Keywords

15.1 Introduction

Food is our fuel. In earlier times, when the gain of nutrients such as vitamins and minerals was much lower, food had a different meaning and function than it does today. Today food is for enjoyment as well as nourishment, and when consuming food, we aim to benefit from bioactive food compounds other than just the essential nutrients that keep our engines running. Nowadays, nutritional research is focused on health promotion, disease prevention, performance improvement, and risk assessment (Kussmann et al. 2007). When we discuss the health-promoting or disease-preventive properties of food, we aim for long-term physiological effects arising from many different bioactive food compounds.

The bioactive food compounds responsible for the positive effects on our well-being are derived mostly from the plant kingdom, but some originate also from animal sources, such as those from dairy products. Multiple epidemiological studies over the course of many years have shown that diets rich in vegetables and fruits promote health and reduce the risk of certain chronic diseases. Through these studies it has been determined that five portions of fruits or vegetables a day is good for our health. Fruits and vegetables contain many bioactive compounds which are secondary metabolites in plants and are considered potential compounds for maintaining human health (Patil et al. 2009). These include both lipophilic compounds such as carotenoids, and hydrophilic compounds such as phenolic acids.

It is a complex task to demonstrate a health benefit of such a vast range of different compounds as the bioactive food compounds can be. Several different assessment steps are needed for a given compound to prove a health benefit. The compound of interest must first be released from the food matrix and become bioaccessible, and must then go through metabolism, and finally reach the target tissue where it can exert its beneficial action on health. The assessments of bioaccessibility and bioavailability of health-associated compounds are critical stages of food nutrition research toward understanding the potentials of these food compounds.

Bioaccessibility is defined as the proportion of a compound which is released from the food matrix during digestion in the mouth and gastrointestinal tract, and hence becomes available for absorption through the gut wall into the bloodstream. Bioavailability is defined as the proportion of a given food that the body can actually take up and use, being a matter of nutritional efficacy (Benito and Miller 1998). Bioavailability addresses several processes including liberation from the matrix, absorption, distribution, metabolism, and elimination phases (LADME). We as food scientists often focus on the end product and its benefits with the bioactive compounds , and overlook the LADME factors that affect the aimed benefit, whether nutritional or functional. For the bioactivity expectation, it is important to note that only a proportion of a nutrient or a bioactive compound from food will be effectively used by an organism (Fernandez-Garcia et al. 2009).

Bioavailability of nutrients is studied through human intervention studies looking at the nutrient pharmacokinetics with area under the curve (AUC), maximum concentration (Cmax), and time of maximum concentration (tmax) measurements. AUC corresponds to the fraction of the ingested nutrient that reaches the systemic circulation. It is therefore measured from plasma samples as a function of time and is expressed in mg*h/L. The area under the curve is dependent on the amount of the ingested food, i.e. digested nutrient, and the rate of elimination of the studied nutrient from the body. In general, the concentration of a nutrient in the blood increases linearly with increasing dose. However, for many nutrients there is a threshold of absorption above which their concentration in the blood reaches a plateau. Cmax is the measurement of the highest concentration of an investigated nutrient in the blood. The time at which Cmax is observed is referred to as tmax.

Many factors influence the bioavailability of nutrients, and they can be broadly divided into external factors, product-related factors, and physiological factors. A major external factor known to affect the bioavailability of an ingredient is the target group. The level of absorption of many compounds varies among age groups, for example, due to the status and composition of the intestinal microbiota and the release of bile. Consumption habits, another example of an external factor, also affect the bioavailability of nutrients. A clear demonstration of this is evident when lipid-soluble nutrients like vitamins A, D, E, and K, and carotenoids are better absorbed when consumed with other dietary lipids.

