As seen in the previous parts, the study of oral bioaccessibility of nutrients is fundamental in order to evaluate their bioavailability. Many phytochemicals, despite the clear effects demonstrated in vitro, could have limited health benefits because they need to be effectively transported to target tissues and organs at effective dose levels in order to have beneficial effects on health. The various physical and chemical characteristics of phytochemicals can in fact cause problems for the effective formulation of an ingredient, polyphenols are a perfect example of this. A perfect example in this case is curcumin in which the low solubility in water leads to poor bioaccessibility which therefore limits its beneficial effects. (Chen L. et al. 2006)

Over the years, there has been a growing interest in the study of bioavailability, and consequently of the possible mechanisms to improve it, and among all the strategies the encapsulation of the active ingredient in a carrier (preferably of natural origin), capable of stabilizing it and improving its bioaccessibility has also been among all the most exploited.

The purpose of encapsulation of an active ingredient is indeed its protection, but it is also to easily allow its release into the enteric tract responsible for absorption. The encapsulated actives therefore obtain better stability and bioavailability.

Among the most important parameters to consider for encapsulating an active or a phytochemical we have the nature of the carrier, the loading capacity, the encapsulation efficiency, the mechanism and the percentage of release from the carrier.

The nature of the carrier is one of the fundamental parameters for creating a good product. Due to their chemical-physical nature they can be divided into protein carriers (gelatin, wheat gluten, soy proteins, casein, keratin, collagen etc.), lipid carriers (oils and waxes, fatty acids and monoglycerides etc.) and polysaccharides carrier (cellulose, chitin, alginates, pectin etc.). Each of these ingredients can be used alone or in combination, but all these elements can potentially also be found in a phytocomplex which can then itself encapsulate phytochemicals. (Frosi I. et al. 2022)

Understanding the base on which the carrier is formulated is important to understand its functionality:

Protein-based carrier

Vegetable proteins are widely used for the development of carriers, especially given their high nutritional value, as well as their low toxicity, biocompatibility, biodegradability and numerous functions capable of producing and stabilizing emulsions and gels. The presence of different functional groups on the different amino acids that make up proteins offer a high capacity to form bonds and furthermore the amphiphilic nature of proteins makes them suitable as emulsifiers and gelling agents. Several studies have also reported that these properties can be modulated through simple technological modifications. (Chen L. et al. 2006)

If proteins are used to create microparticles, they are able to control the release of the incorporated active for correct delivery to the absorption site. This occurs by formulating the carrier in order to obtain a precise structure which can consist of a core containing the active ingredient and other ingredients and the external barrier of variable thickness.

Plant proteins can also be modified or incorporated into other materials to increase their encapsulation functionality and therefore further increase the bioaccessibility of encapsulated actives.

Lipid-based carrier

Just like proteins, lipids can also be an excellent material for encapsulating active ingredients, solid lipid nanoparticles have shown excellent delivery efficiency of lipophilic active ingredients. Vegetable lipids and oils have been much studied for their application as carriers in different encapsulation methods such as liposomes, proliposomes, phytosomes, emulsions and nanostructured lipid particles. Lipid-based encapsulation showed excellent protective, controlled and site-specific release properties.

Lipids are molecules very suitable for being modified to improve their versatility of use, also different types of lipids can be combined together to obtain different products. Examples are the phytosome and the liposome. The phytosome differs from the classical liposome for the encapsulation of the active compound based on the interaction with the hydrophilic heads of the phospholipid through hydrogen bonds. This in fact leads to the formation of a phospholipid phyto-complex in which the lipophilic phosphatidyl group envelops the choline-active compound complex, forming a more stable and efficient vesicle than the classic bilayer vesicle of the liposome. In this way the phytosome manages to implement the bioaccessibility of both fat-soluble and water-soluble compounds, even simultaneously. (Subramanian P. et al. 2021)

Polysaccharide-based carrier

Natural polysaccharides are a class of biopolymers that differ from each other in the monosaccharide units, the molecular weight and the glycosidic bonds between the monomers present in the polysaccharide chain. They are very stable, safe, non-toxic, hydrophilic and biodegradable molecules. Furthermore, thanks to the various functional groups present in the structure, they are excellent carriers for the encapsulation of phytochemicals of various nature. In fact, polysaccharides can be modeled and modified with simple technological approaches and therefore adapted to the encapsulation and delivery of active molecules.

An example of a polysaccharide widely used in this field is chitosan, a natural polysaccharide with good solubility and biodegradability which has been studied for the delivery of polyphenolic molecules. Chitosan nanoparticles can effectively achieve controlled release and increased bioaccessibility of polyphenols.

