Probiotics and Other Bioactive Compounds with Proven Effect against Obesity and Hypertension: Food Design Opportunities from Lulo Fruit ( Solanum quitoense )

This book chapter aims to identify those bioactive compounds that are the most effective in obesity and hypertension prevention and/or treatment, these being the two main disorders associated with metabolic syndrome. Focusing on probiotics and phytochemicals, the document will provide evidences from both in vitro and in vivo studies as well as information about the action mechanisms and how they are affected by the interaction with other food ingredients, the food matrix in which they are placed, etc. Given its high antioxidant capacity, in part due to its spermidine content, lulo fruit has generated considerable interest among health researchers. This, together with its exotic organoleptic properties, offers interesting growth opportunities for the design of new food products from lulo fruit. This book chapter will also discuss some of them.


Foods, technology, and metabolic syndrome
Overweight and obesity are defined as "abnormal or excessive fat accumulation that may impair health" [1]. Since 1975, obesity has almost tripled worldwide so that in 2016, 39% of the adult population and 18% of children and adolescents were overweight. Very often, a high body mass index (BMI ≥ 25-30) is associated with other metabolic abnormalities, such as high blood pressure (hypertension), high blood sugar (hyperglycemia), high serum triglycerides, and low serum high-density lipoprotein (HDL) [2]. The occurrence of at least three of these interconnected physiological, biochemical, clinical, and metabolic factors that directly increase the risk of atherosclerotic cardiovascular disease, cancer, and type 2 diabetes is known as metabolic syndrome [3]. The International Diabetes Federation estimates that one-quarter of the world's adult population suffers from this syndrome, with little

Food components against metabolic syndrome
Main food components considered in the literature as having potential ameliorating effect on any disorder associated with metabolic syndrome may be included in one of the following groups: • Fiber, both soluble (e.g., pectins, beta-glucans, naturally occurring gums, inulin, psyllium) and insoluble (e.g., cellulose, hemicellulose, lignin), is reported to have laxative properties and to mitigate both hypercholesterolemia and hyperglycemia [15]. When fermented by probiotic bacteria, prebiotic fiber (fiber that resists digestion in the stomach and the small intestine and reaches the colon intact) breaks down into short-chain fatty acids (butyrate, acetate, and propionate), which are reported to enhance glucose and fat metabolism [16].
• Monounsaturated fats, polyunsaturated fats (both omega-3 and omega-6), plant sterols, and essential fatty acids instead of saturated fats and trans-fatty acids are proved to be effective in decreasing total cholesterol and increasing the blood level of high-density lipoproteins (HDLs) [17].
• Vitamin E and C consumption is associated to a reduction of vascular risk by decreasing oxidative stress (lipid peroxidation) and proinflammatory cytokines [17]. Improved vitamin C status is hypothesized to alleviate endotoxemia and its consequent proinflammatory responses that are suggested to initiate insulin resistance and related metabolic disorders; on the contrary, inadequate vitamin C status contributes to small intestinal bacterial overgrowth, transcytosis of enteric bacteria, and an elevation of circulating lipopolysaccharide, which elicits a low-grade inflammatory response [18]. As for vitamin D, it reduces the intestinal absorption of fat by increasing that of calcium [17].
• Bioactive peptides, having a size range of 2-50 amino acids, have potential to regulate blood pressure and glycemia, reduce cholesterol level and body mass, and scavenge free radicals [19]. Lactotripeptides isoleucine-proline-proline and valine-proline-proline, whose concentrations increase during the ripening process of cheese, are particularly considered as strong antihypertensive agents [20]. In the case of obesity, foods that contain bioactive peptides provide a satiating effect and lead to appetite suppression.
• Minerals' (selenium, magnesium, and zinc) ability to decline metabolic syndrome is related with their antioxidant properties and their participation in insulin synthesis and regulation [20]. Moreover, calcium intake (1200 mg/ day) has been demonstrated to increase fat mass loss in overweight and obese adults [21]. Mechanisms to explain this effect include that during low calcium intake, more calcium enters adipose tissues cells and subsequently stimulates the expression of lipogenic genes in parallel with suppressing lipolysis [22]. Also, calcium increases fecal fat loss by binding to fat in the lumen and forming non-absorbed complexes [23]. This ability of calcium to decrease lipogenesis may be enhanced due to a synergistic effect with other components in dairy products (vitamin D and angiotensin-converting enzyme inhibitors) [21].
