Inulin: a prebiotic functional food ingredient

The human gut microbiota is increasingly recognized as playing a central role in human health and disease. This dichotomous relationship with the host forms a central theme in this review, which addresses how we may divert the gut microbiota away from some of its more harmful activities towards beneficial interactions with the human host. We describe the concept of prebiotics, which use specific dietary carbohydrates to increase the numbers of what are seen as beneficial bacteria within the colon, in a selective manner. Specifically, the use of β(2-1) fructans or inulin in general, and certain of its fractions in particular as prebiotics, will be described. Prebiotic fructans constitute efficacious functional foods and there is strong evidence supporting the selectivity of their fermentation within the human gut microbiota, resulting in an increase in the relative numbers of Bifidobacterium spp. There is also considerable evidence, mainly from animal studies but also in humans, that dietary supplementation with prebiotic fructans, through modulation of the microbiota, plays a protective role in colon cancer, heart disease and bone health. However, the mechanisms by which this prebiotic microbiota modulation mediates such diverse health outcomes remain unclear. The future challenge facing the field of prebiotic functional foods will be the elucidation of these mechanisms of action. Recent high resolution bioomics technologies, especially metabonomics, provide the tools necessary to define the metabolic consequences of prebiotic microbiota modulation.

Australian sweet lupin kernal fractions as palatable functional food ingredients

Australian sweet lupin kernal fibre addition to foods in the diet beneficially modified serum lipids and the food products palatability was not reduced after repeated consumption. Australian sweet lupin kernal flour addition to white bread lowered the glycaemic index and did not reduce palatability, but increased the insulinaemic index.

Use of Biotechnology to increase the content of bioactive compounds in fermented foods of plant origin

The objectives of this PhD research were: i) to evaluate the use of bread making process to increase the content of β-glucans, resistant starch, fructans, dietary fibers and phenolic compounds of kamut khorasan and wheat breads made with flours obtained from kernels at different maturation stage (at milky stage and fully ripe) and ii) to study the impact of whole grains consumption in the human gut. The fermentation and the stages of kernel development or maturation had a great impact on the amount of resistant starch, fructans and β-glucans as well as their interactions resulted highly statistically significant. The amount of fructans was high in kamut bread (2.1g/100g) at the fully ripe stage compared to wheat during industrial fermentation (baker’s yeast). The sourdough increases the content of polyphenols more than industrial fermentation especially in bread made by flour at milky stage. From the analysis of volatile compounds it resulted that the sensors of electronic nose perceived more aromatic compound in kamut products, as well as the SPME-GC-MS, thus we can assume that kamut is more aromatic than wheat, so using it in sourdough process can be a successful approach to improve the bread taste and flavor. The determination of whole grain biormakers such as alkylresorcinols and others using FIE-MS AND GC-tof-MS is a valuable alternative for further metabolic investigations. The decrease of N-acetyl-glucosamine and 3-methyl-hexanedioic acid in kamut faecal samples suggests that kamut can have a role in modulating mucus production/degradation or even gut inflammation. This work gives a new approach to the innovation strategies in bakery functional foods, that can help to choose the right or best combination between stages of kernel maturation-fermentation process and baking temperature.

Selenized garlic: a future prospect or already a current functional food?

In the last years, functional foods have awakened consumer, scientific and business interest. A commonly found vegetable in such kind of foods includes garlic (Allium sativum). By its ability for selenium (Se) bio-accumulation, garlic can turn into an attractive option of selenized food. Selenium is an essential micronutrient for many organisms including plants, animals, and humans. It is an important trace element due to its antioxidant properties and plays a main role in prevention of cancer and cardiovascular diseases. Nowadays, there is an increasing interest in the study of Se speciation due to the different roles that each species manifests in toxicological and nutrition fields. However, Se exhibits a narrow interval between toxicity and essentiality, which is puzzling toxicologists and alarming nutritionists and legislators. In the present review, an overview on the development of selenized garlic studies and its potential implementation in Argentine production is exposed. The development of novel foods with added value such us selenized garlic could be an attractive alternative for local market. Moreover, it becomes a good offering for factory owners, considering that Mendoza represents about 85% of total garlic production in the country.

Tomato Lycopene: Functional Proprieties and Health

The growing concerns for physical wellbeing and health have been reflected in the way we choose food in our table. Nowadays, we are all more informed consumers and choose healthier foods. On the other hand, stroke, cancer and atherosclerosis may be somehow minimized by the intake of some bioactive compounds present in food, the so-called nutraceuticals and functional foods. The aim of this work was to make a revision of the published studies about the effects of some bioactive compounds, namely lycopene in human health, in the prevention of diseases, thus playing the role of a functional food. Free radical in human body can induce cell damage and consequently can be responsible for the development of some cancers and chronic diseases. Lycopene is one of the most powerful antioxidants known, being the predominant carotenoid in tomato. The respective chemistry, bioavailability, and its functional role in the prevention of several diseases will be object of this work. On the other hand, the inclusion of lycopene in some foods can also be made by biotechnology and represents a way to recover the wastes in the tomato industry with nutritional positive effects in health.

