Functional components and lipid quality assessment in vegetable foods: analytical and technological approaches

Lipids are important components that contribute very significantly to nutritional and technological quality of foods because they are the least stable macro-components in foods, due to high susceptibility to oxidation. When rancidity take place, it makes food unhealthy and unacceptable for consumers. Thus, the presence of antioxidants, naturally present of added to foods, is required to enhance shelf life of foods. Moreover, antioxidant like phenolic compounds play an important role in human health enhancing the functionality of foods. The aim of this PhD project was the study of lipid quality and lipid oxidation in different vegetable foods focusing on analytical and technological aspects in order to figure out the effects of lipid composition and bioactive compounds (phenolic compounds, omega-3 fatty acids and dietary fiber) addition on their shelf life. In addition, bioavailability and antioxidant effects of phenolic compounds in human and animals, respectively, were evaluated after consumption of vegetable foods. The first section of the work was focused on the evaluation of lipid quality impact on technological behaviour of vegetable foods. Because of that, cocoa butter with different melting point were evaluated by chromatographic techniques (GC, TLC) and the sample with the higher melting point showed the presence of fatty acids, triglycerides, 2-monoglycerides and FT-IR profile different from genuine cocoa butter, meaning an adding of foreign fat (lauric-fat) not allowed by the law. Looking at lipid quality of other vegetable foods, an accelerated shelf life test (OXITEST®), was used to evaluate of lipid stability to oxidation in tarallini snacks made up using different lipid matrices (sunflower oil, extravirgin olive oil and a blend of extravirgin olive oil and lard). The results showed a good ability of OXITEST® to discriminate between lipid unsaturation and different cooking times, without any samples fat extraction. In the second section, the role of bioactive compounds on cereal based food shelf life was studied in different bakeries by GC, spectrophotometric methods and capillary electrophoresis. It was examined the relationships between phenolic compounds, added with flour, and lipid oxidation of tarallini and frollini. Both products showed an increase in lipid oxidation during storage and antioxidant effects on lipid oxidation were not as expected. Furthermore, the influence of enrichment in polyunsaturated fatty acids on lipid oxidation of pasta was evaluated. The results proved that LC n-3 PUFA were not significantly implicated in the onset of oxidation in spaghetti stored under daylight and accelerated oxidation in a laboratory heater. The importance of phenolic compounds as antioxidant in humans and rats was also studied, by HPLC/MS in the latter section. For this purpose, apigenin and apigenin glycosides excretion was investigated in six women’s urine in a 24 hours study. After a single dose of steamed artichokes, both aglicone and glucuronide metabolites were recovered in 24 h urine. Moreover, the effect of whole grain durum wheat bread and whole grain Kamut® khorasan bread in rats were evaluated. Both cereals were good sources of antioxidants but Kamut® bread fed animals had a better response to stress than wheat durum fed, especially when a sourdough bread was supplied.

Wheat and low-input agriculture: agronomic, nutritional and nutraceutical implications

Recently, the increasing interest in organic food products and environmental friendly practices has emphasized the importance of selecting crop varieties suitable for the low-input systems. Additionally, in recent years the relationship between diet and human health has gained much attention among consumers, favoring the investigations on food nutraceutical properties. Among cereals, wheat plays an important role in human nutrition around the world and contributes to the daily intake of essential nutrients such as starch and protein. Moreover, whole grain contains several bioactive compounds that confer to wheat-derived products unique nutraceutical properties (dietary fibre, antioxidants). The present research provided interesting insights for the selection of wheat genotypes suitable for low-input systems and the development of specific breeding programs dedicated to organic farming. The investigation involved 5 old not dwarf genotypes (Andriolo, Frassineto, Gentil rosso, Inallettabile, Verna) and 1 modern dwarf variety (Palesio), grown under biodynamic management, over two consecutive growing seasons (2009/2010, 2010/2011). Results evidenced that under low-input farming some investigated old wheat genotypes (Frassineto, Inallettabile) were comparable to the modern cultivar in terms of whole agronomic performance. As regards the nutritional and nutraceutical properties, some old genotypes (Andriolo, Gentil rosso, Verna) emerged for their relevant content of several investigated phytochemicals (such as insoluble dietary fibre, polyphenols, flavonoids, in vitro antioxidant activity) and nutrients (protein, lipid, minerals). Despite of the low technological features, the six wheat varieties grown under low-input management may efficiently provide raw material for the preparation of traditionally processed bread with valuable sensory and nutritional properties. Results highlighted that old wheat varieties have peculiar phytochemical composition and may be a valuable source of nutraceutical compounds. Some of the genetic material involved in the present study may be used in breeding programs aimed at selecting varieties suitable for low-input farming and rich in health-promoting compounds.