• Essential amino acids, mainly histidine and glycine, are associated with a decrease in insulin resistance and blood pressure, respectively, although conclusive evidence is lacking and additional studies are needed [17].
• Probiotics are microorganisms that improve the availability and digestibility of nutrients while maintaining the balance of intestinal microflora in the gut [20]. Mainly belonging to the Lactobacillus and Bifidobacterium genera, probiotics emerge as prospective biotherapies in the management of metabolic disorders including obesity and diabetes by counteracting the adverse effects of a highfat diet [29].

Phytochemicals
Phytochemicals are naturally occurring plant chemicals that, beyond providing plants with color, odor, and flavor, can influence chemical processes within human bodies in a beneficial way. Main phytochemicals under research include carotenoids (β-carotene, lycopene, lutein, zeaxanthin) and polyphenols, which include phenolic acids, flavonoids, and stilbenes/lignans [30]. Flavonoids can be further divided into groups based on their similar chemical structure, such as anthocyanins, flavones, flavanones, isoflavones, flavonols, and flavanols. Flavanols further are classified as catechins, epicatechins, and proanthocyanidins.
Phytochemical compounds have gained popularity in recent years due to their broadly documented effect in cancer prevention among other biological effects, such as the prevention and treatment of obesity, cholesterol, and diabetes [31]. Mechanisms employed by phytochemicals in ameliorating metabolic syndrome risk factors are diverse and dependent on their particular chemical structure. Whereas catechins mainly induce fat oxidation and improve endothelial function, cyanidins and theaflavins inhibit enzymes involved in the synthesis of fatty acids and triglycerides. Other phytochemicals, such as gallic acid, quercetin, and capsaicin, reduce preadipocyte proliferation by induction of cell apoptosis, while low-molecular proanthocyanidins have the ability to inhibit the activity of specific angiotensinconverting enzyme. Having a similar chemical structure, isoflavones can also influence the activity of human estrogens.
For some bioactive components, several in vitro and in vivo (either animals or humans) studies have been performed; however, as evidenced in Tables 1-3, results differ among them. While in vitro or animal studies usually yield positive results, clinical human studies are still inconclusive. The lack of standardization or aspects related to the dose or duration of supplementation may be the cause of these results. Moreover, studies both in vitro and with animals (Tables 1 and 2) have been carried out with synthetic components (only spermidine was obtained directly from lulo fruit), while in vivo studies with humans have been carried out mainly with extracts including the bioactive compounds ( Prevented obesity by decreasing body weight gain, visceral fat-pad weights, and plasma lipid levels; also inhibited visceral adipogenesis and attenuated the production of pro-inflammatory cytokines in visceral adipose tissues [41] Oryzanol and ferulic acid Mice High-fat diet supplemented with 0.5% (w/w) oryzanol or 0.5% (w/w) ferulic acid for 7 weeks Decreased in body weight, improved blood glucose metabolism, may be beneficial for the treatment of diabetic hyperglycemia [42] Probiotics  Table 2.

Fruits, vegetables (or extracts), and phytochemicals endorsed by recent in vivo (preclinical trials) studies.
In vitro studies achieve 50% of cell population growth inhibition with active compound amount of ppm, and, in some cases (some flavonoids, ajoene, catechins), a dose-time-dependent effect is detected. These dose-time-dependent effects, together with the multivariate effects observed in most cases, are largely due to the antioxidant properties of the phytochemicals considered. Flavonoids tested on cell population growth were well correlated with their antioxidant activity [36].
The doses used in the preclinical studies were much higher than those used in the clinical trials, and the limitations in the ingested doses may be the cause of the ineffective results. Kobori et al. [24] concluded that in vitro anti-aggregatory effects of flavonoids are caused by concentrations that cannot be attained in vivo by dietary consumption.
In most preclinical studies, imbalances are induced in mice with a high fructose diet, and the effect that the active component has on these induced disorders is determined. Also in the clinical studies performed with positive results, the component of interest was provided as part of a low-fat diet to patients with physiological alterations associated with metabolic syndrome. In these conditions the results show a palliative effect but in no case a preventive effect. Thus, quercetin supplementation did not affect the antioxidant status under healthy, normal conditions [24].
