Objective: To develop an in vitro digestion method to assess the impact of heat treatment, particle size and presence of oil on the accessibility (available for absorption) of α- and β-carotene in carrots.
Design: Raw and cooked carrots were either homogenized or cut into pieces similar to chewed items in size. The carrot samples, with or without added cooking oil, were exposed to an in vitro digestion procedure. Adding a pepsin–HCl solution at pH 2.0 simulated the gastric phase. In the subsequent intestinal phase, pH was adjusted to 7.5 and a pancreatin–bile extract mixture was added. Carotenoids released from the carrot matrix during the digestion were extracted and quantified on high-performance liquid chromatography (HPLC).
Results: Three percent of the total β-carotene content was released from raw carrots in pieces. When homogenized (pulped) 21% was released. Cooking the pulp increased the accessibility to 27%. Addition of cooking oil to the cooked pulp further increased the released amount to 39%. The trends for α-carotene were similar to those for β-carotene.
Conclusion: The described in vitro digestion method allows a rapid estimation of carotene accessibility in processed carrots, which may reliably predict in vivo behavior.
Sponsorship: This study was supported by the Swedish International Development Cooperation Agency (SIDA) and the International Program in the Chemical Sciences (IPICS), Uppsala University, Sweden.
Vitamin A deficiency is a major public health problem in developing countries (WHO/UNICEF, 1995). Preschool children and women of reproductive age are predominantly affected. For these population groups the primary source of vitamin A is derived from provitamin A carotenoids in plant foods. Food-based strategies for combatting vitamin A deficiency include promotion of a higher production and consumption of dark-green leafy vegetables, yellow and red fruit and vegetables and red palm oil, which are all rich in provitamin A carotenoids (Bloem et al, 1998). However, some studies have indicated that β-carotene may be poorly absorbed from certain vegetables (Brown et al, 1989; Micozzi et al, 1992; de Pee et al, 1995) and thus the justification of such strategies has been questioned. Some epidemiological studies have suggested a lowered risk of developing certain types of cancer or chronic diseases following a diet containing food products rich in carotenoids, due to their anti-oxidative properties (Block et al, 1992; van Poppel, 1993; Steinmetz & Potter, 1996). However, our knowledge about the content of carotenoids in vegetable foods as well as the bioavailability of these is limited.
Bioavailability is defined as the fraction of an ingested nutrient from food that is available for absorption in the intestine and metabolic process and storage (Jackson, 1997). A number of factors have been identified that may affect the bioavailability of carotenoids from foods, eg the matrix in which the carotenoids are incorporated, the content of dietary fat and fiber, the particle size, and the food processing method applied. In carrots the carotenoids are present in crystalline form or associated with proteins embedded in chromoplasts (Byrant et al, 1992), which limit their release during the digestion process. Cooking may enhance the carotenoid release by softening or breaking down the cell walls and by dissociating the protein complex (Poor et al, 1993; Zhou et al, 1996; Rock et al, 1998). Another way of reducing matrix effects is by homogenization or particle size reduction (van Zeben & Hendriks, 1948; Gärtner et al, 1997; Castenmiller et al, 1999; van het Hof et al, 1999). Dietary fat, as well, has a documented positive effect on bioavailability of carotenoids (Roels et al, 1958; Dimitrov et al, 1988; Shiau et al, 1994; Jalal et al, 1998), although the amount of fat required to optimize carotenoid bioavailability has not been clearly identified.
Existing methods to estimate the carotenoid bioavailability include both short- and long-term studies in humans (Micozzi et al, 1992; de Pee et al, 1995; Castenmiller et al, 1999) as well as appropriate animal models, such as ferret (White et al, 1993) and preruminant calves (Poor et al, 1993). The most commonly applied method includes measuring increases in plasma concentrations of carotenoids or retinol in humans following administration of an acute or chronic dose of an isolated carotenoid or a carotenoid-containing food (Brown et al, 1989; Micozzi et al, 1992; Castenmiller et al, 1999; van het Hof et al, 1999). This is a relative measure, as the plasma is highly dynamic and individual responses have been reported to be highly variable (Dimitrov et al, 1988; Törrönen et al, 1996). To obtain a quantitative measure of carotenoid bioavailability, the oral–faecal balance method is a better approach (Roels et al, 1958; Shiau et al, 1994).
