**6.4 Carotenoid increase in heat absorption of oils accelerates the cooking process and renders it healthier**

Increase in thermoconductivity of oil doped by carotenoids may result in an accelerated cooking process. For example, to reach the 84°C temperature of completed cooking for a piece of chicken breast, coated in olive oil at an oven temperature of 180°C, took 15 min. When this oil contained lycopene or astaxanthin, in concentration 0.23 mg/ mL, cooking took 13 and 10 min, respectively (**Figure 5**). For a fillet of wild salmon to

#### **Figure 5.**

*Carotenoids in olive oil accelerate cooking time of chicken breast and preserve vitamin B12 in cooked salmon. \*Astaxanthin concentration was 0.23 mg/mL in olive oil; y-axis—temperature in °C, x-axis—time in seconds. \*\*Yellow—content of preserved intact vitamins, black—lost vitamins.*

reach the temperature of completed cooking at 62°C, it took 9 min for the samples in the control oil and 7 or even 6 min for the oil with lycopene or astaxanthin, respectively.

The observed acceleration of the cooking process and, consequently, reduction in the cooking time may help in preservation in the cooked food of important thermosensitive macronutrients or vitamins. For example, in the above experiment, the concentration of vitamin D3 in the baked salmon was only about one-third of its precooked level. However, when oil contained astaxanthin or lycopene, the remaining level of this vitamin was significantly higher, more than 55 or 82%, respectively. In the same fish, samples of Vitamin B12 were more sensitive to the cooking temperature; by the end of the experiment, only about 6% of these molecules were detected there. Using the oil with astaxanthin did not make much difference, but with lycopene saved more than 96% of this vitamin in the cooked fish (**Figure 5**).

#### **6.5 Carotenoids reduce the digestion rate of lipids and their absorption level**

The industrial process of refining oil production removes all its ingredients, including carotenoids, which are originally present in freshly pressed plant oils and fats. As a result of this, the lipid droplets of these oils have higher viscosity, are significantly smaller, and have a faster digestibility rate leading to an increase in calorie release and absorption. As a result of this, refined vegetable oils alongside refined sugars are the main food sources of easily released calories, the main dietary factor contributing to the development of the global obesity pandemic.

The reintroduction of carotenoids to plant oils and fats increases the size of their droplets or globules (**Figure 1**). Consequently, the time of digestions of these lipids will increase, and subject to the ingested lipid volume, not all lipids would be digested and absorbed. This would lead to a reduction in the amount of the absorbed lipid in the postprandial blood.

#### **Figure 6.**

*Effect of daily ingesting of dairy butter with or without lycopene on the level of serum lipids in the fasting blood of volunteers. Blue—30 g of control dairy butter, red—30 g of the butter with 7-mg lycopene.*

This was confirmed in a crossover clinical trial on healthy volunteers, who were asked to ingest different unmodified fat or fat-rich food products and, after one week's rest, ingest the same products but modified by carotenoids. Postprandial blood was analysed to assess the level and kinetics of the absorbed lipids. For example, after ingestion of 50 g of control dairy butter, area under the curve (AUC) for the first 4 hours for serum total cholesterol was 33±3.9 mg/dL and for triglycerides 25±2.7 mg/ dL (n=10). However, the AUC after ingestion of the same amount of butter with 7 mg of lycopene and these parameters were reduced for cholesterol significantly to 22±2.5 mg/dL (p<0.05) and for triglycerides only as a trend to 19±2.2 mg/dL (p>0.5).

After repeating this experiment with 50 mL sunflower oil, with a slice of white bread, the effect of ingestion of lycopene-modified fat was even stronger. In the control experiment, the AUC for serum total cholesterol was 75±9.2 mg/dL and for triglycerides 56±6.8 mg/dL (n=10). The ingestion of the same amount of this oil but with 7 mg of lycopene resulted in a significant reduction of these parameters to 32±4.3 mg/dL (p<0.01) and 12±3.9 mg/dL (p<0.001).

In the next set of experiments on clinically healthy persons, with borderline hyperlipidaemia, we demonstrated that regular, daily intake of dairy butter with incorporated lycopene could reduce serum triglycerides, total cholesterol and LDL (**Figure 6**).

The butter trial was planned for 2 months. However, its control group was terminated earlier on ethical grounds because there was a significant rise in both blood lipids. In the lycopene butter group, at the end of the trial, the reduction of triglycerides was by 10 mg/dL and total cholesterol by about 20 mg/mL.

The daily ingestion of lycopene chocolate resulted in a significant reduction of both lipids, while in the groups which ingested either the same amount of control chocolate or lycopene in a capsule, there were no changes in these parameters.

## **6.6 Carotenoids reduce the rate of formation cholesterol crystals and facilitate their dissolutions**

The ability of carotenoids to create thermodynamically favourable complexes with lipids, which changes their crystalline properties, can also be observed on their interactions with cholesterol. It was observed that the addition of carotenoids could significantly reduce the rate of cholesterol crystallisation. For example, in the experiment described in the legend to **Figure 7a**, visible cholesterol crystals in the control solution started to appear in 24 hours from the start of the evaporation of the solvent. When lycopene was introduced in a ratio of 1:1000 molecules of cholesterol, it took five times longer before these crystals started to be observed.

#### **Figure 7.**

*Lycopene and cholesterol crystals: reduction of their growth rate (a), disruption of their folding (b) and facilitation of their dissolution in vitro (c) and ex vivo (d). \*2.5 mM of cholesterol in ethanol without or with 2.5-μM lycopene were evaporate at room temperature in a dark room. \*\*Molecular ratio lycopene to cholesterol 1:4000; \*\*\*5 mM of cholesterol in ethanol without or with 2.5-μM lycopene. \*\*\*\*Incubation at room temperature in a dark room in the PBS solution with pre-diluted in ethanol lycopene in concentration 0.1 μg/mL, with NaN3 to prevent microbial growth.*

It was interesting that it was not just the reduction in the rate of crystallisation we observed but also a new type of crystals emerged: they were significantly smaller, and some had needle forms in contrast to the much bigger slab-shaped crystals of unmodified cholesterol (**Figure 7b**). These observations confirmed that that lycopene, like other carotenoids, could create physical complexes with this type of lipids, disrupt their folding, clusterisation and affect their crystal structures.

This ability of carotenoids to disrupt folding in already existing cholesterol crystals was observed in our *in vitro* and *ex vivo* experiments. For example, lycopene could dissolve or help ethanol to dissolve cholesterol crystals in a mole ratio of 1:4000 or below (**Figure 6c**). Lutein was able to do the same, although in a lower concentration range (data not presented).

In the *ex vivo* experiment, a piece of abdominal aorta with massive atherosclerotic lesions and a combination of cholesterol and calcium phosphate crystals were incubated in phosphate-buffered saline containing lycopene pre-dissolved in ethanol. After 12 days of this incubation, not only a substantial amount of fat deposits of atherosclerotic plaques was dissolved, but also the number and size of cholesterol crystals were significantly reduced (**Figure 7d**).

At the same time, this incubation did not affect either the number or the size of calcium crystals in this piece of aorta or cholesterol crystals from a similar type of atherosclerotic lesion (data not presented).