The first preparations of polysaccharide nanoparticles required the use of covalent cross-linkers such as di- and tri-carboxylic acids (tartaric, citric, malic acid for example) which allowed the condensation reaction between the carboxyl groups of the acids and the amino group terminal of the polysaccharide, allowing to obtain biodegradable chitosan nanoparticles. To date, these nanoparticles are obtained by ionic cross-linking which allows for milder and simpler preparation conditions. In fact, for charged polysaccharides, polyanions and polycations can act as ionic cross-linkers to create polyanionic and polycationic polysaccharides respectively. (Liu Z. et al. 2008)

Last but not least, in PART 5: PHYTOCLEW^® - FLANAT’s polysaccharide sustainable carrier to improve bioaccessibility. 

Once understood that INFOGEST is the standard to follow, many companies have adopted this method to determine bioaccessibility. We, at FLANAT, have decided to follow this path to test in vitro bioaccessibility with a static method to be able to have simple studies but be able, at the same time, to get usable data for the purpose of obtaining functional and effective bioaccessible products.

We use in our R&D lab a static in vitro method, based on INFOGEST protocol, optimized by the University of Pavia IT (UNIPV chemical, toxicological and food analysis laboratory). Bioaccessibility tests are performed using an HPLC-UV-Vis analysis with the aim of determining the percentage of bioaccessibility for each phytochemicals. (Ferron L. et al. 2020)

The simulation of the digestion model is made up of several sequential phases in each of which the use of simulated digestive fluids is envisaged, consisting of stock solutions of electrolytes added in different concentrations for each phase of the digestive process. Simulated digestive fluids, i.e. Simulated Salivary Fluid (SSF), Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF) have the role of reproducing physiological digestive fluids for the oral, gastric and intestinal phases respectively.

The INFOGEST in vitro digestion protocol does not take into consideration the colon phase, which is instead studied by other experts. The model developed in 2015 by Hamzalioğlu et al. in order to better mimic the digestive process, introduced the colon phase, and modified some concentrations of the enzymes and bile salts constituting the different simulated digestive fluids. The in vitro static digestion model used in our laboratory combines all the phases together to have a complete model.

Oral phase

The first step of the oral phase is different depending on the formulation or when the food is in liquid or solid form. In the case of liquids this phase can be omitted and move directly to the gastric phase. In the case of a solid form, it is necessary to simulate the chewing process using a kitchen chopper, in order to reduce the size of the food. Only after this phase are the digestive fluids and enzymes added to simulate salivation which allows the food/formula to be lubricated to obtain the bolus. Next step is to weight the appropriate quantity of sample to be analyzed to which the SSF and α-amylase are added, the hydrolase which in the oral phase is capable of splitting the 1,4-α-D-glucosidic bonds and therefore capable of breaking down starch, glycogen and similar molecules. To simulate the oral phase as adequately as possible, the sample thus prepared is incubated in a thermostated bath and with adequate agitation to simulate passage into the mouth. (Minekus M.  et al. 2014)

Gastric phase

To simulate the gastric phase in our system, the SGF, the specific enzyme and HCl are sequentially added to the sample obtained from the oral phase in variable quantities depending on the starting pH of the solution and taking into account that the purpose of the addition of HCl is to bring the pH level to a value of 3. Pepsin enzyme is also a hydrolase activated by the HCl present in the stomach capable of breaking down peptide bonds. The solution obtained, containing the sample, is incubated in a thermostated tilting bath in order to reproduce the digestion time in the stomach. (Minekus M.  et al. 2014)

Intestinal phase

Once the gastric phase is over, the final sample will immediately be subjected to the intestinal phase. To simulate the intestinal environment, SIF, pancreatin and bile are added sequentially. Pancreatin is a mixture of enzymes produced by the pancreas containing α-amylase, trypsin, lipase, ribonuclease and protease, while bile is a non-enzymatic isosmotic solution that collaborates in the digestion and absorption of dietary fats. Suitable quantity of NaOH could be added to bring the solution to the intestinal pH of approximately 7. The sample is then incubated again in a tilting thermostated bath. (Minekus M.  et al. 2014)

Colon phase

The colon phase, is simulated by adding colon bacterial flora proteolytic enzymes to be able to quickly and easily reproduce the environment of the large intestine. In fact, during this phase the hydrolysis of proteins and polysaccharides continues to take place in the sample and it is therefore essential to study this phase too, especially for more complex matrices. For this reason the protease enzyme, a hydrolase are added, and finally Viscozyme® L (Novozymes Corp.) is added. A more prolonged period of incubation is needed to best simulate this phase of digestion. The colon receives all material that has not been digested and absorbed in the upper portions of the gastrointestinal system and has the main function of absorbing water and electrolytes, fermenting polysaccharides and proteins, reabsorbing bile salts, and accumulating and eliminating feces. Studying this phase in an in vitro digestion protocol is also important as some lipids that should be digested in the stomach and intestine could arrive at this phase not completely digested, and therefore not absorbed. An example would be an oil-in-water emulsion surrounded by an indigestible coating, or inserted into an indigestible matrix, which cannot be completely digested by the intestine. To therefore consider the in vitro study of the digestion process complete, it is important to also consider the colon phase. (Hamzalıoğlu A. et al. 2015)

The sample is always heated at the end of each incubation phase to block the reaction of the enzymes. A centrifuge is then carried out in order to collect the supernatant, therefore everything that has passed into solution. The analyses are then performed on HPLC with the analysis method suitable for quantifying the bioaccessible actives.