However, studies based on humans or animals are tedious and costly and are not useful to screen a large number of food samples. There is therefore a need for an in vitro model that simulates the human digestion tract. Such a method should quantitatively assess the amount of carotenoids that becomes accessible both from different food sources and from foods subjected to different food treatments. In connection with this, we use the term bioaccessibility to be defined as the fraction of a nutrient available for absorption, ie the amount of a nutrient that is released from its food matrix during digestion and made accessible for absorption into mucosa. In the present paper the proposed in vitro digestion method therefore measures the amount of carotenoids accessible for absorption from carrots, and is defined as ‘in vitro accessibility’. The objective of the present study was to investigate how different parameters, ie cooking/heating, particle size and presence of fat affect the accessibility of carotenoids from carrots using an in vitro method simulating the human digestion system.
Materials and methods
One batch of carrots (Nandrin) cultivated on Gotland in Sweden was purchased in a local store in Gothenburg, Sweden. All carrots were washed and the excess of water was carefully removed from the surface. The samples were peeled and cut into pieces (about 10×10×40 mm). Half of the samples were boiled in water for 20 min, water:carrot (70:30). The samples were then rapidly frozen at −40°C for 24 h before they were freeze-dried (Lyovac GT 2). The material was stored in amber flasks filled with nitrogen and kept in a freezer at −18°C.
Prior to digestion, the freeze-dried carrots were either finely ground to pass a 250 µm aperture sieve or cut into small pieces. The pieces were fractionated between two sieves of 4.0 and 2.8 mm aperture. An internal standard 20 µg β-apo-8-carotenal (Fluka 10829) dissolved in 100 µl hexane was added to a conical flask and the solvent was evaporated with a flush of nitrogen. About 0.5 g of carrot sample (finely ground or in small pieces) was accurately weighed in the flask and suspended in 10 ml distilled water with 1% (w/vol) ascorbic acid (Merck 100127). Nitrogen was blown into the flask before it was screw capped and left for 30 min to absorb moisture. Different amounts (0, 20, 40 and 60% of dry weight of sample) of a cooking oil (rapeseed oil) stabilized with 1% (w/vol) DL-α-tocopherol (Merck 108283) were then added.
In vitro digestion
The digestion started with addition of 5 ml of 0.5% porcine pepsin solution (Sigma p 6887) in 0.1 mol HCl/l with physiological amounts (Diem & Lentner, 1975) of calcium (3.6 mmol added as CaCl2·2H2O, Merck), magnesium (1.5 mmol added as MgCl2·6H2O, Merck), sodium (49 mmol added as NaCl, Merck), potassium (12 mmol added as KCl, Merck) and phosphate (6.4 mmol added as KH2PO4, Merck). If necessary the pH was adjusted to 2.0 with 2 mol HCl/l. Nitrogen was blown into the flask before the mixture was incubated at 37°C in a water bath (Lab-line 4645-1) with orbital shaking (13 mm diameter) at 250 rpm for 1 h. The pH was adjusted to about 5 by adding 2 mol NaOH/l before 3 ml of a mixture of porcine pancreatin (4 g/l, Sigma P 1750) and porcine bile salt (25 g/l, Sigma B 8631) dissolved in 0.1 mol NaHCO3/l stabilized with 1% (w/vol) DL-α-tocopherol was added. The pH was further increased to 7.5 by adding 2 mol NaOH/l. The mixture was then, after nitrogen had been blown into the flask, incubated again for 30 min. After the incubation the sample was immediately centrifuged (Labofuge M) at 5000 g for 20 min in a centrifuge tube of glass.
To validate the method some critical steps in the digestion procedure were more carefully examined, ie the impact of addition of pancreatic enzymes and different amounts of bile salts (25 and 50 g/l), as well as the shaking conditions (reciprocal and orbital) for the micellarization process.
