J Food Bioact

Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online

Journal website www.isnff-jfb.com

Original Research

Volume 27, September 2024, pages 79-87

First in vitro insights into the digestive stability of polysorbate 80 micelles

Figure 1. Experimental design with (a) all compounds digested with a lipid and different PS80 formulations and (b) Curcumin from PS80 formulations digested following different digestion protocols (excl. lipase or bile extract).

Figure 2. The in vitro solubility (%) and bioaccessibility (%) of curcumin from native curcumin powder digested alone (Native), co-digested with PS80 (Native + PS80) or as pre-micellized PS80/curcumin (Micelles filtered) (n = 6). In vitro digestive conditions were standard or conducted without lipase or bile extract, respectively. Bars with different lowercase letters (abc – in vitro solubility and uvw – bioaccessibility) differ significantly (p < 0.05). Different uppercase letters (ABC) indicate significant differences between overall in vitro solubility and bioaccessibility between different formulations.

Figure 3. The surface charge (mV) of polysorbate 80 (PS80) and curcumin/PS80 micelles and particles from the in vitro solubility fraction* of digestions of curcumin and PS80 (alone and combined) and PS80/curcumin micelles, digested with and without lipase and bile extract. *Supernatant taken after the in vitro digested micelles were centrifuged (4,700 ×g, 60 mins, 4°C).

**Table 1.** Preparation of bioactive + lipid/PS80 solutions for *in vitro* digestion
	Preparation	Bioactive added to digestion	Olive oil/PS80 added to digestion
*Represents the loading capacity (LC) of the micelles: (0.023%), β-carotene (0.014%), naringenin – (2.4%), curcumin (4.0%).
Native	Native powder, olive oil, PS80 added to digestion tube	1.0 mg	–
Native + olive oil	1.0 mg	olive oil – 9.0 mg
Native + PS80	1.0 mg	PS80 – 9.0 mg
Micelles unfiltered	10 mg micelles before filtration	1.0 mg	PS80 – 9.0 mg
Micelles filtered	10 mg micelles after filtration	Dependant on amount remaining after filtration*: CoQ10 – 2.3 µg, β-carotene – 1.4 µg, naringenin – 224.0 µg, curcumin – 397.0 µg	Dependant on amount of PS80 remaining after filtration was not determined).

Table 1. Preparation of bioactive + lipid/PS80 solutions for in vitro digestion

Preparation

Bioactive added to digestion

Olive oil/PS80 added to digestion

*Represents the loading capacity (LC) of the micelles: (0.023%), β-carotene (0.014%), naringenin – (2.4%), curcumin (4.0%).

Native

Native powder, olive oil, PS80 added to digestion tube

1.0 mg

–

Native + olive oil

1.0 mg

olive oil – 9.0 mg

Native + PS80

1.0 mg

PS80 – 9.0 mg

Micelles unfiltered

10 mg micelles before filtration

1.0 mg

PS80 – 9.0 mg

Micelles filtered

10 mg micelles after filtration

Dependant on amount remaining after filtration*: CoQ10 – 2.3 µg, β-carotene – 1.4 µg, naringenin – 224.0 µg, curcumin – 397.0 µg

Dependant on amount of PS80 remaining after filtration was not determined).

**Table 2.** The *in vitro* solubility¹ and bioaccessibility² (µg/mL and percent of amount digested) of compounds from the native powder digested alone, with olive oil or polysorbate-80 (PS80) and from unfiltered/filtered PS80 micelles (solvent evaporation method)
	Coenzyme Q10	β-Carotene	Naringenin	Curcumin
Supernatant taken after the in vitro digests were ¹centrifuged (4,700 ×g, 60 mins, 4°C) and ²centrifuged and filtered (200 nm). ^ABCMeans with different superscripts within each column (compound) indicate significant differences (p < 0.05).
In vitro solubility (µg/ml [% of amount digested])
Native	0.10^A ± 0.05	0.137^A ± 0.004	26.2^A ± 0.4	22.5^A ± 1.1
Native + Olive oil	4.43^C ± 0.65	0.731^C ± 0.039	24.8^A ± 0.8	17.1^A ± 2.2
Native + PS80	0.50^A ± 0.11 [0.2]	0.133^A ± 0.003 [0.04]	71.5^BC ± 2.4 [24]	206.7^E ± 17.8 [69]
Micelles unfiltered	0.31^A ± 0.09 [0.1]	0.132^A ± 0.001 [0.04]	97.4^D ± 8.5 [32]	244.5^F ± 13.9 [81]
Micelles filtered	0.33^A ± 0.04 [49]	0.209^A ± 0.070 [52]	65.4^B ± 0.6 [97]	77.0^D ± 0.9 [65]
Bioaccessibility (µg/ml [% of amount digested])
Native	0.10^A ± 0.02	0.130^A ± 0.001	26.0^A ± 0.4	5.8^A ± 0.7
Native + Olive oil	2.30^B ± 0.35	0.549^B ± 0.026	24.8^A ± 2.2	6.7^A ± 0.1
Native + PS80	0.37^A ± 0.21 [0.1]	0.131^A ± 0.001 [0.04]	67.9^B ± 2.5 [23]	59.3^C ± 5.8 [15]
Micelles unfiltered	0.20^A ± 0.08 [0.1]	0.130^A ± 0.000 [0.04]	81.5^CD ± 21.9 [27]	68.1^C ± 6.2 [17]
Micelles filtered	0.34^A ± 0.07 [49]	0.132^A ± 0.002 [31]	54.9^B ± 4.5 [82]	38.2^B ± 6.1 [32]

Table 2. The in vitro solubility¹ and bioaccessibility² (µg/mL and percent of amount digested) of compounds from the native powder digested alone, with olive oil or polysorbate-80 (PS80) and from unfiltered/filtered PS80 micelles (solvent evaporation method)

Coenzyme Q10

β-Carotene

Naringenin

Curcumin

Supernatant taken after the in vitro digests were ¹centrifuged (4,700 ×g, 60 mins, 4°C) and ²centrifuged and filtered (200 nm). ^ABCMeans with different superscripts within each column (compound) indicate significant differences (p < 0.05).