Stay tuned to know in PART 4 OF 5 how bioaccessibility can be improved

In PART 1 we acknowledge that the bioaccessibility of a compound is the amount that is released from its matrix in the gastrointestinal tract, making it available to enter the bloodstream. We will now look at methods for determining the amount of compound available to be absorbed.

It can obviously be affirmed that the optimal method to determine the bioaccessibility of a compound is through the study of animal or human models (in vivo) capable of providing precise data; however, these studies have limited use as they are economically demanding, time consuming, and have ethical limitations. For this reason, knowledge of digestion is essential to simulate an adequate digestive process in the laboratory (in vitro) considering all the variables involved, such as digestive enzymes, the pH in the gastric and intestinal phase, the digestion time and the concentration of salts present in the digestive tract.

Over the years, various simulations models have been designed simulating the physiological conditions and events that occur during digestion in humans, but given the complexity of the human organism, it has not been possible to recreate an in vitro model that uniquely reflects the physiological conditions and events occurring in human during digestion. In vivo studies, both on animal and human models, generally allow more precise results to be obtained and for this reason they are still considered the "gold standard" for this type of study, although little used given the reasons already described above. For this reason, in vitro models are used to predict the in vivo performance of the formulation, so these digestion protocols must be designed to mimic the performance in the organism as much as possible. At the same time, we also want in vitro models to be fast, labor-intensive, robust and economical. So the protocols used are typically a compromise between complexity and practicality.

In vitro methods can be distinguished as static or dynamic models. Static models, also called biochemical models, are the simplest and are used above all for foods and primary formulations such as powders or liquids, to isolate or purify nutrients or to isolate allergenic proteins. Static models involve the incubation of the food or formulation in a series of bioreactors in which the physiological and enzymatic environment of each digestive compartment is mimicked. Dynamic models can be mono- or multicompartmental and allow the incorporation of physical processes and temporal changes in the conditions of the gastrointestinal tract, in order to better mimic in vivo conditions. (Dupont D. et al. 2019)

An advanced multi-organ cellular model, capable of reproducing the gastro-intestinal system, has been developed at the University of Pavia from the research group of Professor Papetti. The dynamic millifluidic model is based on the LiveFlow® bioreactor, (IvTech Srl - Massarosa - LU Italia), a versatile platform for recreating different cellular or tissue models, even in a multi-organ approach. The multicompartmental system associate gastric (GIST-882) and intestinal (Caco-2) cells, causing molecules to continuously flow onto the cells. The gastric and intestinal environment is therefore reproduced in the different compartments in order to determine the gastric and intestinal absorption and the metabolic profile of the sample flowing within the system. (Colombo R. et al. 2019)

In general, all in vitro digestion processes can be divided into several phases (oral, gastric and intestinal) and can be studied separately or in combination with each other based on the aim of the study. Several models have been so developed and all different from each other. The first ones developed, for example, do not contemplate the oral phase due to the complexity to simulate the mastication and the formation of the bolus, some other models have simplified this phase just homogenizing the food. Other more advanced methods have instead added the colon phase to the 3 main phases (oral, gastric and intestinal), in order to comprehensively study the digestion process. (Minekus M.  et al. 2014; Hamzalıoğlu A. et al. 2015)

The result obtained with an in vitro digestion can be significantly influenced by many factors: the sample characteristics, the digestive fluids composition, the mechanical stress, the incubation times and above all the enzymatic activity. This last is, in fact, in turn influenced by many factors such as the enzymes concentration, the pH of fluids in which they are found, the incubation temperature, their stability, the presence of activators or inhibitors and the incubation times. In vitro static digestion methods have attracted the interest of many researchers who have understood their potential application in various fields for the purpose of evaluating the bioavailability of different samples.

Although the static digestion may appear to be an easy method, the lack of consensus regarding the physiological conditions to be used has led to the creation of several models, very different to each other’s, which results are impossible to compare.

INFOGEST, an international research network, developed in 2015, thanks to a group of 200 researchers from all over the world, a static in vitro digestion model with the aim of harmonizing existing models. INFOGEST method is still recognized today as the official standard regarding these experiments. This standardization makes it possible to apply the same conditions even in different laboratories, thus obtaining excellent reproducibility of the results.

