**1. Introduction**

Functional foods are those with health benefits that must demonstrate that: a) they have a beneficial effect on one or more specific functions of the organism, beyond their usual nutritional effect, b) they improve the state of health and wellbeing, and c) they reduce disease risk [1]. Functional food must contain functional ingredients or bioactive compounds (BC), which are natural constituents that are generally found in small amounts in food. These compounds provide health benefits beyond the essential nutritional value of the product and for that reason, they are intensively studied to evaluate their effects on human health [2, 3].

BC include flavonoids, phytoestrogens, isoflavones, resveratrol, lycopene, organosulfur compounds, soluble dietary fibers, and isothiocyanates monoterpenes, plant sterols, olive oil. Among the BC of hydrophobic nature are carotenoids, tocopherols, flavonoids, polyphenols, and phytosterols stand out [3].

Regarding the importance of particular BC is the folic acid (FA) or vitamin B9, which belongs to the folates family and act as cofactors in carbon transfer reactions (formyl, hydroxymethyl, and methyl) in nucleotide biosynthesis (purine and pyrimidine bases), amino acid metabolism (methionine, histidine) and metabolism neurotransmitters (serine, choline) [4]. Animals and humans cannot synthesize folates. Thus, it is necessary to incorporate them into the diet from plant sources or food FA fortified [5].

The consumption of FA is critical since the deficiency of this vitamin is related to neural tube defects, coronary heart disease, and megaloblastic anemia, similar to that generated with the deficiency of vitamin B12, which occurs more frequently in pregnancy [5–7].

Scientific studies link low folate intake with neurocognitive dysfunctions. Folates play an essential role in developing the central nervous system and in the metabolism of some neurotransmitters; low folate concentrations may also be related to dementia and decreased cognitive function [6].

Various studies explain that FA is being used as a potential agent for preventing cancer and reducing the risk of heart disease [8, 9]. Epidemiological studies have shown that folate supplements can significantly reduce pancreatic cancer and breast cancer [10]. Besides, some studies link the intake of FA with a decrease in colon cancer and neurological diseases such as Alzheimer's [11]. The protective effect of FA on cardiovascular diseases, hematological and neurological diseases, and cancer has been associated with the antioxidant activity of this vitamin [12].

Another important BC is vitamin E (VE), also called α-tocopherol [13]. There are also foods such as eggs, seeds, nuts, and whole grains, which also contribute to VE's daily intake [5].

VE is the main fat-soluble antioxidant in the body. Its action is based on capturing peroxide radicals produced in cells by oxidative metabolism [7, 14]. Hence, it prevents the formation of hydroperoxides, delaying the initial phase of the oxidative process [13, 15]. This action protects cell membranes and other lipids from severe alterations produced by peroxidation [7, 14]. Various clinical studies also describe the beneficial effects of VE, alone or in combination with other vitamins, in some types of tumors such as prostate, gastric and lung. This fact is based on experimental studies that show the role of free radicals as a critical factor associated with the development of cancer. It is precisely the effectiveness of antioxidants from the diet such as tocopherols that have an essential role in the prevention of the development and progression of this disease [16].

Another group of BC are polyunsaturated fatty acids (PUFAs). There are two families of PUFAs: the n-6 family and the n-3 family. The PUFAs n-6 family is derived from linoleic acid, with two double bonds, and is characterized by having its first double bond at the carbon chain number 6, counting from the methyl at the end of the chain [17]. The n-3 PUFAs family derives from α-linolenic acid (with three double bonds), whose fatty acids have a first double bond at carbon number 3 in the chain. Besides being a source of energy, the PUFAs n-6 and n-3 families incorporate into cell membranes. They are precursors of eicosanoids like prostaglandins, prostacyclins, thromboxanes, and leukotrienes, that intervene in several physiological processes, like blood clotting or inflammatory and immune responses [17].