Lipid quality and oxidative stability in food products from animal origin, as affected by breeding factor

Nowadays it is requested more investigations on alternative rearing systems that are able to improve poultry welfare and to warrant high-quality and safe meat products. This thesis work was focused on the evaluation of the oxidative stability of poultry meats, obtained with different rearing systems, diets (supplemented with bioactive compounds), and packaging conditions. The thesis work was divided into the following parts: - Evaluation of the effects of different rearing systems on the quality, fatty acid composition and oxidative stability of poultry thigh and breast meat belonging to different product categories (“rotisserie” and “cut-up” carcasses); - Evaluation of the effects of different rearing systems and packaging conditions on the shelf-life of poultry thigh meat stored at 4°C for 14 days, and the effects of feed supplementation with thymol (control diet and diet with 2 different concentration of thymol) and packaging conditions on lipid oxidation of poultry thigh meat shelf-life (stored at 4°C for 14 days). The oxidative stability of poultry meat was studied by means of the spectrophotometric determinations of peroxide value and thiobarbituric acid reactive substances. - Evaluation of anti-inflammatory effects of different flavonoids (thymol, luteolin, tangeretin, sulforaphane, polymethoxyflavones, curcumin derivates) to detect their biological activity in LPS-stimulated RAW 264.7 macrophage cells in vitro, in order to study more in depth their action mechanisms. It was evaluated the cell vitality (MTT assay), nitrite concentration and protein profile. The study was focused on the identification of potential dietary bioactive compounds in order to investigate their biological activity and possible synergic effects, and to develop new suitable strategies for long-term promotion of human health, in particular against cancer.

Milk and dairy products: evaluation of bioactive components by analytical techniques

Milk and dairy products are important source of bioactive compounds useful to satisfy the nutritional and physiological needs of any newborns of mammalian species and useful to guarantee adequate growth and development of infants as well as provide a complete nourishment of adults. Physico-chemical, nutritional and organoleptic properties of the main constituents and the “minor” components have a crucial role in the quality of milk and milk products. Although in the past decades dietary milk fat was often regarded as harmful for the human health, recent researches suggest that milk contains specific fatty acids with nutritional and physiological health benefits. For these reasons, a major attention is given to the quantity and quality of total fat intake. In the recent years, as a result of the new concept of multifunctional agriculture and the changing behaviours about diet, consumer demands in favor of high-quality, security and safety dairy products are increased. Moreover, milk proteins and milk-derived bioactive peptides are recognized to have a high nutritive value, several health-promoting functional activities and excellent technological properties. Accordingly, growing interest in the development of functional dairy products and preparation of infant formulae for babies who cannot be breast-fed, has been give in order to meet the specific consumer’s requests. This manuscript presents the main results obtained during my PhD research aimed to evaluate the main bioactive lipids and proteins in milk and dairy products using innovative analytical techniques. The experimental section of this manuscript is divided in two sections where are reported the main results obtained during my research activities on dairy products and human milks in order to characterize their bioactive compounds for functional food applications.