A product-related factor often found to affect the bioavailability of nutrients can be linked to the amount of a nutrient in a product and the molecular structure of the particular nutrient, i.e. the physical form (solid vs. liquid), chemical form (crystalline vs. salt), and whether the molecule is coupled to another nutrient or a protein. These factors can significantly affect the solubility and stability of the nutrient, and therefore its bioavailability. Furthermore, the chemical structure of the nutrient might also affect the site of absorption in the gut, which is linked to physiological factors affecting bioavailability—for example, whether a nutrient is absorbed by passive or active transport into the systemic circulation. In some cases, the stereo configuration of the nutrient similarly affects the distribution of the compound in the body, because some transport proteins have a higher affinity to one stereoisomer form than the other. Physiological factors are also important in terms of bioavailability. For example, some individuals are described as being either high or low absorbers of a specific compound. This has been linked to the composition and activity of the intestinal microbiota, but also to the presence and activity of the different digestive enzymes, the ability to emulsify lipophilic nutrients, and the activity of the transport systems.

The limited bioavailability of bioactive compounds hinders their use as functional ingredients. It is only by understanding the mechanisms of the absorption of bioactive compounds , the possibilities of different delivery systems that can be used, and the ways to protect these molecules during the whole chain from ingredient manufacturing to product formulation and storage conditions, that the bioavailability of bioactive compounds can be enhanced and their efficiency improved.

15.2 Improvement of Bioaccessibility and Bioavailability

Food bioaccessibility has not been investigated to a great extent, and it seems that scientists up to now have put more effort into studying and understanding nutrient bioavailability than bioaccessibility (Patil et al. 2009). This is understandable from the perspective that bioavailability is the limiting step in bioefficacy . However, that is not the case if the bioactive compounds are not liberated from the food matrix in the first place. Aiding the bioaccessibility of bioactive compounds from foods through different means of delivery systems, processing , and molecular modifications can promote both the bioavailability and bioefficacy of bioactive compounds (Fig. 15.1).

Fig. 15.1
figure 1

Basic steps involved in the bioavailability/bioaccessibility of bioactive food compounds. a Regular absorption of bioactive compounds from the food matrix that generally results in lower efficacy. b Methods/techniques for improving absorption of food compounds aiming to increase efficacy

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15.2.1 Food Processing

Food processing takes many different forms, from mixing to milling, drying to cooking, fermenting to roasting, and so forth. Various processing steps can affect the bioactive food compounds, whether of plant or animal origin, either destructively or beneficially.

Processing of plant foods can influence the bioaccessibility of digestible starch, liposoluble vitamins and carotenoids, and other nutrients. For example, changes in cell wall structure and properties or isomerization and other structural changes in bioactive molecules can occur due to processing steps such as heating (Colle et al. 2013). These changes can then alter the bioaccessibility and bioavailability of the different food compounds, carotenoids having been studied extensively in this respect. It has been shown that cooking and pureeing carrots improves in vitro bioaccessibility and bioavailability of carotenoids (Netzel et al. 2011), and significantly more β-carotene is absorbed by human ileostomy patients from meals containing cooked, pureed carrots than from meals containing the raw vegetable (Livny et al. 2003). Processing raw tomatoes into tomato paste has been shown to increase the bioavailability of lycopene, a major carotenoid in these vegetables. The addition of lipids prior to processing of tomatoes also enhances lycopene bioaccessibility (Colle et al. 2013); however, it seems that the effect of fat is selective for the type of carotenoid and appears to be dependent on the food matrix (Victoria-Campos et al. 2013). Homogenization of wolfberry in hot skimmed milk was found to significantly improve the bioavailability of zeaxanthin, another carotenoid pigment in plants, in healthy humans when compared to conventional homogenization with hot water or with warm milk. The presence of milk proteins and high temperature improved zeaxanthin bioavailability through incorporation into mixed micelles, uptake to enterocytes, and release in lipoproteins, although the exact mechanism still remains unclear (Benzie et al. 2006). A majority of studies relating to processing effects on carotenoids have demonstrated a positive effect of cooking on bioaccessibility and bioavailability. In the case of red peppers, however, the opposite was observed. Different carotenoid pigments were more bioaccessible in in vitro experiments when digested raw, and grilling and boiling reduced the bioaccessibility of most free and esterified pigments of green and red peppers (Victoria-Campos et al. 2013). The same observation was made with fruit juice beverages containing milk, where high-pressure temperature and thermal treatment did not facilitate carotenoid bioaccessibility. It was thus concluded that higher amounts of total carotenoids in beverages as a consequence of processing do not ensure an increase in bioaccessibility. The controversial bioaccessibility results found with carotenoids from different plant origins indicate that the effects of food processing on bioactive food compound bioaccessibility and bioavailability are much more complex than the largely positive effects expected in relation to mechanical or thermal processing involving plant cell wall disruption (Cilla et al. 2012).