It is essential to highlight the contribution benefits of essential fatty acids. Man needs these fatty acids for normal functioning but must ingest them since body cannot produce them. That is why they are called essential [5]. Both linoleic and α-linolenic are essential fatty acids that cannot be synthesized by the body. Therefore, both fatty acids must be provided in the diet [17]. Research published has established that these fatty acids are very beneficial in the prevention of

*Why Produce Food-Bioactive Compounds to Generate Functional Grade Foods? DOI: http://dx.doi.org/10.5772/intechopen.96421*

cardiovascular diseases [18–20], schizophrenia [21], and cancer [22], among others. Also, they have vasodilator, antihypertensive, anti-inflammatory, and anti-atherothrombotic properties [23].

The problem is that many of these BC are not consumed in the necessary amounts for the body's normal functioning. According to the recent 2015–2020 Dietary Guidelines for Americans, VE and choline consumption is below the daily requirements, so it must be compensated with the intake of supplements in 50% of adults [24].

### **2. Developing formulations with bioactive compounds**

The BC amount that must be added to food and its nutritional values detected are useful in preventing certain diseases. Then there is a need to study and bring to the industry the incorporation of matrices/carriers that permit BC in food. Various types of matrices in the food industry, like liposomes, nanoemulsions, microemulsions, solid lipid nanoparticles, and polymeric micro and nanoparticles have been studied for BC encapsulation [25]. A whole new market has even been generated related to nano-foods associated to nanotechnological techniques or tools, or to which manufactured nanomaterials should be added, either during their starting point or production processing or packaging [26].

Liposomes are considered within an emerging trend in the market called nanofeeding. They offer a series of competitive advantages compared to other matrices. For example, their production on a larger scale is of good feasibility. They are easy to obtain and scale-up, which allows the incorporation of BC compounds in this type of matrices in food production lines. Besides, their characterization and physicochemical, microbiological, and sensorial stability can be studied with different techniques. Also, liposomes components are low cost, and natural food can be easily obtained [25, 27].

Liposomes are microscopic spherical vesicles, formed by lipids that enclose liquid compartments in their structure [28], allowing the encapsulation of molecules, whether they are liposoluble or water-soluble BC [29, 30].

The liposomes can be formulated with phospholipids, which are polar lipids characterized by having hydrophilic and lipophilic groups on the same molecule [30]. These spherical vesicles are formed under certain conditions. After flash evaporating solvent, phospholipids are hydrated and organized into lipid bilayers. These lipid bilayers called lamellae, unite to form the phospholipid sphere that encloses the water [31]. Liposomes can have one or multiple concentric lamellae called a vesicle or multilamellar liposome [30].

These types of matrices have broad applications in the industry to transport BC or other types of compounds. For example, in accelerating cheese ripening, the vesicles offer a uniform distribution of hydrophilic enzymes [30]. In the encapsulation of flavorings, acidulants (citric acid, ascorbic acid, buffer, and alkalis), antioxidants, colorants, essential oils, vitamins, and minerals. Furthermore, these systems are used to encapsulate lactoferrin, a bacteriostatic glycoprotein, and nisin z, an antimicrobial polypeptide, to increase dairy products' shelf life. Liposomal systems are also used to trap Phosvitin (antioxidant), which inhibits lipid oxidation in various dairy products and ground pork. Besides, they are used to capture antioxidants like vitamin C, maintaining 50% of activity after 50 days in refrigerated storage and non-encapsulated vitamin C, which loses its activity after 19 days [32].

In this way, liposomes can encapsulate all kinds of BC. Besides, specific BC can be part of the carrier itself if a strategy is applied in these matrices' design.

To implement liposomes with BC in the food industry, the research and development of these matrices should be deepened to ensure the compounds' stability to be encapsulated and incorporated into food. It is essential to mention that for an industrial application, membrane stability and structure are important factors when designing liposomes [30], and must always ensure that they are food grade [33].