Sometimes solubility and bioavailability are a major limitation factor. As an example, pentacyclic triterpenes such as ursolic acid are poorly bioavailable because of poor aqueous solubility and permeability through biological membranes [26]. These limit their biological effects. Some strategies have increased bioavailability. For example, on comparing the oral bioavailability of ursolic acid microcrystals and nanocrystals to its coarse suspension in rats, ursolic acid microcrystals and nanocrystals exhibited 1.40-and 2.56-fold enhancement, respectively. Also, a new  product from polyphenols of lychee has been developed. Oligonol, a unique lowmolecular-weight polyphenol, was developed to enhance absorption of polyphenols from the intestines. It contains 15.7% polyphenol monomer ((+)-catechin, (−)-epicatechin, etc.) and 13.3% polyphenol dimer (procyanidin B2, etc.), while lychee fruit-derived polyphenol contains 6.4% polyphenol monomer and 9.9% polyphenol dimer [46]. Concluding from above, translational studies from animal observations to human clinical trials and ultimately community interventions are needed to further confirm the effects of phytochemicals and foods rich in these bioactive compounds. The technological development should be aimed at the implementation of strategies that increase the bioavailability of active components with proven activity and that allow to provide the adequate doses avoiding toxicity problems.

Probiotics
Probiotics are defined as live microorganisms which, when administered in adequate amounts, confer a health benefit on the host [50]. Evidence from the latest studies in which probiotic efficiency in metabolic disorder management is assessed by both in vitro and in animal or human subjects is compiled in Tables 4 and 5.
Regarding the strains employed in the management of several inflammatory diseases, they usually belong to the Lactobacillus and Bifidobacterium genera, although Pediococcus pentosaceus LP28, Bacteroides uniformis CECT 7771, and Akkermansia muciniphila have also been proved to have anti-obesity effects [65]. In general, doses NTU 101-fermented tea had a more significant effect on the reduction of body weight gain and body fat content than the unfermented tea greater than 10 8 CFU/day were orally administered to the drinking water or by oral gavage of the lyophilized bacterial powder in water (in animal studies) as well as in the form of capsules or fermented milk products (in human studies). Probiotic supplementation in rat and mouse studies usually applies to both diet-induced obese individuals and lean individuals fed on a high-fat diet, thus showing the effect of probiotics in both the treatment and the prevention of several metabolic abnormalities. However, human studies are basically applied to overweight or obese healthy individuals, submitted or not to energy restriction and/or regular exercise.
As regards in vitro studies, treatment of 3T3-L1 pre-adipocytes with test substances during their differentiation stage is the most common technique.   Among the analytical determinations, the most representative are changes in the body weight and the body fat content as well as main serum biochemical parameters (glucose, insulin, leptin, lipids, lipoproteins, and inflammatory indicators). Postmortem determinations, such as the liver weight or the adipose tissue histology, are also common in animal studies. Finally, since probiotics are known to increase the bacterial diversity of intestinal microflora, in vivo studies usually include viable counts in fecal samples and evaluation of intestinal survival. In fact, a lot of recent research relates gut microbiota composition with almost every chronic disease: from gastrointestinal diseases to obesity, diabetes, cancer, and even neurological and neurodegenerative disorders such as depression, autism, anxiety, and Parkinson's disease [66]. Not only does a certain microbiota predispose to suffer certain diseases, but also the incidence of a certain disorder modifies the gut microbiota of an individual. In overweight/obese subjects, Bacteroides, Parabacteroides, Ruminococcus, Campylobacter, Dialister, Porphyromonas, Staphylococcus, and Anaerostipes are the dominant genera linked to a low diversity of species, while Faecalibacterium, Bifidobacterium, Lactobacillus, Butyrivibrio, Alistipes, Akkermansia, Coprococcus, and Methanobrevibacter are predominant in lean individuals with a high bacterial diversity [67]. Apparently, the intestinal microflora of obese subjects is more efficient at extracting energy from a given diet than that of lean individuals, thus leading to increased energy storage and adiposity [65]. Moreover, beneficial intestinal microflora is known to produce short-chain fatty acids (e.g., acetate, butyrate, and propionate) from indigestible polysaccharides, which may act as energy substrates as well as regulators of satiety and food intake. Last but not least, Lactobacilli and Bifidobacteria are known to synthesize bioactive isomers of conjugated linoleic acid with antidiabetic, anti-atherosclerotic, immunomodulatory, and anti-obesity properties [68]. In other words, low bacterial diversity in obese individuals is associated with a reduction in butyrate-producing bacteria, a reduction in hydrogen and methane production, an increase in mucus degradation, and an increase in the potential to manage oxidative stress. Since intestinal microflora composition is strongly affected by dietary patterns, studies evaluating the effect of certain food components on the growth of bacteria with beneficial effect on metabolic syndrome and obesity, particularly Akkermansia muciniphila and Faecalibacterium prausnitzii, are of great interest. Increasing the intestinal population of these two species has become a real opportunity to decrease alterations associated with obesity and metabolic disorders [69]. In addition to this, high-fat diet treatment has been proven to induce metabolic changes that impair gut barrier function in rats [55].
