (1%
w/
v) after 14 h of incubation, determined in absorbance units by DNS assay [
31]. (nd = no detected activity).
Enzyme | Activity (Abs/14 h) |
---|
endo-arabinase | nd |
α-amylase | 2.615 |
Cellulase | nd |
xylanase | 0.038 |