15.2.2 Matrix Effect

Interactions between different food components influence the bioaccessibility and bioavailability of the digested food (Fernández-García et al. 2009; Shen et al. 2011). The physicochemical properties of the food and the surrounding environment in the digestive system such as pH, concentrations, and intermolecular forces affect the bioaccessibility and bioavailability of the food compounds (Neilson and Ferruzzi 2011; Serra et al. 2013). In a simulated gastrointestinal digestion of cooked seafood products, the bioaccessibility of fluoride was increased by a lower pH, the presence of cations, and a higher concentration of bile salts (Rocha et al. 2013).

There is increasing evidence that dietary fibers play an important role in the bioaccessibility of nutrients and bioactive compounds during digestion. In a simulated digestion, delivery of microencapsulated tuna oil from a cereal bar was hindered in comparison to that from orange juice or yogurt, most likely due to the high content of fat and dietary fiber in the matrix (Shen et al. 2011). In studies comparing the digestion of bioactive compounds from vegetables such as broccoli or cabbage with the digestion of pure bioactive compounds, the vegetable matrix improved the bioavailability in an in vitro model with the latter and in a rat model with the former vegetable (Keck et al. 2003; Lee et al. 2014). On the other hand, plant cell walls are largely resistant to degradation in the upper gut, representing an important barrier for the release of bioactive compounds . One such example is ferulic acid, a phenolic acid in whole grains, which has limited bioavailability due to its high binding to polysaccharides, i.e. dietary fibers. Mateo Anson et al. (2009) studied the bioaccessibility of ferulic acid from wheat fractions and baked breads. The authors observed that wheat ferulic acid had low bioaccessibility (<1 %), but that bioaccessibility was high when free ferulic acid was added to baking flour (~60 %), because it was not bound to polysaccharides. Similarly, when cocoa cream was enriched with proanthocyanidins, a condensed tannin type of polyphenol, and fed to rats, the metabolite profile of rat plasma was different from the plasma metabolite profile of non-enriched food given to rats, indicating that formulation or fortification of bioactive compounds was a potential means of improving bioavailability (Serra et al. 2013).

Improved bioavailability through improved bioaccessibility can be seen in the following example of phytosterols, which are plant sterols with a chemical structure similar to that of cholesterol. When phytosterols were added to food products, changes in product texture appeared due to recrystallization. The crystalline phytosterols cannot be absorbed in the intestine, thus resulting in very low bioaccessibility; however, synthesized colloidal phytosterols have better solubility, which improved their bioavailability (Rossi et al. 2010). More recently, crystalline deposition of bioactive molecules was studied and the bioavailability of β-carotene and lycopene from carrots, tomatoes, and papaya were compared in a randomized crossover trial with healthy subjects. The bioavailability of β-carotene from papaya was found to be ca. three times as high as that from carrots and tomatoes. The possible explanation is related to the liquid crystalline deposition of β-carotene, and the storage of lycopene in very small crystalloids in papayas was found to be associated with their high bioavailability (Schweiggert et al. 2013).