Extraction and quantification
After centrifugation the supernatant was collected with a plastic pipette in a separatory funnel containing 20 ml of 25% (w/vol) NaCl water. Twenty milliliters of 95% ethanol and 15 ml of petroleum ether (stabilized with 0.1% (w/vol) butyl hydroxytoluene, BHT) were added and the funnel was swirled before being left to allow the two layers to separate. The lower phase was drawn off and re-extracted with 15 ml petroleum ether and 10 ml 95% ethanol. This procedure was repeated until the organic phase was colorless. The upper organic phases were combined and evaporated in a rotary vacuum evaporator with nitrogen flush at 35°C to dryness. The residue was dissolved in a tert-butyl-ether and methanol solution (1:1, stabilized with 0.1% (w/vol) BHT), made up to 10 ml and passed through a disposable cellulose membrane filter with 0.45 µm pore size (Sartorius Filtration AB) into an amber vial. The preparation procedure was made in dim light.
The carotenes were separated and quantified by high-performance liquid chromatography, Water 600 HPLC with a multi-solvent delivery system, provided with a 996 photodiode array detector with a Millennium Chromatography manager to acquire and process spectral and chromatographic data. A YMC carotenoid column (YMC Inc., Wilmington, NC, USA) was used prepared on high purity silica (5 µm) with a C30 polymer ligand. The column is made of stainless steel 4.8×250 mm in size. The isocratic mobile phase was methanol:tert-butyl methyl ether:water (56:40:4; Lichrosolv grade, Merck). The flow rate was 1 ml/min and injections were made with a 20 µl loop. Analysis of each sample was performed in duplicate.
Determination of total α- and β-carotene content of carrots
The freeze-dried carrot samples were ground into a fine powder and 0.25 g was accurately weighed and put into a conical flask and mixed with 10 ml of 1% (w/vol) ascorbic acid dissolved in distilled water. One hundred microliters of internal standard (200 µg β-apo-8-carotenal/ml) were added before the flask was left for 30 min. The flask was swirled vigorously after 10 ml of a mixture of acetone and 95% ethanol (1:1) had been added. The upper phase was decanted into a separatory funnel with 20 ml 25% (w/vol) NaCl water. Another 10 ml of the acetone and ethanol mixture was added and decanted until the residue was colorless. The carotenoids were extracted with petroleum ether (stabilized with 0.1% (w/vol) BHT) and prepared for quantification on the HPLC in the same manner as the in vitro samples.
Differences in mean values of in vitro accessible carotenes were tested by analysis of variance (ANOVA) and determination of the significance of difference among samples using P-values obtained by Tukey's HSD multiple rank test.
The total amount of α- and β-carotene in the cooked carrots was 468±11 and 1357±52 µg/g dry matter (n=4), respectively. All samples were performed in duplicates and replicated once or twice. In samples without added cooking oil, raw carrot pieces released about 3% of the total β-carotene after in vitro digestion and about 6% was released from cooked pieces (Table 1). When the carrots were homogenized (pulped) the percentage accessible β-carotene increased to 21% in the raw sample and to 27% in the cooked sample. The amount of released β-carotene after digestion was 40±9 and 82±18 µg/g dry matter from raw and cooked carrot pieces, respectively, and from pulped samples 290±28 and 367±23 µg/g dry matter of β-carotene were released, respectively (Figure 1). Addition of 20% oil per gram dry matter carrot resulted in a significant increase (P<0.05) of the amount of accessible β-carotene in the homogenized samples. The percentage increase in the raw pulped sample was 30% and in the cooked pulped sample 39%. Increasing the amount of added oil (up to 60%) resulted in a further slight increase of accessible β-carotene, although not significant. Adding oil to the carrot piece samples had no significant effect on the amount of released β-carotene, although there was a tendency for a slight increase.
The released amounts of α-carotene are shown in Figure 2. The trends for α-carotene were similar to those for β-carotene, except that the absolute values were about 60% lower.