Together with increasing gut microbiota diversity, the production by fermentative action of those bioactive molecules involved in the metabolic pathways that trigger the metabolic syndrome has also taken a lot of interest in the last years. In particular, many studies focus on the phenolic compound bioconversion by food fermentation into other components with greater beneficial effect on the abnormalities associated with metabolic syndrome. As an example, Wang et al. [53] proved that the use of Lactobacillus paracasei subsp. paracasei strain NTU 101 in the fermentation of green tea and Houttuynia cordata leaves increased the levels of epigallocatechin gallate (EGCG), epigallocatechin (EGC), and chlorogenic acid, which enhanced the probiotic effect on body fat reduction. These results show the synergistic or complementary effect between the two bioactive compounds: the probiotic strain increases gut microbiota diversity and enhances intestinal absorption, while the EGCG acid promotes the lipolysis process. Zarrati et al. [70] also reported a synergistic effect between a weight loss diet and probiotic yogurt in overweight and obese individuals.
It should be noted that in order to exert their health benefits, probiotics do not necessarily have to be alive. In fact, heat-killed Lactobacillus reuteri GMNL 263 was as effective as live Lactobacillus reuteri GMNL 263 in attenuating obesity-induced metabolic abnormalities in high-fat diet-induced rats by reducing insulin resistance and hepatic steatosis formation [55]. Also both heat-killed Lactobacillus plantarum strain Ln4 and freeze-dried cultured MRS broth significantly reduced lipid accumulation and stimulated glucose uptake in 3T3-L1 adipocytes [51]. Finally, Plovier et al. [56] found that pasteurization enhanced the capacity of Akkermansia muciniphila to reduce fat mass development, insulin resistance, and dyslipidemia in mice. It seems that a specific protein isolated from the outer membrane of Akkermansia muciniphila is stable at temperatures used for pasteurization and improves the gut barrier, thus being the main responsible factor of the beneficial effect of the bacteria on health.
Of all the studies analyzed, it is concluded that many microorganisms have the potential for development as therapeutic probiotics for obesity and associated disorders. However, due to the strain specificity of probiotic microbes in exerting their beneficial effects, bacterial strains of the same species have different effects on adiposity and insulin sensitivity.