In vitro solubility (µg/ml [% of amount digested])

Native

0.10^A ± 0.05

0.137^A ± 0.004

26.2^A ± 0.4

22.5^A ± 1.1

Native + Olive oil

4.43^C ± 0.65

0.731^C ± 0.039

24.8^A ± 0.8

17.1^A ± 2.2

Native + PS80

0.50^A ± 0.11 [0.2]

0.133^A ± 0.003 [0.04]

71.5^BC ± 2.4 [24]

206.7^E ± 17.8 [69]

Micelles unfiltered

0.31^A ± 0.09 [0.1]

0.132^A ± 0.001 [0.04]

97.4^D ± 8.5 [32]

244.5^F ± 13.9 [81]

Micelles filtered

0.33^A ± 0.04 [49]

0.209^A ± 0.070 [52]

65.4^B ± 0.6 [97]

77.0^D ± 0.9 [65]

Bioaccessibility (µg/ml [% of amount digested])

Native

0.10^A ± 0.02

0.130^A ± 0.001

26.0^A ± 0.4

5.8^A ± 0.7

Native + Olive oil

2.30^B ± 0.35

0.549^B ± 0.026

24.8^A ± 2.2

6.7^A ± 0.1

Native + PS80

0.37^A ± 0.21 [0.1]

0.131^A ± 0.001 [0.04]

67.9^B ± 2.5 [23]

59.3^C ± 5.8 [15]

Micelles unfiltered

0.20^A ± 0.08 [0.1]

0.130^A ± 0.000 [0.04]

81.5^CD ± 21.9 [27]

68.1^C ± 6.2 [17]

Micelles filtered

0.34^A ± 0.07 [49]

0.132^A ± 0.002 [31]

54.9^B ± 4.5 [82]

38.2^B ± 6.1 [32]

**Table 3.** Solubility of compounds¹ in water, olive oil, or polysorbate-80 (PS80) compared to the changes in *in vitro* solubility² after digestion with olive oil or PS80
		Coenzyme Q10	β-Carotene	Naringenin	Curcumin
¹Values obtained from literature. ²Calculated from data in Table 2. Only information for soybean oil available, but β-carotene has very low solubility in vegetable oils at room temperature (Ribeiro and Cruz, 2005). –, no significant change in in vitro* solubility.
Solubility (µg/ml) in:	Water	9 × 10⁻⁵ (Fir et al., 2009)	Insoluble	5 (Shulman et al., 2011)	3 (NCBI, 2021)
Olive oil	14 × 10⁴ (Thanatuksorn et al., 2009)	100* (Roohinejad et al., 2015)	2 × 10⁴ (Yeo et al., 2021)	450 (Takenaka et al., 2013)
PS80	2 (Shah et al., 2018)	0.04 (Roohinejad et al., 2015)	7 × 10⁴ (Yeo et al., 2021)	1 × 10⁴ (Anjana et al., 2012)
Significant (p < 0.05) increases in in vitro solubility after digestion with:	Olive oil	44-fold	5-fold	–	–
PS80	–	–	2.5-fold	9-fold

Table 3. Solubility of compounds¹ in water, olive oil, or polysorbate-80 (PS80) compared to the changes in in vitro solubility² after digestion with olive oil or PS80

Coenzyme Q10

β-Carotene

Naringenin

Curcumin

¹Values obtained from literature. ²Calculated from data in Table 2. *Only information for soybean oil available, but β-carotene has very low solubility in vegetable oils at room temperature (Ribeiro and Cruz, 2005). –, no significant change in in vitro solubility.

Solubility (µg/ml) in:

Water

9 × 10⁻⁵ (Fir et al., 2009)

Insoluble

5 (Shulman et al., 2011)

3 (NCBI, 2021)

Olive oil

14 × 10⁴ (Thanatuksorn et al., 2009)

100* (Roohinejad et al., 2015)

2 × 10⁴ (Yeo et al., 2021)

450 (Takenaka et al., 2013)

PS80

2 (Shah et al., 2018)

0.04 (Roohinejad et al., 2015)

7 × 10⁴ (Yeo et al., 2021)

1 × 10⁴ (Anjana et al., 2012)

Significant (p < 0.05) increases in in vitro solubility after digestion with:

Olive oil

44-fold

5-fold

–

PS80

–

2.5-fold

9-fold

**Table 4.** Difference (ΔmV) in surface charge of particles/micelles of digestions of curcumin loaded and blank formulations
	PS80 only formulation subtracted from curcumin containing PS80 formulation
–, No measurement.
(Native + PS80) – (PS80)	–	10.9	9.7	−0.1
(Micelles filtered) – (PS80 micelles)	3.4	11.7	6.4	2.1

Table 4. Difference (ΔmV) in surface charge of particles/micelles of digestions of curcumin loaded and blank formulations

PS80 only formulation subtracted from curcumin containing PS80 formulation

Undigested

In vitro digestion

In vitro digestion without lipase

In vitro digestion without bile extract

–, No measurement.

(Native + PS80) – (PS80)

–

10.9

9.7

−0.1

(Micelles filtered) – (PS80 micelles)

3.4

11.7

6.4

2.1