Qualitatitive and quantitative analysis of fish spoilage processes for development of new food monitoring systems

This study investigates the growth and metabolite production of microorganisms causing spoilage of Atlantic cod (Gadus morhua) fillets packaged under air and modified atmosphere (60 % CO2, 40 % O2). Samples were provided by two different retailers (A and B). Storage of packaged fillets occurred at 4 °C and 8 °C. Microbiological quality and metabolite production of cod fillets stored in MAP 4 °C, MAP 8 °C and air were monitored during 13 days, 7 days and 3 days of storage, respectively. Volatile compounds concentration in the headspace were quantified by Selective ion flow tube mass spectrometry and a correlation with microbiological spoilage was studied. The onset of volatile compounds detection was observed to be mostly around 7 log cfu/g of total psychrotrophic count. Trimethylamine and dimethyl sulfide were found to be the dominant volatiles in all of the tested storage conditions, nevertheless there was no close correlation between concentrations of each main VOC and percentages of rejection based on sensory evaluation. According to results it was concluded that they cannot be considered as only indicators of the quality of cod fillets stored in modified atmosphere and air.  

Nutritional and chemical characterization of food products enriched with biological produced fruits of Vaccinum myrtillus L

Vaccinum myrtillus L. belongs to Ericaceae family, being commonly known for its sweet small fruits: the blueberries. Widely consumed in fresh, these fruits are also used in jams and marmalades due to their digestive and hypoglycemic properties and also due to the presence of several bioactive compounds [!]. Therefore, it has become a very appealing matrix in the development of functional products that, beyond their nutritional properties, will add a long-term beneficial physiological/health effect [2]. In the present work, three novel blueberry based products developed by RBR Foods Company (Portugal), were characterized in terms of their nutritional and chemical properties: carbohydrates, ash, proteins, fat and energetic value (following official methods of food analysis), fatty acids profile (by CG-FID), soluble sugars (by HPLCRI), organic acids (by HPLC-DAD) and tocopherols (by HPLC-fluorescence). The products result from a mixture of the fruits with rose petals (PI), marigold petals (P2) and apple and goji berries (P3). The blueberry fruits were used as control sample. The nutritional profile of the novel products was very similar to the control sample: the carbohydrates were the most abundant macronutrient, followed by proteins and total fat. Regarding sugars, fructose, glucose and sucrose were identified in all the samples. P 1 and P2 didn't show significant differences in comparison to the control, however, P3 revealed a lower concentration of sugars. In terms of fatty acids composition, all the studied samples presented higher contents in polyunsaturated fatty acids, especially due to the contribution of linoleic and alinolenic acids. The results of tocopherols revealed that the control sample only presented two isoforrns of tocopherols, a- and y-tocopherol, being the same observed in P3. However, P 1 revealed the presence of all the isoforrns of tocopherols, while P2 was lacking otocopherol; which is related with the contribution of rose and marigold petals, respectively. The a-tocopherol isoforrn was the most abundant in all the studied samples. Overall, this work contributed to the nutritional characterization of novel blueberry based products and is a part of a wider project that aims the detailed study of these products, namely their potential to be used as functional foods.

Chromatographic analysis of antioxidant and related compounds in Polyporus squamosus from different origins

The antioxidant potential of mushrooms is mainly attributed to their composition in polysaccharides, phenolic compounds, tocopherols and some organic acids [1]. Phenolic compounds contribute directly to the antioxidative action and play an important role in stabilizing lipid peroxidation [2]; exhibit a wide range of bioactive properties such as anti-allergenic, anti-inflammatory and antimicrobial, which have been in part related to their antioxidant activity [3]. Tocopherols are important fatsoluble antioxidants, acting in the cellular membrane; due to their role as scavenger of free radicals protecting human cells against degenerative malfunctions [4]. Some organic acids are very common in natural matrices; malic acid contributes to a pleasantly sour taste and is often used as a food additive; citric acid is known due to its antibacterial and antioxidant properties and fumaric acid is important because of its antioxidant, anti-inflammatory, antimicrobial and acidifying properties [5]. The purpose of the present study was to analyze antioxidant and related compounds (phenolic compounds, tocopherols and organic acids) of Polyporus squamosus (Huds.) Fr. samples originated from two different origins (Portugal and Serbia). Specimens of P. squamosus were collected in Bragança (Northeast Portugal) and Jabučki rit (Northern Serbia) during April 2015 and 2012, respectively. Phenolic compounds, organic acids and tocopherols were determined by high performance liquid chromatograph (HPLC) coupled to a diode array detector (DAD), in the two first cases, and a fluorescence detector in the last one. With respect to phenolic and related compounds, p-hydroxybenzoic and cinnamic acids were identified in both samples; the first one predominates in the sample from Portugal, while cinnamic acid was more abundant in the sample from Serbia. Tocopherols (α-, β and γ-isoforms) were found in the sample from Serbia, but in the sample from Portugal, γ-tocopherol was not identified. This sample showed the highest total tocopherols content, and revealed the highest level of β-tocopherol; γ- tocopherol predominated in the sample from Serbia. Among organic acids, it was possible to quantify oxalic, malic and fumaric acids in both samples. Malic acid was found in higher amounts in the sample from Serbia. Overall, the present study shows that mushroom samples from different origins have dissimilar results, but are both rich in bioactive compounds, being a valuable source for the development of natural medicines and nutraceuticals.