Diet, cell signalling, and tumourigenesis in multiple intestinal neoplasia mice

The incidence of colon cancer is high in Western societies, and in Finland it is among the three most common cancer types in both females and males. Environmental factors, including diet, affect colon cancer development. During the last few years, a vast amount of new, functional foods have been introduced to the consumers. Several products are already available that are marketed as promoting intestinal health. To be able to reliably call a dietary compound a chemopreventive substance it is of fundamental importance to understand the mechanism by which it affects tumour formation and the integrity of the epithelial cells. In this thesis, three different dietary compounds were studied in an experimental model of colon cancer. Inulin is a non-digestible fibre found naturally in chicory roots, artichokes and onions, amongst others. Nowadays it is widely used as an added dietary fibre in several food products. Conjugated linoleic acid (CLA) is a conjugated form of the fatty acid linoleic acid. CLA is formed by bacterial fermentation of linoleic acid in the rumen of cows and other ruminants. Concomitantly, it can naturally be found in milk and meat of ruminants. White currant is a colourless berry low in phenolic compounds that are believed to prevent cancer formation. Contrary to what was expected, inulin and the conjugated linoleic acid isomer trans-10, cis-12, were tumour growth promoting dietary constituents when fed to Min mice. Both diets decreased the NF-kappaB levels in the mucosa, but physiological adenoma development did not affect NF-kappaB. Diet altered beta-catenin and p53 signalling in the adenomas, confirming their involvement in adenoma growth. White currant, on the other hand, was chemopreventive, despite its low contents of phenolic compounds. The chemopreventive effect was accompanied by increased p53 levels in the mucosa, and decreased beta-catenin and NF-kappaB levels in the adenoma. This could explain the reduced adenoma number and size. The results underline the importance of carefully testing new dietary compounds in different settings to reliably confirm their health benefits. In this study two compounds that are consumed and believed to add to our health proved to be cancer promotive. A berry with low phenolic contents, on the other hand, was chemopreventive.

Consumption of anthocyanin-rich Queen Garnet plum juice reduces platelet activation related thrombogenesis in healthy volunteers

The anti-thrombotic properties of an anthocyanin-rich Queen Garnet plum juice (QGPJ) and anthocyanin-free prune juice (PJ) were studied in this randomised, double-blind, crossover trial. Twenty-one healthy subjects (M = 10, F = 11) consumed QGPJ, PJ or placebo, 200 mL/day for 28-days followed by a 2-week wash-out period. Only QGPJ supplementation inhibited platelet aggregation induced by ADP (<5%, P = 0.02), collagen (<2.7%, P < 0.001) and arachidonic acid (<4%, P < 0.001); reduced platelet activation-dependent surface-marker P-selectin expression of activated de-granulated platelets (<17.2%, P = 0.04); prolonged activated-partial thromboplastin clotting time (>2.1 s, P = 0.03); reduced plasma-fibrinogen (<7.5%, P = 0.02) and malondialdehyde levels, a plasma biomarker of oxidative stress ( P = 0.016). PJ supplementation increased plasma hippuric acid content ( P = 0.018). QGPJ or PJ supplementation did not affect blood cell counts, lipid profile, or inflammation markers. Our findings suggest that QGPJ but not PJ has the potential to significantly attenuate thrombosis by reducing platelet activation/hyper-coagulability and oxidative stress.

羟基自由基降解琼胶及其产物的活性研究

琼胶是一种从石花菜等红藻中提取的，目前生产工艺和结构等方面研究比较成熟的海藻多糖，广泛应用于医药、仪器等行业。但是，海藻多糖因为具有分子量大，粘度大，溶解度较小的等特点，而使其应用范围受到限制。利用降解的手段对其进行修饰，降低分子量和粘度，改善溶解性，可以拓展其应用范围。并且根据文献报道，琼 胶寡糖具有一些特殊的生物活性，如抗氧化性，抗炎症等。因此，对琼胶降解的研究具有生要意义。本研究中，为了选择一种合适的降解方法，进行了几种水解方法的尝试，其中包括在不同湿度和酸度下盐酸水解，过氧化氢和醋酸催化水解，Fenton体系羟基自由基降解。对于酸水解和Fenton体系氧化还原降解方法，通过粘度法对反应的速度进行了比较，表明氧化还原降解反应中琼胶的粘度降低比较快，并且具有代表性和新意，确定为本实验的降解琼胶的方法并对氧化还原降解所得的产物进行了活性实验。通过模仿自然界普遍存在的氧化还原降解反应，利用Vc诱导的Fenton体系产生的羟基自由基氧化还原降解琼胶得到低分子量的琼胶。降解产物经过高速离心、60％乙醇沉淀，除去分子量比较大的降解产物和磷酸盐，得到可溶于60％乙醇的分子量估计小于3000的降争产物，其产率为85％。利用经Sephadex-G25凝胶色谱分离所香的不同分子量的级分进行分子量和α－葡萄糖苷酶抑制活性关系的实验。降解产物对α－葡萄糖苷酶的抑制率和各级分的浓度呈线性正相关，并且各级分的IC_(50)则随着分子量的降低而降低。另外，对所得的降解产物混合物进行了红外吸收光谱、质子去偶核磁共震碳谱和负离子基质辅助激光诱导－飞行时间质谱结构分析。结果表明，氧化还原降解反应的专一性差，在得到寡糖的同时，在光谱图中出现一些比较复杂的副产物的结构信息。最后，根据MTT法的原理，以有体皮肤成纤维细胞为材料，通过紫外线辐射产生自由基造成氧化损伤，研究降解产物对成纤维细胞的保护作用。当无紫外线辐射时，降解产物对成纤维细胞具有显著的促进生长增殖作用：当经UVa、UBb辐射时则可以显著地表现出对损伤的保护作用，并且这种促进生长和保护作用呈显著的量效关系，表明降解产物具有清除基自由基的作用。但是，因为氧化还原降解以应的机理尚不十分明的以及琼羟胶的特殊结构，使得反应的副产物很难预测，也就使得分离工作难以进行，所以，根据目前所得的信息，尚不能确定是降解产物的什么级分产生的以上两种生物活性。