It has been recognized that foods with high caloric and fat content promote improved bioavailability through their effects on intestinal physiology (Mullen et al. 2008a, b; Welch et al. 1988a, b). For example, Walsh et al. (2003) proposed through an in vitro stimulation that the bioavailability of isoflavonoids from foods containing fat and protein exceeded that of isoflavonoid supplements consumed without food. In a study using a sophisticated gastrointestinal model (TIM-1 model) investigating the effects of a high-fat meal matrix and protein complexation on blueberry anthocyanin bioaccessibility , it was shown that blueberry-enriched defatted soybean flour anthocyanin samples were protected during the upper gut transit by the co-existing proteins, resulting in higher recovery from the TIM-1 system compared to plain blueberry juice anthocyanin samples (Ribnicky et al. 2014). The addition of olive oil as an oil-in-water emulsion during digestion has also been demonstrated to increase carotenoid bioaccessibility in their micellar uptake from tomato- and carrot-derived purées (Moelants et al. 2012). Fat also increased the bioaccessibility of chlorophyll in a digestive model (Victoria-Campos et al. 2013).

Few scientific publications have focused on the effect of the food matrix in modulating the bioavailability of coffee phenolics and flavonoids present in cocoa and tea products. Some studies have looked at the effect of milk proteins on modulating the bioavailability of coffee chlorogenic acids. Using in vitro/ex vivo modeling, Dupas et al. (2006) showed that 40 % of added 5-caffeoylquinic acid binds to casein. Renouf et al. (2010) studied the effect of adding milk or sugar/non-dairy creamer to coffee on the bioavailability of phenolic acid equivalents in humans. When 10 % whole milk was added to coffee, no significant difference was observed in AUC, Cmax, or tmax of caffeic acid, ferulic acid, or isoferulic acid equivalents compared to coffee alone. However, when sugar/non-dairy creamer was used, some differences in Cmax (lower) and tmax (longer) were observed compared to plain coffee. The overall delivery of chlorogenic acid metabolites was not significantly different between treatments, but the addition of sugar/non-dairy creamer to coffee led to significant changes in the plasma appearance of those metabolites.

The food matrix does not influence the bioaccessibility and bioavailability of bioactive compounds in all cases, as was shown with hesperidin, a citrus fruit bioactive belonging to the flavanone subgroup of flavonoids. Consumption of orange juice with full-fat yogurt did not significantly affect the Cmax or tmax values of hesperidin (Mullen et al. 2008a, b). Food processing also had no effect on hesperidin bioaccessibility and bioavailability when comparing the consumption of orange fruits with the consumption of orange juice (Brett et al. 2009). Controversial results have been reported with regard to the effect of milk protein on the bioaccessibility of epicatechin, the flavan-3-ol subgroup of polyphenols present in significant quantities in foods such as cocoa, teas, and apples (Rein et al. 2013). Serafini et al. (2003) reported an unfavorable effect of milk on the bioaccessibility of epicatechin, showing a lower AUC when chocolate was ingested with milk. However, several other studies have demonstrated the nonexistence of such an effect of milk (Schroeter et al. 2003; Keogh et al. 2007; Roura et al. 2007, 2008). It was recently proposed that milk reduces the urinary excretion but not plasma pharmacokinetics of cocoa flavan-3-ol metabolites in humans (Mullen et al. 2009). In addition to the effect of milk, Neilson et al. (2009) concluded that the physical form of the food and the sucrose content may influence the tmax and Cmax of cocoa flavan-3-ols. With regard to tea, in general, it is suggested that tea with milk has a negligible effect on the bioavailability and bioaccessibility of the flavan-3-ols (Reddy et al. 2005; Van der Burg-Koorevaar et al. 2011).

When addressing the bioaccessibility and bioavailability of bioactive food compounds, it is necessary to evaluate not only the digested source of these nutrients, but the effect of the whole meal ingested at the same time. Such bioaccessibility data can be used to support the risk/benefit estimation of vitamin and mineral intake (Rocha et al. 2013) as well as that of other bioactive compounds .