Part 3 will explain which method are used in our laboratory to evaluate bioaccessibility

The action associated with the intake of phytochemicals is linked to their bioavailability: the ability to be absorbed at an enteric level to reach the bloodstream and therefore reach the target of interest.

Plant secondary metabolites, or phytochemicals, are a large group of structurally and chemically different molecules known not to be essential for humans, but it is otherwise known that their intake has beneficial effects on the human organism, promoting the prevention of various pathologies.

Most of these compounds consumed daily through the diet do not reach the tissue concentrations necessary to exert the expected beneficial effects due to their low bioavailability. A prerequisite for the absorption of a compound at the intestinal level, and therefore for its bioavailability, is in fact its bioaccessibility: the fraction of the compound that is available for absorption after being released from the food matrix and having solubilized in the gastrointestinal fluids. (Alminger 2014)

Bioaccessibility is then a factor limiting the absorption, and consequently the bioavailability, of molecules and depends both on the characteristics of the food matrix and on the physiological conditions of the digestive process.

A 2020 study compared the bioaccessibility index of polyphenols present in different types of fruits and in the extracts obtained from them, observing that the food matrix plays a key role in the release of the molecules. In fact, the polyphenols present in the extracts are released more easily, while the fruit polyphenols have greater bonds with the matrix which prevent them from passing into solution. Important differences have been noted between the different types of fruit as the different bonds that can be established between the molecule and the matrix influence its release. (Tarko 2020)

The release of the molecules can be influenced by the food matrix and also by the type of process to which it is subjected. Some phytochemicals are in fact more susceptible to oxidation and degradation following matrix breakdown. In some other compounds, however, the destruction of the matrix is responsible for their release since they are normally enclosed in cellular compartments that prevent this. During the crushing of the garlic bulb, for example, alliinase is released, the enzyme that allows the production of those sulfur compounds responsible for the beneficial effects.

The chemical characteristics of the molecules are also a factor influencing bioaccessibility and carotenoids are another group of molecules whose release from the food matrix is difficult and depends on interactions with proteins; these bonds can be degraded with high temperatures (for example with cooking). Carotenoids, as lipophilic compounds, are absorbed following emulsification and incorporation into micelles, so it is possible to establish a competition between carotenoids and other lipophilic nutrients. (Alminger 2014)

The chemical characteristics of the molecules are also a factor that influences bioaccessibility; carotenoids, for example, are another group of molecules whose release from the food matrix is difficult and depends on interactions with proteins; these bonds can degrade at high temperatures (for example during cooking). Carotenoids, as lipophilic compounds, are absorbed after emulsification and incorporation into micelles, therefore competition may arise between carotenoids and other lipophilic nutrients. (Alminger 2014)

Other lipophilic compounds, such as quercetin and rutin, show low bioaccessibility due to their poor solubility. Resveratrol is also a non-water-soluble compound, however its solubility in ethanol suggests increased absorption and plasma concentration in the presence of a lipophilic food matrix. Despite this, its bioaccessibility remains limited: a study shows that it would be necessary to consume approximately 3500 L of red wine, 2600 kg of grapes, up to 35000 kg of peanuts or 2500 kg of apples per day to reach the correct daily doses. Furthermore, the different chemical characteristics of polyphenols also influence their stability to factors such as temperature, pH and enzymes.

Another fundamental element in the absorption of phytocomplexes is the digestive process and all the factors related to it. Anthocyanins, for example, are degraded before they even reach the intestine (their specific site of absorption). (Ozkan 2020) In-depth knowledge of the human digestive process and therefore of all the factors that intervene during the intake of a phytocomplex, allows us to evaluate the appropriate solutions to overcome all the critical points that limit its bioaccessibility.

In this way it is possible to formulate foods or supplements capable of releasing molecules that can be absorbed by the body in the necessary doses and manifest the desired beneficial effects. In fact, everything that is taken orally undergoes the digestion process before being absorbed systemically. The process of digestion of food is complex and involves two sub-processes that occur simultaneously: a mechanical process that starts from the mouth and continues up to the stomach in which the food is destroyed into smaller pieces, and an enzymatic process that also begins in the mouth and continues to the intestine where the macromolecules are broken into smaller molecules in order to be absorbed into the bloodstream. (Lucas-Gonzales 2018)

We can state that the bioaccessibility of a compound is the amount that is released from its matrix in the gastrointestinal tract, making it available to enter the bloodstream. Evaluating the bioaccessibility of compounds is therefore the primary objective in the development of a functional food or a nutraceutical formulation.

In Part 2 we will review how bioaccessibility is evaluated.