Extraction, identification, fractionation and isolation of phenolic compounds in plants with hepatoprotective effects

The liver is one of the most important organs of human body, being involved in several vital functions and regulation of physiological processes. Given its pivotal role in the excretion of waste metabolites and drugs detoxification, the liver is often subjected to oxidative stress that leads to lipid peroxidation and severe cellular damage. The conventional treatments of liver diseases such as cirrhosis, fatty liver and chronic hepatitis are frequently inadequate due to side effects caused by hepatotoxic chemical drugs. To overcome this problematic paradox, medicinal plants, owing to their natural richness in phenolic compounds, have been intensively exploited concerning their extracts and fraction composition in order to find bioactive compounds that could be isolated and applied in the treatment of liver ailments. The present review aimed to collect the main results of recent studies carried out in this field and systematize the information for a better understanding of the hepatoprotective capacity of medicinal plants in in vitro and in vivo systems. Generally, the assessed plant extracts revealed good hepatoprotective properties, justifying the fractionation and further isolation of phenolic compounds from different parts of the plant. Twenty-five phenolic compounds, including flavonoids, lignan compounds, phenolic acids and other phenolic compounds, have been isolated and identified, and proved to be effective in the prevention and/or treatment of chemically induced liver damage. In this perspective, the use of medicinal plant extracts, fractions and phenolic compounds seems to be a promising strategy to avoid side effects caused by hepatotoxic chemicals.

Production of bioactive metabolites by intestinal bacteria

The adult intestinal microbiota comprises a microbial ecosystem of approximately 100 trillion microorganisms, with specific bacterial communities holding distinct metabolic capabilities. Bacteria produce a range of bioactive compounds to survive unfavourable stimuli and to interact with other organisms, and generate several bioactive products during degradation of dietary constituents the host is not capable of digesting. This thesis addressed the impact of feeding potential probiotic bacteria and other dietary strategies such as pure fatty acids and prebiotics, on gut microbiota composition, short chain fatty acid (SCFA) production and modulation of metabolism in animal models. In the first experimental chapter (Chapter 2) a gas chromatography method for the quantification of SCFA was optimized and applied in the analysis of caecal samples obtained in animal studies described in other chapters of this thesis. In Chapter 3, t10, c12 CLA supplementation was shown to significantly alter murine gut microbiota composition and SCFA production rather than no supplementation. These changes were suggested to be extra factors affecting host lipid metabolism. Chapter 4 described the contrasting effects of CLA-producing strains, Bifidobacterium breve DPC 6330 and B. breve NCIMB 702258, on murine fat distribution/composition and gut microbiota composition, suggesting that these changes were most likely strain-dependent. In Chapter 5, dietary GABA-producing strain Lactobacillus brevis DPC 6108 was shown to significantly increase (p<0.05) serum insulin in healthy rats, leading to a second experiment using a type 1 diabetes rat model. Lb. brevis DPC 6108 administration did not change insulin levels in diabetic rats, but attenuated high levels of glucose when compared to diabetic control. However, an auto-immune-induced diabetes model was suggested as a better model to study GABA-related effects on diabetes. In Chapter 6 bovine milk oligosaccharides, 6’sialyllactose and Beneo Orafti P95 oligofructose supplementations were associated with depletion or reduction of less favourable bacteria, demonstrating that ingestion of these oligosaccharides might be a safe and effective approach to modulate populations of the intestinal microbiota. In Chapter 7 (General discussion) the major findings of all studies were reviewed and discussed.

