**3. Incorporating bioactive compounds into different types of food**

Liposomes have the particularity that they require an aqueous medium for their stability [30]. Based on the above, it is essential to consider that the choice of food to incorporate the liposomes with the BC must have a high water-activity (aw) to maintain the liposomal formulations' stability. After considering this factor, the second thing is how the functional food to be produced will be positioned in the market. Alternatively, may be the product production has a social focus related to the country's nutritional deficiencies.

Our line research, based on the social requirements of our country and the market niche that we want to cover, the incorporation of liposomal formulations with BC in the following types of food was proposed:


Thus, chocolate milk fortified with VE and FA was implemented with essential omega-3 and omega-6 FAs and choline (from the SPC-based liposome). Besides, when using the liposomal formulation SPC:CaS, calcium is added to the product. This type of food will provide energy and nutritional contribution to the consumer.

This research was complemented by studying the same liposomal formulations but with 5 mM of VE and 50 mM of vitamin C (VC).

In all cases, the focus of incorporating vitamins was towards the food fortification with the incorporation of these vitamins that are considered as BC.

The objective will be to offer a massive product such as milk, with an essential nutritional addition, in response to a growing demand for products with high nutritional value and nutritional deficiencies in the country's specific sectors.

Another type of food prepared with the addition of liposomal formulations-BC was orange juice. In this case, FA could not be incorporated; as explained in the previous section, considering it is not stable at extreme pHs. In this case, liposomal formulations were used with VE and VC in the concentrations already mentioned.

The liposomes in the food were incorporated in the volumetric ratio 1/100. Furthermore, they did not modify the base food's density, pH, or visual characteristics [27, 29, 33].

Once the essential foods have been established, the first phase of the study in the research and development of these functional foods is the characterization and physicochemical stability on a laboratory scale of the liposomal formulations with BC. At this stage, the following parameters should be studied: size, shape, electrical charge, encapsulation efficiency, oxidative stability, packing and lipid membrane stability, and rheological behavior of liposomal formulations with and without the addition of vitamins. Also, the heat treatment of the selected food must be considered, in our research line LTLT process was selected. LTLT is low temperature and longtime pasteurization process, that is 65 ºC for 30 minutes applied to milk and juices.

There is a question of whether all liposomal studies with BC can successfully fortify the base food. Food has components that can interfere with analytical

determinations. It is for this reason that in this study stage, food simulants were used. Milk and flavored milk are found within foods with a pH > 5. Therefore, the food simulant distilled water was used instead of milk, while fruit juices are within pH <5, so the food simulant of 3% m/v acetic acid was employed [46].

Among the studies to be carried out at this stage, transmission electron microscopy is one of the most suitable methodologies to obtain information on the morphology of liposomes [47]. This technique must be complemented with optical microscopies that allow analyzing the shape and distribution of the liposomes, respectively. In the formulations developed, explicitly mentioning those with VE-FA, the liposomes presented a structure in which an outermost zone and a central nucleus were differentiated (**Figure 1**). They also presented a variety of sizes with spherical shapes and non-spherical related to the method of preparation and composition of liposomes. Moreover, isolated and aggregated liposomes were also observed [29, 33]. Data discussed coincide with those obtained by other authors [48], where the unilamellar liposomes of egg Lecithin at pH 7.2 showed liposomal aggregation. Nacka and collaborators [49] also demonstrated liposome aggregation of mainly phosphatidylcholine and phosphatidylethanolamine at various pH. In liposomal systems, aggregation is a physicochemical mechanism that can occur under certain conditions influenced by pH, heat treatment, external load, and cations' presence, among others [29, 33, 49].

It is important to mention the surface and viscosity behavior study of the matrices that will encapsulate the BC. The matrix that encapsulates the BC has a behavior like that of the product. It represents a great advantage to apply to the food industry, if visualizing an industrial-scale production. The size and shape of the liposomes with the BC are related to the final product's stability. But there is also another aspect that is related to the composition, shape, morphology distribution, tendency of aggregation, and membrane packing of the liposomal formulation with BC, and that is the rheological behavior and viscosity that they will contribute to the food product [29, 34]. Newtonian behavior occurs in almost all common liquids such as water, milk, orange juice, apple juice, and corn syrup. Furthermore, the pseudoplastic behavior is also present in common foods such as sauces and orange juice concentrate [50, 51].