15.2.3 Molecular Interactions

Factors influencing bioavailability include physical and chemical properties such as hydrophobicity, pK a, and solubility. On the other hand, bioavailability has some connection with permeability, efflux transporters (e.g. P-glycoprotein, P-gp), and enzyme induction or inhibition on intestinal epithelial cells. The molecular structure of a compound has a considerable effect on its absorption and therefore its bioavailability (Scholz and Williamson 2007). Molecular weight is an important factor to consider; for example, high molecular weight compounds such as the oligomeric proanthocyanidins and complex lipids do not pass through the intestinal cells unless they are broken down (Deprez et al. 2000; Appeldoorn et al. 2009).

According to Leucuta (2014), the requirements for absorption of chemical compounds are solubility and permeability, both aspects highly influenced by lipophilicity. The same is applied to bioactive food compounds; McClements (2013) reported that the bioavailability of many lipophilic bioactive compounds was relatively low due to their poor solubility, permeability, and/or chemical stability within the human gastrointestinal tract. In a double-blind, randomized, placebo-controlled crossover study using healthy volunteers, Ramprasath et al. (2013) reported that due to structural differences between krill oil, a phospholipid-based oil, and fish oil, a triglyceride-based oil, the bioavailability of krill oil was higher than that of fish oil, resulting in a more effective increase of n-3 polyunsaturated fatty acid (PUFA) levels. Cruz-Hernandez et al. (2012) reported improved bioavailability of eicosapentaenoic acid (EPA) when delivered by structured and free monoacylglycerols (MAG) in a preclinical model of lipid malabsorption. Moran et al. (2013) studied the metabolic differences in an animal model between lycopene, the major tomato carotenoid, and phytoene, a minor tomato carotenoid. Although phytoene is a minor component in tomatoes, it is found in human blood and tissues in concentrations similar to those of lycopene. The two carotenoids differ not only structurally by saturation, but also in terms of bioavailability, tissue deposition, and clearance. The results of this study suggest that phytoene has greater bioavailability and is cleared more slowly than lycopene.

Some bioactive food compounds are attached to sugar moieties such as β-glucosides that can be absorbed and metabolized by enzymes (e.g. β-glucosidases and lactase-phlorizin hydrolase [LPH]) in the small intestine (Hollman et al. 1999). However, when the compounds are attached to an additional rhamnose moiety, they need to reach the large intestine to have the sugar moieties cleaved off by the intestinal microbiota before absorption (Erlund et al. 2000). Nielsen et al. (2006a, b) studied the improvement in the bioavailability of hesperidin by enzymatic modification in a randomized double-blind crossover trial in humans. Hesperidin was reported as having low bioavailability due to its rutinoside moiety. The authors noted that the consumption of orange juice treated with hesperidinase, leading to the conversion of hesperidin to hesperetin-7-glucoside, significantly and favorably affected bioavailability parameters such as AUC, Cmax, and tmax.

Apart from the chemical structure of food bioactive compounds , their isomeric configuration can also affect their absorption. This is the case for (−)-epicatechin and (+)-catechin bioavailability (Ottavianni et al. 2011) as well as cis-isomers and all-trans isomer of lycopene bioavailability (Boileau et al. 2002), for the biological activity of (R-, S-) equol (Muthyala et al. 2004), and for the metabolism of (R-, S-) hesperidin (Lévèques et al. 2012). Jensen et al. (2006) studied the bioavailability of α-tocopherol stereoisomers in a rat model fed for 10 days a diet containing either α-tocopherol—also referred to as RRR-α-tocopherol (2,5,7,8-tetramethyl-2R-(4’R,8’R,12-trimethyltridecyl)-6-chromanol)), and which has the highest reported biological activity—or all-rac-α-tocopheryl acetate (2,5,7,8-tetramethyl-2RS-(4’RS,8’RS,12-trimethyltridecyl)-6-chromanol), a mixture of isomers of synthetic origin and widely used to supplement food . The authors observed that increasing dietary levels of all-rac-α-tocopheryl acetate led to a decrease in the proportion of RRR-α-tocopherol found in plasma, whereas the other stereoisomers were not affected. This indicated that in a racemic mixture, the presence of other stereoisomers hindered the absorption of RRR-α-tocopherol.