Antioxidant Activity Of Hydrophilic And Lipophilic Extracts Of Brazilian Blueberries.

Hydrophilic and lipophilic extracts of ten cultivars of Highbush and Rabbiteye Brazilian blueberries (Vaccinium corymbosum L. and Vacciniumashei Reade, respectively) that are used for commercial production were analysed for antioxidant activity by the FRAP, ORAC, ABTS and β-carotene-linoleate methods. Results were correlated to the amounts of carotenoids, total phenolics and anthocyanins. Brazilian blueberries had relatively high concentration of total phenolics (1,622-3,457 mg gallic acid equivalents per 100 g DW) and total anthocyanins (140-318 mg cyanidin-3-glucoside equivalents per 100 g DW), as well as being a good source of carotenoids. There was a higher positive correlation between the amounts of these compounds and the antioxidant activity of hydrophilic compared to lipophilic extracts. There were also significant differences in the level of bioactive compounds and antioxidant activities between different cultivars, production location and year of cultivation.

Comparison Of Techniques For The Isolation Of Volatiles From Cashew Apple Juice.

The aim of this study was to compare the performance of the following techniques on the isolation of volatiles of importance for the aroma/flavor of fresh cashew apple juice: dynamic headspace analysis using PorapakQ(®) as trap, solvent extraction with and without further concentration of the isolate, and solid-phase microextraction (fiber DVB/CAR/PDMS). A total of 181 compounds were identified, from which 44 were esters, 20 terpenes, 19 alcohols, 17 hydrocarbons, 15 ketones, 14 aldehydes, among others. Sensory evaluation of the gas chromatography effluents revealed esters (n = 24) and terpenes (n = 10) as the most important aroma compounds. The four techniques were efficient in isolating esters, a chemical class of high impact in the cashew aroma/flavor. However, the dynamic headspace methodology produced an isolate in which the analytes were in greater concentration, which facilitates their identification (gas chromatography-mass spectrometry) and sensory evaluation in the chromatographic effluents. Solvent extraction (dichloromethane) without further concentration of the isolate was the most efficient methodology for the isolation of terpenes. Because these two techniques also isolated in greater concentration the volatiles from other chemical classes important to the cashew aroma, such as aldehydes and alcohols, they were considered the most advantageous for the study of cashew aroma/flavor.

Mecanismos de ação de compostos bioativos dos alimentos no contexto de processos inflamatórios relacionados à obesidade

É indiscutível o papel da dieta e dos alimentos na manutenção da saúde e na redução do risco de DCNT. Estudos epidemiológicos mostram que o aumento do consumo de alimentos de origem vegetal influencia positivamente a saúde, enquanto estudos in vitro e in vivo em modelo animal elucidam os mecanismos pelos quais compostos bioativos não nutrientes, presentes nos alimentos, atuam na manutenção da saúde e na redução do risco de doenças. A modulação da expressão de genes que codificam proteínas envolvidas em vias de sinalização celular ativadas em DCNT é um dos mecanismos de ação dos compostos bioativos, sugerindo que estes possam ser essenciais à manutenção da saúde. A biodisponibilidade dos compostos bioativos de alimentos, as suas rotas metabólicas e o modo de ação de seus metabólitos são importantes fatores no seu efeito nas DCNT. Todos esses aspectos são temas de investigações recentes, cujos resultados contribuem para a compreensão da ocorrência e desenvolvimento das DCNT e da sua relação com a dieta. Essa revisão visou discutir alguns dos mecanismos envolvidos na resposta inflamatória induzida pela obesidade, apresentar os compostos bioativos de alimentos que modulam essa resposta inflamatória e sua relação com o metabolismo desses compostos.