In this study an in vitro method that simulates the human intestinal digestion process was developed in order to estimate the amount of carotenoids from carrots that becomes accessible for absorption by the human mucosa. Garrett et al (1999) developed an in vitro digestion method with subsequent examination of uptake of micellarized carotenes by cultures of Caco-2 human intestinal cells. In contrast to that method, the present method estimates the maximum amount of carotenoids released, not necessarily micellarized, from the food matrix that at optimal physiological and dietary conditions could be absorbed by the human mucosa.
In vitro method considerations
Carotenoids are susceptible to oxidation owing to the numerous double bonds. To avoid oxidation of carotenoids during the in vitro digestion, ascorbic acid and DL-α-tocopherol were added to serve as antioxidants. The ascorbic acid would serve as an antioxidant in the water phase and the α-tocopherol is thought to be located at the surface of the oil droplets and bile salt micelles where the carotenoids are incorporated. During extraction of the digesta with organic solvents the carotenoids were protected by BHT. As an extra precaution against oxidation, the analyses were performed under dim light and whenever possible nitrogen gas was used to minimize the contact with oxygen.
Some critical steps in the present in vitro method were investigated more carefully, ie the significance of bile salts and pancreatic enzymes, as well as shear forces for the micellarization process. A mixture of homogenized cooked carrots with added cooking oil (40% of dry weight) was used to study the significance of bile salts and pancreatin enzymes. The amount of released β-carotene decreased by ∼80% when bile salts were omitted from the digestion mixture and was probably dissolved within lipid droplets originating from the carrot mixture. The pancreatin mixture with bile salts is necessary for the formation of bile salt micelles and carotenoid absorption is also known to be minimal when intraluminal bile salts are below the concentration required for aggregation into micelles (El-Gorab & Underwood, 1973). However, adding the double amount of bile salts into the pancreatin mixture (from 25 to 50 g/l) revealed no further increase in carotenoid accessibility. Remarkably, pancreatin showed less importance, since the amount of β-carotene released into the aqueous phase was only about 10% lower with pancreatin absent in the digestion mixture. From samples only subjected to the gastric phase, the amount of β-carotenes released into the aqueous phase was ∼90% lower, which means that the intestinal phase with bile salts is of major importance.
Once the lipophilic carotenoids are released from the food matrix in the stomach, they dissolve in an oily phase of lipid droplets. The formation of bile salt micelles requires mechanical disruption of the large lipid droplets into smaller droplets. This disruption is, in the in vitro method, brought about by shaking during incubation. Different shaking conditions were tested with cooked and homogenized carrot samples with added cooking oil (60% of dry weight sample). With the use of a waterbath (Heto, TB VS-02) with reciprocal shaking set at a moderate speed of ∼100 rpm, the coefficient of variation for β-carotene could be as high as 30% between assays. When we changed to a waterbath with orbital shaking set at a speed of 250 rpm we received reproducible results. The coefficient of variation within one assay was 3.7% (n=2) and the coefficient of day-to-day variation was 5.9% (n=4).
Parameters affecting the bioaccessibility of carotenoids
The effects of heat treatment, particle size and fat content on accessibility of carotenoids from carrots were studied with the present in vitro method. The in vitro accessibility of each carotenoid is expressed as percentage of the total amount present in the carrot and is thus an estimation of the amount accessible for absorption. The obtained values are compared with results from other studies in order to validate the in vitro method.
In the present in vitro study 3% of the β-carotene content was released from raw carrots in pieces, which is in agreement with results from a balance study in humans conducted by Roels et al (1958), where less than 5% of the carotene content in raw grated carrots was absorbed. However, cooking may bring about a more efficient release of carotenoids from the food matrix by softening the cell structure so that the digestive enzymes may work more efficiently. In the present study the heat treatment improved the release of β-carotene from carrots in pieces about two-fold and about 1.3 times in homogenized carrots. In a study on preruminant calves, accumulation of β-carotene in different tissues was 1.6 times higher after consumption of cooked carrots compared with consumption of raw carrots (Poor et al, 1993). Consumption of thermally processed carrots and spinach resulted in a plasma β-carotene response in women three times that associated with the consumption of these vegetables in the raw form (Rock et al, 1998).