Other important factors affecting the bioavailability of bioactive food compounds are the different transport mechanisms taking place in the intestinal lumen, including passive diffusion, facilitated diffusion, and active transport (Rein et al. 2013). Therefore, potential ways of improving the bioavailability of bioactive food compounds could emerge through the competition and inhibition of intestinal cell transporters (Scheepens et al. 2010). The ATP-binding cassette (ABC) family of transporters, including the intestinal P-gp (P-glycoprotein) efflux pump, have been reported as a major contributor to the low bioavailability of a number of compounds (Xie et al. 2011). For example, in vitro experiments suggest that the bioavailability of the flavonoid hesperidin may be enhanced by inhibiting the ABC transporters by competitive exposure to other flavonoids such as quercetin, resulting in a decrease in the efflux of hesperidin (Brand et al. 2008). The same approach has been suggested for (−)-epigallocatechin 3-gallate (EGCG), where the combination of the bioactive with naturally occurring inhibitors of efflux proteins resulted in increased cytosolic levels of the compound (Hong et al. 2003).

Membrane transporters are involved in two mechanisms related to the permeability of compounds: uptake and efflux. Vitamin transporters, the glucose transporter (GLUT) family, sodium-glucose linked transporter (SGLT) family, and organic anion transporter 1 (OAT1), amongst others, are involved in the uptake of compounds, enhancing their transport across the intestine (Kerns and Di 2008). Whitley et al. (2005) studied the ability of organic anion transporters (OATs) and organic anion-transporting polypeptides (OATPs) to transport ellagic acid, a phenolic acid found in berries and nuts. OATs play a critical role in the distribution and elimination of a diverse array of exogenous and endogenous compounds. The authors reported that the interaction between hOAT1 and rOat1 with ellagic acid was of high affinity, suggesting the potential combination of ellagic acid with other compounds in order to improve their bioavailability.

Cytochrome (CYP) enzymes are responsible for the breakdown of endogenous and exogenous compounds into metabolites, with various CYP forms present in the human liver. Flavonoids have been identified as inhibitors of cytochrome P450 enzymes (Dresser and Bailey 2003). Kimura et al. (2010) studied the inhibitory effects of polyphenols on human cytochrome P450 3A4 and 2C9 activity by in vitro assays. The results showed that three coumarins and 12 flavonoids significantly suppressed CYP3A4 or CYP2C9 activity, and among these compounds, galangin, chrysin, and apigenin, which are present in propolis, had the most potent inhibitory effect against these CYP isoforms. These findings suggest that dietary compounds, and particularly flavonoids, may have the potential to inhibit the metabolism of other compounds such as clinical drugs.

As bioactive food compounds are present in our diet in many different forms, understanding and elucidating their chemical properties is crucial when studying the bioavailability and potential bioefficacy of these compounds with regard to human health.

15.2.4 Delivery Systems

The use of nanoparticles has been reported as a promising means of improving the delivery of compounds with low bioavailability, leading to enhanced efficacy (Li and Huang 2008). However, the main emphasis in the design of nanosystems is improving local bioavailability. For example, nanotechnology is used to increase compound delivery to the diseased target tissue (e.g. vessel wall, tumor, or brain) in addition to improving the therapeutic index or avoiding toxicity (Yliperttula and Urtti 2008).

