In the previous parts we talked about the importance of studying the bioaccessibility of phytochemicals and how to maximize it to improve the bioavailability of our ingredients. The most common and effective method to enhance this parameter is to encapsulate the active molecules to protect them from the digestion process and therefore improve their bioaccessibility.

Different natural compounds can work as carrier capable of encapsulating active ingredients such as lipids, proteins and polysaccharides. As explained in Part 4, each carrier interacts differently with molecules in accordance to its own chemical-physical nature.

In FLANAT we decided to study an innovative formulation to increase bioaccessibility of our botanical products, paying attention on sustainability. We at FLANAT do believe that working applying the Life Cycle Thinking approach is the future of sustainability, we then decided to study and develop a new carrier from the Camelina sativa L. cake, the co-product of the cold press seed extraction of Camelina oil.

The result is a patented phytocomplex, which mainly contains polysaccharides, obtained with a simple aqueous extraction of the Camelina cake.

𝐏𝐇𝐘𝐓𝐎 𝐂𝐋𝐄𝐖®, 𝐅𝐋𝐀𝐍𝐀𝐓’𝐬 𝐈𝐍𝐍𝐎𝐕𝐀𝐓𝐈𝐕𝐄 𝐏𝐎𝐋𝐘𝐒𝐀𝐂𝐂𝐇𝐀𝐑𝐈𝐃𝐄 𝐏𝐇𝐘𝐓𝐎𝐂𝐎𝐌𝐏𝐋𝐄𝐗

PhytoClew®, our innovative polysaccharide phytocomplex, acts as an incapsulating agent thanks to its gum-like composition that can trap molecules into its matrix and then release them by swelling and erosion of the matrix itself.

With PhytoClew® its possible to formulate phytocomplexes with improved stability and bioaccessibility without any addition of further solvents or excipients. Phyt

The encapsulation of polyphenolic phytocomplexes has been widely investigated because of their low stability during the digestion process. Tests performed with PhytoClew®, in a formulation with anthocyanin, underlined an improvement of anthocyanins bioaccessibility of about 40%. (Tests performed in collaboration with the chemical, toxicological and food analysis laboratory of University of Pavia).

PhytoClew® is an innovative solution for the improvement of bioaccessibility of phytocomplexes, obtained from the best combination of technological enhancement and sustainability.

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