Case study: functional food development from lulo fruit with potential effect against metabolic syndrome
The lulo fruit (Solanum quitoense Lam.), also known as "naranjilla," is an important native Andean crop. Grown and consumed mainly in Colombia, Ecuador, and Central America, the plant produces a spherical, 3-8-cm-diameter fruit with orange skin (epicarp) covered by short, stiff, and thorny hairs or spines. The internal structure of the fruit is similar to that of the tomato fruit: a very juicy, acidic, and translucent yellow-green pulp (mesocarp and endocarp) that is located in four compartments separated by membranous partitions [71]. In Colombia, lulo is an economically important crop which, in 2015, was grown in a total area of 10,623 ha, with a total yield of 82,354 tons and an average yield of 9.6 tons/ha [72]. Although the principal market of this crop is in the producing countries themselves, it has gained interest in recent years in national and international markets due to its organoleptic properties and its nutritional value. In fact, lulo has an intense and refreshing taste and is rich in proteins, vitamin C, fiber, and antioxidant compounds, such as alltrans-β-carotene, lutein and zeaxanthin, chlorogenic acids, and flavonol glycosides [73][74][75][76], in addition to iron, calcium, phosphorus, and some precursors of vitamin A [77] (Table 6 [78]). In particular, fruit carotenoids present in lulo fruit have been associated to the prevention of several illnesses, including hypertension, obesity, and cardiovascular diseases [79][80][81]. Also the potential of lulo as an antihypertensive agent is related to its content in N1,N4,N8-tris (dihydrocaffeoyl) spermidine and N1,N8-bis (dihydrocaffeoyl) spermidine (actually bioactive amines), which are bitter active compounds with inhibitory activity against the angiotensin-converting enzyme (ACE-1) that indirectly increases the blood pressure by causing blood vessels to constrict [35]. In turn, when evaluating the antihypertensive activity of some compounds of the lulo fruit by means of chemical computation techniques, the researchers of the Natural Additives of Aroma and Color group from the Chemistry Department of the Universidad Nacional de Colombia found that this was between 10 and 20 times higher than that of the drugs traditionally used to treat hypertension.
Based on the considerations made above, the lulo fruit comes to be a promising alternative with regard to the prevention and relief of hypertension-related diseases. However, being a highly perishable fruit, technological transformation processes are indispensable to take advantage of its beneficial properties by as many consumers as possible. According to this, the group of Functional Foods of the University Institute of Food Engineering for Development of the Polytechnic University of Valencia (Spain), in conjunction with the group of Biodiversity Evaluation and Use of the Technological University of Chocó (Colombia), is working on the development of new functional foods from lulo fruit (Solanum quitoense Lam). On the one hand, a stable lulo juice with improved antioxidant properties has been obtained by means of the application of moderate high homogenization pressures (from 50 to 150 MPa) instead of traditional thermal pasteurization. The same juice has proved to be a suitable impregnation liquid for the enrichment of other fruits with a porous structure, such as Granny Smith apples. The lulo fruit itself was found to have a high impregnation capacity, which implies susceptibility to be enriched with different bioactive compounds. Finally, after fermentation with Lactobacillus reuteri, selected for being one of the strains with proven effect against the metabolic syndrome, the number of viable counts in lulo juice resulted to be high enough to claim that it also may exert a probiotic effect. Most relevant results in relation to these advances are shown next.

Enhancing antioxidant properties of lulo juice by means of moderate high-pressure homogenization
This section shows the effect that homogenization pressures in the range of 50-150 MPa have on main physicochemical properties, including the total content of phenols and flavonoids and the antioxidant activity measured by both DPPH and ABTS methods. To obtain the juice, washed and without peduncle lulo fruits were crushed for 10 min in a blender (Phillips Avance Collection Standmixer, 800W 2L). The liquefied product was then filtered with a stainless steel sieve of 500 μm nominal aperture. When necessary, the juice was homogenized at 50, 100 or 150 MPa in a laboratory scale high-pressure homogenizer (Panda Plus 2000, GEA-Niro Soavi, Parma, Italy).  As it can be observed in Table 7, neither the soluble solid content nor the pH or the density of the lulo juices was significantly affected by homogenization pressure. On the contrary, the consistency index (K) increased significantly after the homogenization step, which is directly related to particle size reduction. As regards the average size of particles, it was maximum in the non-homogenized juice (251 ± 5 μm) and minimum in the juice homogenized at 150 MPa (57.94 ± 0.14 μm). Therefore, homogenization increased the amount of solids in suspension and, consequently, the stability of the cloud.
As regards the antioxidant properties of lulo juice, the fruit's own transformation into juice significantly reduced both total phenol and total flavonoid contents, which were probably separated from the juice together with the bagasse during the filtration step. However, the concentration of such compounds increased slightly (from 1.03 ± 0.16 to 1.28 ± 0.07 mg GAE/g for phenols and from 0.35 ± 0.24 to 0.570 ± 0.011 mg QE/g for flavonoids) after juice homogenization at 150 MPa and the subsequent reduction in the average particle size. Similar trends were observed when analyzing the total antioxidant activity by both the ABTS and the DPPH methods and when quantifying spermidine by HPLC analysis. For the latter compound, concentration increased from 1.86 ppm in non-homogenized lulo juice to 2.04 ppm in lulo juice homogenized at 150 MPa.