In principle, the use of nanosystems could facilitate the transport of the compound across biological barriers, as well as avoiding the metabolic modifications that could lead to low absorption. However, it is very important to understand from an efficacy perspective that the choice of carrier material in the oral delivery system is extremely important, because it significantly affects the pharmacokinetics and pharmacodynamics of the ingested compounds (EFSA Guidelines 2011; Xie et al. 2011). Xie et al. (2011) developed PLGA [poly (lactic-co-glycolic acid)] nanoparticles to improve the bioavailability of curcumin, a low molecular weight hydrophobic polyphenol that is extracted from turmeric, using a rat model. The bioavailability of curcuminoids is low due mainly to their poor water solubility and rapid metabolism. After oral administration of curcumin nanoparticles, relative bioavailability was 5.6-fold higher and had a longer half-life compared with the native curcumin. Neves et al. (2013) reported the use of lipid nanoparticles for improving the bioavailability of resveratrol, a polyphenol found in grapes and red wines. This compound has been described as having poor bioavailability and low water solubility, and as being chemically unstable. The authors conducted in vitro release studies on conditions of storage, and concluded that the lipid nanoparticles could be considered suitable carriers for oral administration, conferring protection to the incorporated resveratrol and allowing a controlled release after uptake. Tzeng et al. (2011) reported the use of a nanoparticle engineering process to enhance the dissolution and antioxidant activity of kaempferol, a flavonoid widespread in food sources but with limited clinical application due to poor dissolution properties. The authors suggested that kaempferol nanoparticles could be considered a low-dose alternative to kaempferol in health food and future clinical research.

The application of either encapsulation or emulsions is also an effective strategy for achieving several objectives, including improved bioaccessibility and stability of the bioactive compounds . For example, Yu et al. (2012) developed a food-grade curcuminoid organogel with high bioaccessibility and high loading of curcumin. Organogel-based delivery systems are relatively new in food science. Organogels are formed by liquid oils trapped by the extensive crystalline networks of organogelators, such as monoglycerides, fatty acids, and fatty alcohols. In another study, Rossi et al. (2010) reported the synthesis of colloidal phytosterols and evaluated their bioaccessibility in an in vitro model that resulted in effective solubilization in model dietary mixed micelles, and the micellar cholesterol concentration was effectively reduced by almost 50 % within 2 h. Fernandez-Garcia et al. (2008) developed an emulsifier system to improve the bioaccessibility of carotenoids. Bioaccessibility of these compounds from natural sources (mainly fruits and vegetables) is often low and is conditioned by different factors, mainly the processing state of the food and the matrix composition. One of the key factors affecting bioaccessibility is the amount and type of fat present in the food. A minimum amount of fat is required for increasing absorption, so formulation of carotenoids in an oily matrix (vegetable oils) may provide high bioaccessibility. However, as was previously discussed in this chapter, it has been shown that the bioaccessibility of carotenoids from fatty food formulations is not as high as expected. Walde et al. (2013) compared the bioavailability of dietary tocotrienols from barley and palm oils in an animal model. The main component of tocotrienols from barley is α-tocotrienol, while palm oil tocotrienols are particularly rich in γ-tocotrienol. Nanoemulsification of barley oil led to higher tocotrienol levels than with non-emulsified oil, resulting in high proportions of α-tocotrienol. In addition, the authors observed that tocotrienol content in hens fed barley oil was significantly higher than in those fed palm oil, suggesting that α-tocotrienol is better absorbed than γ-tocotrienol.

Since the application of nanoparticles is still a new field in the nutrition and food science areas, it is important to emphasize that it must be evaluated in preclinical toxicological tests and in human clinical trials. Nonetheless, many authors have reported that safety problems are expected to be no greater with nano-delivered compounds than with regular compounds (Yliperttula and Urtti 2009).

