ABSTRACT
   In this thesis the technological development and functional assessment by in vivo studies of snacks from apple and mandarin juice by vacuum impregnation has been carried out. Specifically, two foods were developed, one with effect on cardiovascular risk in obese children of school age and the other one effective against Helicobacter pylori in infected children of school age. The interdisciplinary research has been carried out in collaboration with the Department of Food Science at the Institute of Agrochemistry and Food Technology (CSIC), with the Department of Obstetrics and Gynecology at University of Valencia and with the Paediatric Service of the Hospital Universitario Dr. Peset in Valencia.
   Firstly, a literature review was conducted in order to identify the current situation and the evolution of the technologies used in the development of functional foods during last years. The technologies used to develop functional foods have evolved, increasing considerably those technologies that form a structure aimed to prevent deterioration of physiologically active compounds. Literature review resulted in a review article titled “Functional foods development: Trends and technologies”, included on results section, and published in a Elsevier journal “Trends in Food Science & Technology” with impact factor 3.672. The review is included into 5 most downloaded articles list since september 2011.
   Secondly, a pilot plant was designed in order to produce the functional food in quantities to carry out the in vivo studies conducted in this thesis. While it has not been carried out at industrial level scaling, is a first step in this direction and a mathematical model has been developed establishing the relationship between all the variables involved.
   In the development of low humid functional food from apple and mandarin juice, with effect on cardiovascular risk in obese children of school age, technological, economic and environmental aspects associated with the manufacturing process were taken into account. The functional assessment of the food was carried out by in vivo studies.
- The effect of homogenization pressure on the physicochemical and functional properties of mandarin juice was studied. The suitability of the juice to be used in the vacuum impregnation operation was assessed. Mandarin juice was homogenized at pressures of 0, 5, 10, 15, 20, 25 and 30 MPa and the particle size, suspended pulp, turbidity, flavonoid content, colour and antiradical capacity were determined in the juice at each homogenization pressure  as well as the volume of the juice incorporated in the structural matrix of apple by vacuum impregnation operation was calculated. The results obtained allowed to conclude applying homogenization pressures in obtaining process of mandarin juice is favourable for a subsequent implementation of a vacuum impregnation operation because homogenization pressures led to greater incorporation of juice into the structural matrix of apple, decreased particle size and increased the stability of the pulp without adversely affecting the colour, the flavonoid content and the antiradical activity of the juice. This research resulted in two articles, one titled “Effects of pressure homogenization on particle size and the functional properties of citrus juices” and the other one titled “Homogenization pressures applied to citrus juices manufacturing. Functional properties and application”, both published in “Journal of Food Engineering” with impact factor of 2.414.
- The technological development of the functional food was carried out. Vacuum impregnation operation was analyzed; the uniformity of the operation was determined in juices homogenized at different pressures and, in those cases in which vacuum impregnation was regular, the amount of flavonoid incorporated into the structural matrix of apple was calculated. To stabilize the impregnated apple different dehydration methods and conditions were used. Specifically, we used hot air drying at 40 ºC for 24 hours and at 70 ºC until the same final moisture content was achieved and freeze-drying for 12 and 24 hours. The optical and mechanical properties of all the products obtained were determined and sensory analysis was also conducted. Due to the relation cost-acceptance, hot air drying at 40 ºC was used to develop the functional food. The flavonoid content was estimated and the effect of processing on the antiradical activity as well as the composition of the impregnated and dried apple was determined. The main conclusion from this study was bioactive components of mandarin juice were successfully incorporated into the structural matrix of apple by vacuum impregnation operation, without adversely affecting the antiradical activity after drying by hot air. So that, 40 g of dry product contained the same amount of flavonoid as 250 ml fresh mandarin juice. This study resulted in article titled “Technological development and functional properties of an apple snack rich in flavonoid from mandarin juice” published in journal “Innovative Food Science and Emerging Technologies” with impact factor 3.030.
- The effect of the functional food developed on health was studied. Two in vivo studies were conducted, one with experimental animals and the other with obese children of school age. In the in vivo study with experimental animals, the effect of functional food developed on biomarkers of oxidative stress in the liver injury induced by the action of tamoxifen was studied. Plasma aminotransferase levels were determined as an indicator of hepatocellular damage and interleukin-6 as a measure of inflammation. Carbonyl groups were measured in plasma and liver, as indicative of protein oxidation, ?-tocopherol as antioxidant defense evaluation and the modified base 8-hydroxydexyguanosine was determined as indicative of DNA damage. The results obtained in this study allowed to conclude tamoxifen induced an increase in aminotranferase, markers of oxidative stress and DNA damage in both plasma and liver that were significantly reduced, following consumption of apple snacks; in most of cases they even reach normal values as those achieved previous to induce the injury with tamoxifen. The effect of the mandarin juice incorporated into the structure of apple mainly accounts for increased levels of ?-tocopherol in both plasma and liver. This research resulted in article titled “Dried apple enriched with mandarin juice by vacuum impregnation counteracts the liver oxidative effect of tamoxifen in rats” submitted to the journal “Food and Chemical Toxicology” with impact factor 2.999. 
In the in vivo study with obese school-age children, the effect of incorporating snacks made from apple and mandarin juice in the mild energy-restricted diet was studied. This study attempted to provide useful information about antioxidant and anti-inflammatory potential of the product. In this way, we conducted an intervention study to examine whether the dietary intake of the snacks developed could be associated with a modification in oxidative status and biomarkers of systemic inflammation such as high sensitivity C-reactive protein (CRP), tumour necrosis factor ? (TNF-?), ? interleukin 1 (IL-1 ?) and interleukin 6 (IL-6).The results obtained in this study led to conclude that incorporation to the diet of the functional food developed improved systolic blood pressure and lipid profile of children. A significant increase in the antioxidant capacity of serum, a reduction in DNA oxidative damage and in inflammatory markers was found. Overall, short-term intake of the products ameliorates several risk factors for atherosclerosis. This research resulted in article titled “Dried apple snacks enriched with mandarin juice improves antioxidant capacity and decreases inflammation in obese children” submitted to journal “Food Chemistry” with impact factor 3.655.
   Moreover, in the development of functional food low humidity from apple and mandarin juice, effective against Helicobacter pylori infection in children of school age, we assessed the growth of two probiotic microorganisms (Lactobacillus acidophilus and Lactobacillus salivarius spp. salivarius), effective against Helicobacter pylori, in two commercial juices (mandarin juice and pineapple/grape juice) to three different pHs (5, 5.5 and 6) and incubated at two different times (24 and 48 hours). Mandarin juice with Lactobacillus salivarius spp. salivarius at pH 6 and incubated for 24 hours resulted in higher growth of microorganisms. This juice was used for the manufacture of functional food. Physicochemical properties and microorganism content were determined at each stage of the process, vacuum impregnation operation was analyzed and finally a preliminary in vivo test with 5 children infected by Helicobacter pylori was carried out. To complete the study of the manufacturing process, the impregnated apple was stabilized by different dehydration methods and conditions. Specifically, hot air drying at 30 and 45 ºC for 24 hours and freeze-drying for 12 to 24 hours were used. The optical and mechanical properties of all the products obtained were determined as well as the microorganism content. The results obtained in this study showed that after the vacuum impregnation operation and different dehydration methods and conditions employed the microorganisms levels reached in the product were enough to have probiotic effect. Similarly, the results obtained in the in vivo test confirmed the potential effects of the developed product against Helicobacter pylori infection. This research resulted in article titled “No invasive methodology to produce a probiotic low humid apple snack with potential effect against Helicobacter pylori” published in “Journal of Food Engineering” with impact factor 2.414.