In our research line, we sought to incorporate saturated fatty acids that can act as membrane stabilizers and intervene against lipid oxidation processes [33]. Based on the research carried out by Hsieh and collaborators [28], stearic acid (SA) is an excellent alternative to prepare stable liposomes, structural benefits, and increase the efficiency of liposomal encapsulation, as well as oxidative stability. SA is an 18-carbon saturated fatty acid, insoluble in water, so it is located between the hydrophobic chains of the fatty acids in the bilayer. The authors carried out studies on liposomes formed with egg phosphatidylcholine (EPC) and SA in the molar ratio of 1: 0.25. The problem was that EPC has a much higher cost than soy phosphatidylcholine (SPC) in Argentina since EPC is a specific raw material obtained in the bench lab at a laboratory scale.

In comparison, SPC is a lipid product of the country's intense soy farming activity. It is a raw material that is easily obtained at a large scale and has a low cost. Therefore, SPC with SA was used in the molar ratio 1:0.25. A second strategy is developing liposomal formulations using the SPC base system and incorporating calcium stearate (CaS), with the double benefit that CaS can act as a possible stabilizer of the liposomal bilayer. CaS also incorporates a mineral such as calcium that increases the nutritional value [27, 29, 33, 34]. CaS is a salt composed of two 18-carbon saturated fatty acids linked to a calcium cation. The concentration used was the same as in SPC and SA. In this way, the possible effects of the stability provided to the liposomal bilayer incorporating saturated fatty acids are preserved, and at the same time, extra calcium is added to the formulation.

The formulations proposed to obtain liposomes that encapsulate and protect BC were the following [27, 29, 33, 34]:


Besides, SPC is a natural lipid that generate the liposome's transporter, has essential fatty acids such as linoleic acid (omega-3) and linolenic acid (omega-6).

**Table 1** shows the percentage composition of fatty acids in SPC and EPC considered essential fatty acids. Furthermore, SPC is also the source of choline, an essential nutrient needed to synthesize neurotransmitters (acetylcholine). It plays an essential role in the fetus's brain and memory development, and some researchers have indicated that choline and methionine intake may be necessary for reducing the risk of neural tube defects [35].

Multilamellar liposomes were prepared by the dehydration–rehydration method [36]. Briefly, 40 μmol of lipids were dissolved in 500 μL ethanol in a round bottom flask, and the solvent was dried in a rotary evaporator at 37 °C. Dry lipid film composed of SPC, SPC:SA (1:0.25, mol ratio), or SPC:CaS (1:0.25, mol ratio) was rehydrated with 2 mL distilled water to a final 50 mM lipid concentration.

To prepare liposomes with VE, a stock solution of this vitamin diluted in ethanol was prepared. Stock concentration was 22.4 mM. Then, 0.445 mL of this stock was mixed with a proper amount of lipids. The solvent was evaporated until the lipid film was obtained. Any liposoluble BC to be incorporated into the liposome


*Why Produce Food-Bioactive Compounds to Generate Functional Grade Foods? DOI: http://dx.doi.org/10.5772/intechopen.96421*

#### **Table 1.**

*Composition of fatty acids in SPC and EPC (\*essential fatty acids).*

must always be done in this step. Moreover, it is essential to use only ethanol to dissolve lipids since it is a solvent that is approved as an additive at a national and international level of the Food Committee with concentrations (possible trace) that do not exceed the maximum permitted [37–39]. When the film was rehydrated in 2 mL of distilled water, a final concentration of 5 mM was reached, and this is the step to incorporate the hydrosoluble BC's. In the case of FA, recently prepared solutions of this vitamin needed when the experiment is on the rehydration step. FA was weighed and diluted with distilled water to reach a 0.136 mM concentration.

Samples were prepared with the primary goal of fortifying food with the mentioned vitamins. According to Argentina regulations [40], the percentage of recommended daily intake (RDI) in a portion of fortified food must be between 20% and 50% for fat-soluble vitamins and between 20% and 100% for hydro-soluble vitamins. The RDI of VE is 10 mg and for FA is 400 μg. In order to fortify aqueous food like chocolate milk, regular milk, or juice, 2 mL of liposome suspension (50 mM) with vitamins was added to each serving of food (200 mL), which implies that it was fortified with 4.3 mg of VE (5 mM) equal to 43% of the RDI and 120 μg of FA (0.136 mM) equivalent to 30% of the RDI. Thus, 1 L of aqueous food will contain, for example, liposomes of SPC:CaS and BC in the proportion seen in **Table 2**.