Regarding the ability of the homogenized lulo juice treated at 150 MPa to impregnate Granny Smith apples sliced, it was found to be similar to that of an isotonic sucrose solution. In this way, the bioactive compounds present in lulo juice may become part of the vacuum impregnated fruit composition. To be more precise, around 0.22 m 3 of lulo juice homogenized at 150 MPa could be incorporated to every m 3 of fresh apple.

Lulo fruit as a food matrix for vacuum impregnation and food property improvement
In this section, impregnation properties of lulo fruit are discussed. Vacuum impregnation being a matrix engineering technique allows to introduce desirable compounds into the porous structure of foods by applying a pressure gradient [14]. Among the impregnation parameters, the volume of the external liquid that can be incorporated into the cellular tissue in a controlled way (i.e., X, in m 3 /m 3 ) stands out, which informs about the feasibility of incorporating physiologically active compounds into its porous structure for the formulation of new products with enhanced functional properties. Hence, unpeeled lulo fruit was cut into 5 mm thick slices and immersed in an isotonic sucrose solution (a w = 0.994 ± 0.003). Vacuum impregnation was carried out in a pilot plant scale equipment located at the University Institute of Food Engineering for Development of the Universitat Politècnica de  Table 7.
Effect of homogenization pressure on pH, soluble solid content (brix), apparent density (ρ), and rheological properties of lulo juice.
València (Spain). This equipment consists of a stainless steel vacuum chamber connected to a liquid ring pump (SIHI model LOHE-25007). The vessel containing the impregnating solution was placed into the vacuum chamber, and the lulo samples were immersed in the liquid by means of a pneumatic arm operated by a compressor (COMBA, 1,5 HP de 25 L). The working conditions were set at 50 mbar for 10 min and atmospheric pressure for 10 min more. In each trial, the weight change of the samples was recorded according to the procedure described by [82], thus allowing to calculate the characteristic impregnation parameters of the lulo fruit.
As it is shown in Table 8, the different batches analyzed behaved in a similar way during the vacuum impregnation step. Positive values of parameters X 1 (between 1 and 5%) and X (between 8.6 and 16%) indicate that the impregnating liquid entered the porous structure after both the vacuum and the atmospheric steps. Likewise, positive values of parameters γ 1 (between 3.9 and 7.1%) and γ (between 2.9 and 6.6%) indicate a volumetric expansion of the lulo matrix after both the vacuum and the atmospheric steps. Compared to other fruits and vegetables [83], the volume fraction of fresh lulo that was filled with the impregnating solution at the end of the process (X, in m 3 /100 m 3 ) was significantly lower than that of Granny Smith apple (21.0 ± 0.9) or Soraya aubergine (64 ± 2) but considerably higher than that of Chandler strawberry (6.4 ± 0.3), Hayward kiwifruit (0.7 ± 0.5), or Bulida apricot (2.2 ± 0.2). Despite such differences, the lulo matrix can be considered as suitable to be enriched with other active compounds by means of the vacuum impregnation technique.