15.3 Human Studies and Efficacy

Although numerous studies have reported the potential health benefits of the diverse bioactive food compounds, many aspects related to their bioavailability , and particularly their metabolism, have been poorly investigated. In recent years, there has been an increase in the number of human intervention studies in the nutrition area; however, until now the majority of studies on the bioefficacy of bioactive food compounds have typically been based on in vitro models evaluating the parent compound at concentrations far higher than the physiological range. Therefore, when considering their potential beneficial effects, there are still many aspects that need a better understanding, especially with regard to the main compounds and their metabolites responsible for the health benefits and mechanisms of action (da Silva Pinto 2013).

The fact is that, today, there is an even stronger need for well-controlled long-term human studies in order to fully comprehend the metabolic and potential health effects of bioactive food compounds. It is important to understand that when planning any study, the choice of experimental design and statistical methods of analysis are crucial, since this choice will be reflected in the outcome of the analysis and the conclusions drawn (Chow and Liu 2009). Another important point that researchers should consider when planning human intervention studies to evaluate potential health benefits associated with bioactive compounds is that not only must care be taken in the experimental design, but the hypothesis must be very well defined prior to the study, as well as the biological relevance in the context of overall diet.

Because most food sources contain a mixture of potential bioactive compounds, it is also important to highlight the current need to identify and investigate the bioefficacy of the circulating metabolites, since it is most likely these compounds, and not the parent compounds, that will reach the various sites of action. In addition, since individual subjects differ widely in their physiological and metabolic responses to the compounds, the knowledge of inter- and intra-subject variability provides valuable information for assessing bioavailability . In general, in order to improve the intra-subject variability in bioavailability comparisons, a crossover design is often considered.

Indeed, care should be taken not only to consider statistical significance as the aim of the intervention study, but also the biological relevance of the primary objective/outcome that must be clearly defined prior to the study. For example, a statistically significant difference in the comparison of bioavailability between compounds does not necessarily imply that there is a clinically significant difference between them, since they can still achieve the same therapeutic effect (Chow and Liu 2009).

15.4 Conclusions and Future Perspectives

Bioaccessibility and bioavailability are crucial steps in assessing the potential health benefits of bioactive food compounds, and evaluating these steps is important in understanding the relationship between food and nutrition. Bioavailability addresses several phases, including liberation from the matrix, absorption, distribution, metabolism, and elimination (LADME). Scientists up to now have put more effort into studying nutrient bioavailability than bioaccessibility, although aiding the bioaccessibility of nutrients and bioactive compounds from foods through different processing techniques, delivery systems, and molecular modifications are excellent means of promoting bioavailability, and consequently bioefficacy . The various methods of improving bioaccessibility and bioavailability are all aimed at enabling the bioactive compound to exert a health benefit, improving performance, or managing risk.

Food processing can enhance the bioaccessibility of different compounds, for example, through disruption of plant cell walls, inducing molecular isomerization and other structural changes, influencing solubility, and improving micellar uptake of lipophilic bioactive compounds. Similar effects take place through changes in molecular interactions , which can influence the absorption of bioactive compounds through different transport mechanisms or impact metabolism and enzyme activity. The food matrix also plays an important role in bioaccessibility and bioavailability . The content of fat and dietary fiber in a given food can have a matrix effect, either hindering or improving nutrient bioavailability. Therefore, understanding the bioaccessibility and bioavailability of bioactive food compounds and the complexity of these processes is a key aspect in the formulation of functional foods.

The available data from bioaccessibility and bioavailability clinical studies can support the benefit and risk estimations of nutrients and other bioactive compounds. Although nanosystems have been investigated recently in terms of improving the bioavailability of clinical drugs, for nutrition we are still in the first stages of in vitro and preclinical investigations. As food sources are a mixture of bioactive compounds , well-designed, long-term human studies are needed in order to gain an understanding of the potential health effects of these bioactive compounds in the context of overall diet. Human studies are also needed to validate the observations made in in vitro and preclinical studies on bioaccessibility and bioavailability before any claims of bioefficacy with bioactive food compounds can be considered.