Cálcio dietético: estratégias para otimizar o consumo

O cálcio é um nutriente essencial necessário em diversas funções biológicas. Estudos têm demonstrado a associação entre o baixo consumo de cálcio e doenças crônicas, entre elas osteoporose, câncer de colón, hipertensão arterial e obesidade. Entretanto, grande parte da população brasileira apresenta consumo de cálcio abaixo do recomendado. Este artigo objetiva revisar os fatores endógenos (idade e estado hormonal) e exógenos (fitatos, oxalatos, sódio, compostos bioativos e vitamina D) que influenciam a absorção do cálcio, bem como as principais metodologias utilizadas para avaliar a absorção e biodisponibilidade desse nutriente. Discorre-se sobre os possíveis fatores para o baixo consumo de cálcio: 1) Hábito alimentar - substituição de leite por bebidas com baixo teor de cálcio como o refrigerante, refeições realizadas fora de casa e a não realização de refeições como o café da manhã; 2) Alto custo dos alimentos fontes de cálcio. Além disso, este artigo discute as estratégias para otimizar o consumo do cálcio, que incluem: 1) Aumentar o conhecimento sobre a importância do consumo de cálcio para a saúde e as principais fontes alimentares desse nutriente; 2) Aumentar a disponibilidade de alimentos fortificados com cálcio; 3) Uso de suplementos em grupos específicos - quando e como administrar os sais de cálcio.

Yerba Maté (Ilex paraguariensis) aqueous extract decreases intestinal SGLT1 gene expression but does not affect other biochemical parameters in alloxan-diabetic wistar rats

Yerba mate´ (Ilex paraguariensis) is rich in polyphenols, especially chlorogenic acids. Evidence suggests that dietary polyphenols could play a role in glucose absorption and metabolism. The aim of this study was to evaluate the antidiabetic properties of yerba mate´ extract in alloxan-induced diabetic Wistar rats. Animals (n ) 41) were divided in four groups: nondiabetic control (NDC, n=11), and diabetic yerba maté (DY, n = 10). The intervention consisted in the administration of yerba mate´ extract in a 1 g extract/ kg body weight dose for 28 days; controls received saline solution only. There were no significant differences in serum glucose, insulin, and hepatic glucose-6-phosphatase activity between the groups that ingested yerba maté extract (NDY and DY) and the controls (NDC and DC). However, the intestinal SGLT1 gene expression was significantly lower in animals that received yerba maté both in upper (p = 0.007) and middle (p < 0.001) small intestine. These results indicate that bioactive compounds present in yerba maté might be capable of interfering in glucose absorption, by decreasing SGLT1 expression

Evaluation of Antiproliferative, Anti-Type 2 Diabetes, and Antihypertension Potentials of Ellagitannins from Strawberries (Fragaria x ananassa Duch.) Using In Vitro Models

Strawberries represent the main source of ellagic acid derivatives in the Brazilian diet, corresponding to more than 50% of all phenolic compounds found in the fruit. There is a particular interest in the determination of the ellagic acid content in fruits because of possible chemopreventive benefits. In the present study, the potential health benefits of purified ellagitannins from strawberries were evaluated in relation to the antiproliferative activity and in vitro inhibition of alpha-amylase, alpha-glucosidase, and angiotensin I-converting enzyme (ACE) relevant for potential management of hyperglycemia and hypertension. Therefore, a comparison among ellagic acid, purified ellagitannins, and a strawberry extract was done to evaluate the possible synergistic effects of phenolics. In relation to the antiproliferative activity, it was observed that ellagic acid had the highest percentage inhibition of cell proliferation. The strawberry extract had lower efficacy in inhibiting the cell proliferation, indicating that in the case of this fruit there is no synergism. Purified ellagitannins had high alpha-amylase and ACE inhibitory activities. However, these compounds had low alpha-glucosidase inhibitory activity. These results suggested that the ellagitannins and ellagic acid have good potential for the management of hyperglycemia and hypertension linked to type 2 diabetes. However, further studies with animal and human models are needed to advance the in vitro assay-based biochemical rationale from this study.