#### **Table 2.**

*Composition of SP:CaS liposomal formulation as carries of BC: VE, FA, choline and essential fatty acids in 1 L of product for example chocolate milk [27, 29, 33, 34].*

The design, research, and development of carriers for BC, including all aspects, like the final quality of the food product and its feasibility, have to be considered until reaching the consumer's market. Thus, the liposomal formulation must be stable from a physicochemical, microbiological, and sensory perspective. The interaction of these matrices with BC must ensure their stability and protection until consumption. This data is not minor given that in the food industry, a series of treatments, usually thermal, must be applied to ensure the useful shelf life and safety of the product.

This fact presents a challenge in the development and research of foods with BC because many of these degrade or lose activity before reaching the industry's scale up regular treatments. VE is heat stable but oxidizes quickly in the air, with consequent loss of vitamin activity, especially in the presence of ferric ions and other metals [13]. Furthermore, VE is destroyed by exposure to UV light and to a great extent, during oil refining process [7]. Also, concerning food storage, during the storage of plant foods, VE has a weak antioxidant character, being much more active against animal fats, especially in the presence of synergistic substances [13]. On the other hand, FA is stable to alkalis under anaerobic conditions. However, under aerobic alkaline conditions, its hydrolysis occurs, separating the side chain and yielding glutamic acid, and pterin-6-carboxylic acid. Acid hydrolysis under aerobic conditions yields 6-methylpterin [41]. For this reason, to favor the stability of the FA, it must remain at a pH close to neutrality [42]. None of the degradation compounds mentioned shows biological activity; therefore, during the formulation of pharmaceuticals, nutraceuticals, or foods enriched with FA, it is necessary to protect this vitamin against environmental factors such as extreme light and pH [7, 43]. Specifically, in acidic media, it was shown that FA is unstable [44]. Furthermore, FA solutions decompose when exposed to light, forming glutamic acid, and pterin-6-carboxylic acid [41].

The milk pasteurization by the high temperature and short time method (2–3 seconds, 92 ºC) causes a loss of around 12% of total folates, and the loss caused by boiling the milk for 2–3 minutes is in the order of 17%. Sterilization of milk in bottles (13–15 minutes at 119–120 ºC) is the treatment that causes the most significant losses, about 39% [41].

Based on those aspects mentioned above of unfavorable conditions, if the objective is to add BC such as FA and VE, the liposomes must have oxidative stability not to affect the vitamin antioxidant activity, and they must protect the FA from the applied food heat treatments. Also, the liposomes can be food-incorporated at a neutral pH due to FA's stability.

The application of advanced liposomal formulation has shown that they can be applied in pH foods such as chocolate milk and orange juice. They demonstrate that liposomes can protect thermolabile vitamins such as vitamin C and FA [29, 33]). Other authors demonstrated that the capture of antioxidants like vitamin C in liposomes maintains 50% of activity after 50 days in refrigerated storage, and non-encapsulated vitamin C loses its activity after 19 days [32].

It should also be mentioned that the liposomal formulations of our research line presented values of oxidative stability under quality food parameters [45]. According to what is established by the authors [45], for a food to have a good quality, it must have an oxidative value below 0.2 mg of malondialdehyde (MDA) per Kg of food. In SPC, SPC:SA and SPC:CaS with VE-FA after pasteurization thiobarbituric acid reactive species (TBARS) value were 0.2380 ± 0.0248 μM, 0.2017 ± 0.0645 μM, 0.1816 ± 0.0581 μM, respectively. The results are shown as the mean ± SD of three independent assays; as published in Marsanasco and collaborators [29]. Taking as a reference the average value of SPC of 0.2380 μM that was the highest of the three formulations, if the transition from μM of TBAR to mg of

MDA/Kg of food is performed, it gives a value of 0.0166 mg per 1 Kg of chocolate milk (density was 1,033 Kg/L). Thus, is below 0.2 mg of MDA/Kg established by the authors, complying with the excellent quality parameter.