To make carotenoids accessible for absorption, mechanical as well as chemical disruption of the food matrix is important (Furr & Clark, 1997). By mechanical disruption, the surface area for the digestive enzymes to attack enlarges and thereby the carotenoids are more easily released from their matrix. A seven-fold and an almost five-fold improvement of the β-carotene accessibility were shown in the present study after homogenization of raw and cooked carrot samples, respectively. Van Zeben and Hendriks (1948) showed an almost five-fold increase in concentration of plasma carotenes when women consumed cooked carrots homogenized in a mixer compared with unhomogenized carrots. In a study conducted by Törönnen et al (1996), the serum β-carotene response in women was almost twice as high after consumption of carrot juice compared with raw carrots. Huang et al (2000) evaluated the relative bioavailability of β-carotene in adult males after a single ingestion of stir-fried (with oil) shredded carrots using the serum response method. Compared with pure β-carotene in a capsule the bioavailability from the carrot meal was 33%. This figure is comparable with our in vitro measurement of cooked homogenized carrot with added oil (20, 40 and 60% of dry weight) that had a β-carotene accessibility of 38–45% (Table 1). Improved carotenoid (lycopene and β-carotene) bioavailability after disruption of the food matrix has been shown in human studies using other vegetable sources than carrots. The peak lycopene concentration in chylomicrons was reported to be about twice as high after consumption of tomato paste compared with fresh tomatoes (Gärtner et al, 1997). The plasma β-carotene response was twice as high in humans after consumption of enzymatically liquefied spinach compared with whole spinach (Castenmiller et al, 1999).
Ingestion of fat along with carotenoids is thought to be crucial in the absorption process (Prince & Frisoli, 1993). Dietary fat provides a hydrophobic environment for the released carotenoids that would be dissolved into small lipid droplets facilitated by mechanical mixing. With addition of bile salts these lipid droplets are transformed into mixed bile salt micelles from which the carotenoids are thought to be absorbed in the intestine. A positive effect of adding oil was measureable with the present in vitro method. Depending on the carrot sample, a 30–60% increase of in vitro β-carotene accessibility was obtained when cooking oil was added. Adding 20% oil per gram dry matter, which meant about 2 g per 100 g fresh sample, as the water content of the carrots was about 90%, was enough to detect a significant improving effect. Roels et al (1958) showed a five-fold increase in absorbed carotenes after supplementation of oil to raw grated carrots in a human balance study. In the present in vitro study, addition of 6 g instead of 2 g of cooking oil to the carrot samples had no improving effect on accessible β-carotene, although there was a slight increase (see Figure 1). From highly soluble β-carotene in the form of beadlets, about 17% was absorbed when added to meals containing no fat. Added to a low-fat diet, on average 32% was absorbed measured by the total wash-out method in young men (Shiau et al, 1994). Doubling the amount of fat in the meal had no further effect on the carotene absorption. No significant difference in plasma β-carotene response was shown in humans when spinach was ingested with 5 or 10 g of oil (Jayarajan et al, 1980) or when a β-carotene capsule was ingested with a low-fat (3 g) or a high-fat (32 g) diet (Roodenburg et al, 2000).
Although results of β-carotene accessibility using the in vitro digestion method are not strictly comparable with results using animal models or plasma β-carotene as a biomarker, still the trends found are comparable. Human balance studies reveal results similar to those obtained with the present in vitro method.
In conclusion, with the described in vitro digestion method, it has been shown that pulping is more important than cooking for release of carotenoids in carrots, and addition of oil is more effective in enhancing accessibility when carrot is pulped rather than in pieces. The in vitro digestion method described in the present paper appears to be useful for rapid screening of carotenoid accessibility in plant foods. The method gives an estimation of the amount of carotenoids that are released from the food matrix after digestion and absorbed into the human mucosa. Effects of different processing and preparation methods as well as dietary factors on carotenoid accessibility can be compared with the proposed in vitro digestion method.
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