For example, liposomal formulations SPC, SPC:SA, all with VE-FA and pasteurized, presented a behavior similar to that of a Newtonian fluid. While the SPC:CaS formulation with VE-FA pasteurized presented a behavior with a tendency towards a pseudoplastic fluid. This result is related to various factors as the membrane packing with the viscosity of formulation and rheology. According to the results obtained by other authors [52], a greater rigidity of the membrane of the phosphatidylcholine L-αdipalmitoyl liposomes increased in viscosity. In the system with CaS, FA's addition would favor the association of two adjacent phosphatidylcholines with the calcium cation. It must be considered that FA (pka1 = 2.3) was shown to decrease the pH of distilled water from 6.0 to 3.88 by releasing the protons to the medium. Moreover, the

**Figure 1.**

*Light micrographs of the liposomal system with 5 mM of VE and 0.136 mM of FA in distilled after pasteurization: a) SPC; b) SPC:SA, c) SPC:CaS.*

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

low pH favors the dissociation of calcium from CaS, which generates bonds between calcium cations and two adjacent phosphatidylcholines [53, 54]. This greater stiffness of the membrane would also explain the pseudoplastic behavior of the SPC: CaS system [29]. Newtonian or pseudoplastic behavior in liposomal formulations with BC represents an advantage if, in the future, it is desired to apply liposomes on an industrial scale, especially the foods to be fortified, such as milk and orange juice.

Other studies carried out are the membrane packaging and its behavior, for this type of study; techniques are implemented that allow detecting the hydrophobicity factor (FH). This factor determines the degree of hydrophobic sites exposed at interface level and the possibility that the probe used, which is merocyanine MC540, is inserted into the bilayer. For this reason, the higher the value of this parameter, the higher the number of membrane surface defects [29].

In liposomes obtained, specifically those with VE and FA, considering that MC540 is slightly located above the domain of the main glycerol chain of phospholipids [55]. It can be inferred that the addition of vitamins favored membrane fluidity (specifically in the phospholipid polar zone) with the consequent greater probe entry and a greater number of defects in a said membrane appearance (**Table 3**).

Regarding VE, the result would be related to the effect of this vitamin that produces a general increase in the mobility of the headgroup in the lipid bilayer [56], which would favor the entry of the probe MC540 [57]. In the case of the SPC:SA (1: 0.25 molar ratio) or SPC: CaS (1: 0.25 molar ratio) system, the polar part of the fatty acids alternates with 21% SA or CaS, respectively. As the amount of saturated fatty acids increases, it is expected a membrane stiffness increment and fewer probes entering. But considering the membrane, the MC540 location; the addition of the SA or CaS could be favoring their income. In the case of SPC:SA, a possible explanation would be related to the fact that the polar heads of phospholipids such as phosphatidylcholine bind to water molecules through hydrogen bonds, and the polar heads of fatty acids such as SA also interacting with water molecules [58]. Although SA is an anion (as occurs in the pH of distilled water), it can also affect the hydrogen bonds with water [59]. The greater the hydration and formation of hydrogen bonds in the polar heads, the greater the water's penetration, which promotes the formation of more fluid domains [60]. In the case of the SPC:CaS system, the presence of FA (pka1 = 2.3) and the pH descent in distilled water with the consequence of the release of calcium ions from CaS and the association with close next phosphatidylcholines, increase the surface fluidity of the membrane and favors the entry of the probe [29].


*Values of the membrane packing of SPC, SPC:SA and SPC;CaS with 5 mM of VE and 0.136 mM of FA after pasteurization in distilled water. Results are shown as the mean ± SD of three independent tests for MC540 hydrophobicity factor (HF). Statistical comparison was made in each system with vitamin / s compared to the same system without vitamins (control), by means of the Dunnett's Test. The significant differences with respect to the control are shown as \* p <0.5; \*\* p <0.01; \*\*\* p <0.001 [29].*

#### **Table 3.**

*Values of the membrane packing of SPC, SPC:SA and SPC;CaS with 5 mM of VE and 0.136 mM of FA after pasteurization in distilled water.*

In this case, the incorporation of SA or CaS increased the lipid membrane's fluidity and defects, but this does not imply ruling out the developed carrier. Because it can generate this effect but have important oxidative stability, protection, and encapsulation of BC, that is why all the characterization and stability studies must be carried out to have a precise knowledge of how the carrier interacts with the BC and, based on that, define which ones will continue in the second study stage.