Probiotic food development from lulo fruit
The growing number of consumers with lactose intolerance, high cholesterol levels, and/or following vegetarian or vegan diets has encouraged the recent use of fruits and vegetables as probiotic carriers in the development of new functional foods. Fruit and vegetable juices are especially suitable for the growing of probiotic microorganisms since they inherently contain beneficial nutrients and have taste profiles that are pleasing to all the age groups [84]. In addition, due to their fast passage through the digestive tract, the viability of probiotic cells in the juices is hardly affected by the harsh acidic environment of stomach [85]. However, these food matrices do not always fulfill the pH or the essential amino acids and vitamins required for the optimum growth of most LAB with proven probiotic effect. This section evaluates the possibility of using the non-homogenized lulo juice as a medium for the growth of Lactobacillus reuteri CECT 925T. For this purpose, the lulo juice obtained by the procedure described above was pasteurized at 75°C for Batch X 1 γ 1 X γ ε e 1 5 ± 7 a 5 ± 4 a 8.8 ± 1.6 a 3 ± 3 a 6 ± 4 a 2 2.1 ± 1.8 a 6 ± 5 a 10 ± 3 a 5 ± 4 a 6 ± 5 a 3 2 ± 4 a 5 ± 2 a 11 ± 2 ab 3.7 ± 0.9 a 8 ± 2 a 4 1 ± 1.4 a 3.9 ± 0.9 a 16 ± 6 b 6.6 ± 1.0 a 9 ± 6 a 5 2.5 ± 1.3 a 7.1 ± 1.0 a 8.6 ± 0.9 a 2.9 ± 0.8 a 6.3 ± 1.2 a abc… different superscripts in the same column indicate statistically significant differences (p ≤ 0.05). X 1 and X stand for the volume fraction of fresh sample impregnated at the end of the vacuum step and at the end of the atmospheric step, respectively; γ 1 and γ stand for the relative volume deformation of fresh sample at the end of the vacuum step and at the end of the atmospheric step, respectively; ε e stands for the effective porosity. 2.5 min before being inoculated with 4 mL/L of MRS broth containing the active microorganism in a concentration of 10 8 CFU/mL. After 24 hours of incubation at 37°C, viable counts in the juice were of the order of 10 6 CFU/mL. Although this value was high enough to make an EU-based health claim [86], it was significantly lower to that obtained in mandarin juice inoculated with either Lactobacillus salivarius spp. salivarius CECT 4063 or Lactobacillus acidophilus CECT 903 [87].
In a further step, the lulo juice containing the probiotic was employed as impregnating liquid for the vacuum impregnation of Granny Smith apple slices (5 mm thick). In this way, the probiotic was introduced into a solid matrix without disturbing its organized cellular structure. However, since only 20% of the initial volume of the apple is filled with the impregnation liquid during the vacuum impregnation step, the probiotic content in the impregnated apple was not greater than 10 5 CFU/g. Subsequent lyophilisation of the vacuum impregnated apples did not increase the Lactobacillus reuteri content as expected by water removal and subsequent weight loss, it being lower than 10 6 CFU/g in the liophylized sample. Probiotic counts in both the lulo juice and the impregnated apple snack could be improved by adding certain ingredients (e.g., prebiotics, cryprotectants, soygerm powder, yeast extract, etc.) and/or applying specific processing technologies that can improve microorganism survival such as microencapsulation or sublethal homogenization. In any case, it should be interesting to evaluate through both in vivo and in vitro studies the antihypertensive activity of Lactobacillus reuteri in the products designed, since it could be enhanced due to a synergistic effect with the spermidine from the lulo juice.

Market and consumer trends toward functional foods
Revolution in living standard, eating habits, and increased health awareness has shifted consumer's acceptance toward nutritious, healthy, and disease-preventive food with wider health benefits. Consumer is becoming more and more conscious about the role of food in life extension, well-being, and prevention of chronic diseases [87].
Specific consumer characteristics, such as demographic background or personal motivation to participate in pro-health activities, play a remarkable role in functional food acceptance and consumption. Some sociodemographic characteristics such as gender, education, and age are the most important factors related to the acceptance of functional food. In addition, apart from health benefits, the carrier and the origin of functional components play an important role in making the decision to purchase functional products, consumers being more likely to purchase those functional components found naturally in foods. Other factors, such as organoleptic attributes, convenience, or label information, are found to be essential for consumer's acceptance. In his study, Kraus [88] concludes that consumers are not willing to sacrifice taste and general pleasure of eating and also states that naturalness of a product is very important.
Particularly for probiotics, a major challenge for these products is product acceptability by consumers with regard to sensory criteria. Traditionally, health benefits of probiotics were based in the consumption of fermented dairy products; however, lactose intolerance, cholesterol content, and allergic milk proteins have limited the growth of dairy probiotics. Besides, the increase in vegetarian consumers in both developed and developing countries has also contributed to a growing demand for plant-based probiotic products [87]. According to Panghal et al. [89], fruits are healthy and refreshing and have good taste and flavor profile and can be suitable for probiotics. They are an ideal medium to develop functional foods and have more nutritional values due to the presence of various phytochemicals, antioxidants, no cholesterol, vitamins, mineral content, and dietary fibers. Besides, economic reasons for the developing countries also require the search for an alternative to dairy products with good nutrients along with health-promoting factors, e.g., fruits, vegetables, cereal, legume, etc., and products which lack cholesterol content but are rich in protein, starches, minerals, fiber, vitamins, and antioxidants.
Nowadays an increasing trend in the Western society is consumer interest and focus toward natural and organic products, where the use of synthetic additives is limited. It has been suggested that natural ingredients with strong antioxidant activity could be used to design novel functional beverages. An increased interest relies upon the fortification with polyphenols due to their beneficial role against cardiovascular diseases, type 2 diabetes, and obesity, among other conditions. The combination of prebiotics, and also phenols with probiotic microorganisms, represents an innovative biotechnology to enlarge the functional food market and especially beverages [90].
According to Grand View Research [91], the global functional food market was higher than 129 billion dollars (US) in 2015, and it is expected to increase up to 250 billion in 2024. Growing consciousness among consumers on their health and proper diet, together with the prospect of reducing or even eliminating nutrition-related diseases, is responsible of this market trend. Society is becoming more and more conscious on the impact that changing dietary patterns may have in the incidence of type 2 diabetes, coronary heart disease, cancer, periodontal disease, and obesity. In this regard, functional foods are believed to play an outstanding role. In addition, increasing healthcare cost, along with the desire of improving later years among the geriatric population, has driven the growth of the functional food industry worldwide.
The global functional food market includes that of carotenoids, dietary fibers, fatty acids, minerals, prebiotics and probiotics, vitamins, minerals, phytochemicals, enzymes, and antioxidants in general. Market revenue of all these products separately is also expected to increase in the coming years. For example, dietary fibers, which are considered to prevent obesity and diabetes, are expected to grow by 8.4% in the next 8 years. Other phytochemicals, such as flavonoids, held a share of over 30% in terms of market value. Although these have been commonly consumed in their natural form, consumer's habits have led to their use in the form of functional food products which are aimed at preventing diet-related chronic diseases including those related to the metabolic syndrome. North America accounts for the largest market in flavonoids, the Asia Pacific demand was over 110 million US Dollars in 2015, and Europe is expected to grow, although at a slower pace. In any case, prevalence of diabetes, obesity, and chronic diseases is likely to propel demand for these nutritional foods and beverages in Europe.
With regard to probiotics, there is also a growing concern on awareness in their functional health benefits against different conditions, including those related to the metabolic syndrome such as obesity or type 2 diabetes [92]. The global probiotic market was thought to be worth 35.5 billion dollars in 2016, with predictions of this increasing up to 65 billion dollars by 2024 [93]. As reported by Lumina intelligence [92], a survey of Ganeden on consumers concluded that almost 80% of consumers preferred to consume probiotics in food and beverage products than in supplements. This is of special value taking into account that consumer preference is a key currency for measuring product success and predicting upcoming tendencies. North America demand for probiotics is expected to increase by 7.9% from 2016 to 2025, whereas the European market will grow at a pace of 7.3%. As for Asia Pacific countries, the probiotics industry is also expected to increase significantly.
Probiotics have achieved a prominent position in the global food market. Among the countries that have shown growth in the probiotic market, Europe represents the largest and fastest growing market, followed by Japan. Currently, there is a wide range of probiotic products offered by companies such as BioGaia Biologics AB, Christian Hansen A/S, ConAgra Functional Foods, Danisco, Groupe Danone, or Lifeway [87].
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Conclusions
The huge increase in obesity and consequently of physiological disorders associated with this has led to a massive increase in research work conducted in this area over the past 10 years. The relationship between diet and the incidence of metabolic syndrome is clearly contrasted. Although this relationship is tremendously complex and it is hardly affected by other variables related to lifestyle, specific works establish phytochemicals and probiotics as two of the active components present in food, which have the greatest effect on prevention and in the reduction of symptoms associated with metabolic syndrome.
Currently, the technological development achieved by the food industry allows both the design and development of specific foods that include active components in their composition as well as the application of specific techniques that increase the functional value of natural foods. The use of these advances in the right direction can be decisive in the solution of health problems related to obesity. Specifically, the applications of moderate homogenization pressures or food formulation techniques, such as vacuum impregnation, are presented as possibilities to develop liquid and/ or solid foods that combine the presence of phytochemicals and probiotics with demonstrated effectiveness against obesity in natural foods such as lulo fruit.