Once the studies of physicochemical stability, encapsulation efficiency, and characterization of the BC liposomal formulations have been carried out, start the second study stage, the final product application. For that type of study, two points of great importance must be evaluated: the final functional food's microbiological and sensory stability. The purpose of microbiological tests is to analyze whether the composition, preparation, and incorporation of matrices with BC in food contributes to microbial load. It is crucial to ensure that certain factors such as manufacturing techniques, acceptable manufacturing practices and especially heat treatment contribute to the fact that liposomal formulation with BC does not provide bacterial load and is safe.

The other aspect of studying is the sensory evaluation of the product with the carrier and BC. Sensory evaluation is needed because changes in food can be physical or chemical and can affect the product's appearance, texture, taste, smell, aroma, taste, and safety.

In these studies, the product's heat treatment and shelf life must be considered, and in these cases, they must be carried out on the base food and not on a food simulant as applied in the previous stage of characterization.

The sensory analysis allows knowing the organoleptic properties of food because it is done through the senses. Sensory evaluation is innate in man since from the moment a product is tried, a judgment is made about it, whether it likes or dislikes, and describes and recognizes its characteristics of taste, smell, and texture. Discriminatory tests should be used when it is necessary to determine if two samples are significantly different. Within this type of test is the triangular one that consists of presenting to the evaluator three suitably coded samples, of which two are the same, and the third is different [61]. This test was used in our research line, considering that there were three liposomal formulations used with the vitamins: SPC, SPC:SA, and SPC:CaS. For that reason three triangles were used for each combination of vitamins, for example VE and FA. Each rater was presented with three coded samples in each triangular trial, two of which were identical, and one was different, all numbered with random three-digit numbers for identification. The same samples could be the product (e.g., chocolate milk) with or without liposomes-BC, and the difference could be the product (chocolate milk) without or with liposomes-BC, respectively [29, 34]). The results obtained concluded that the addition of the liposomal formulations with the BC produced significant sensory changes in some products, such as chocolate milk. For this reason, it was proposed to carry out global acceptability tests to analyze whether the differences found were positive or negative in the base food.

The global acceptability test is within the affective tests, which are those in which the evaluator expresses his subjective reaction to the product, indicating whether he likes or dislikes it, whether he accepts or rejects it, or whether he prefers it to another. The primary purpose of effective methods is to evaluate the response (reaction, preference, or acceptance) of a product's actual or potential consumers. Hedonic scales are used to carry out these tests. The word hedonic comes from Greek and means pleasure. In this sense, hedonic scales are instruments for measuring the pleasant or unpleasant sensations produced by a portion of food to those who taste it. Hedonic scales can be verbal and graphic, and the choice of the type of scale depends on the age of the evaluators and the number of samples to be

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

evaluated. These scales are those that present a verbal description of the sensation that the sample produces. They must always contain an odd number of points, and the central point, "I neither like nor dislike," must always be included [61, 62].

Objectivity is achieved in the evaluators' responses to the sensations caused by a food product using the hedonic scales [61]. The evaluators were summoned, and two samples were presented. One was the chocolate milk without liposomes-BC, and the other was the chocolate milk with the liposomes and BC. In this case, they did not know what they were evaluating. **Table 4** shows the global acceptability test results where the SPC and SPC:CaS liposomes in the chocolate milk do not modify its acceptability concerning the raw food. Only the SPC:SA formulation in chocolate milk generates a decrease in its acceptability, coinciding with the fact that it had shown significant differences in the triangular test.

This global acceptability test can be performed using the pairs of product samples with or without BC-liposomes but informing the evaluator who the functional food is and the base food—obtaining very positive results that demonstrate the excellent acceptability of potential consumers regarding wanting to consume a food with different nutritional properties [29].

Thus, the implementation of sensory tests that are already effective to evaluate matrices with BC in food allow inferring the impression that a consumer would have of this functional food, and the achievement is that it is positive, which implies a significant advantage at the moment for producing BC-fortified food on an industrial scale. Because if we consider the importance and above all the cost that would imply launching a product for sale, this task cannot be carried out without being sure that the incorporation of BC matrices does not modify the base foods' sensory aspect.