Sugar matrices in stabilization of bioactives by dehydration

Development of functional foods with bioactive components requires component stability in foods and ingredients. Stabilization of sensitive bioactive components can be achieved by entrapment or encapsulation of these components in solid food matrices. Lactose or trehalose was used as the structure-forming material for the entrapment of hydrophilic ascorbic acid and thiamine hydrochloride or the encapsulation of oil particles containing hydrophobic α-tocopherol. In the delivery of hydrophobic components, milk protein isolate, soy protein isolate, or whey protein isolate were used as emulsifiers and, in some cases, applied in excess amount to form matrices together with sugars. Dehydrated amorphous structures with bioactives were produced by freezing and freeze-drying. Experimental results indicated that: (i) lactose and trehalose showed similar water sorption and glass transition but very different crystallization behavior as pure sugars; (ii) the glass transition of sugar-based systems was slightly affected by the presence of other components in anhydrous systems but followed closely that of sugar after water plasticization; (iii) sugar crystallization in mixture systems was composition-dependent; (iv) the stability of bioactives was better retained in the amorphous matrices, although small losses of stability were observed for hydrophilic components above glass transition and for hydrophobic components as a function of water activity; (v) sugar crystallization caused significant loss of hydrophilic bioactives as a result of the exclusion from the continuous crystalline phase; (vi) loss of hydrophobic bioactives upon sugar crystallization was a result of dramatic change of emulsion properties and the exclusion of oil particles from the protecting structure; (vii) the double layers at the hydrophilic-hydrophobic interfaces improved the stability of hydrophobic bioactives in dehydrated systems. The present study provides information on the physical and chemical stability of sugar-based dehydrated delivery systems, which could be helpful in designing foods and ingredients containing bioactive components with improved storage stability.

Novel ingredients from brewers' spent grain - bioactivity in cell culture model systems and bioactivity retention in fortified food products

Functional food ingredients, with scientifically proven and validated bioactive effects, present an effective means of inferring physiological health benefits to consumers to reduce the risk of certain diseases. The search for novel bioactive compounds for incorporation into functional foods is particularly active, with brewers’ spent grain (BSG, a brewing industry co-product) representing a unique source of potentially bioactive compounds. The DNA protective, antioxidant and immunomodulatory effects of phenolic extracts from both pale (P1 - P4) and black (B1 – B4) BSG were examined. Black BSG extracts significantly (P < 0.05) protected against DNA damage induced by hydrogen peroxide (H2O2) and extracts with the highest total phenolic content (TPC) protected against 3-morpholinosydnonimine hydrochloride (SIN-1)-induced oxidative DNA damage, measured by the comet assay. Cellular antioxidant activity assays were used to measured antioxidant potential in the U937 cell line. Extracts P1 – P3 and B2 - B4 demonstrated significant (P < 0.05) antioxidant activity, measured by the superoxide dismutase (SOD) activity, catalase (CAT) activity and gluatathione (GSH) content assays. Phenolic extracts P2 and P3 from pale BSG possess anti-inflammatory activity measured in concanavalin-A (conA) stimulated Jurkat T cells by an enzyme-linked immunosorbent assay (ELISA); significantly (P < 0.05) reducing production of interleukin-2 (IL-2), interleukin-4 (IL-4, P2 only), interleukin-10 (IL-10) and interferon-γ (IFN-γ). Black BSG phenolic extracts did not exhibit anti-inflammatory effects in vitro. Hydroxycinnamic acids (HA) have previously been shown to be the phenolic acids present at highest concentration in BSG; therefore the HA profile of the phenolic extracts used in this research, the original barley (before brewing) and whole BSG was characterised and quantified using high performance liquid chromatography (HPLC). The concentration of HA present in the samples was in the order of ferulic acid (FA) > p-coumaric acid (p-CA) derivatives > FA derivatives > p-CA > caffeic acid (CA) > CA derivatives. Results suggested that brewing and roasting decreased the HA content. Protein hydrolysates from BSG were also screened for their antioxidant and anti-inflammatory potential. A total of 34 BSG protein samples were tested. Initial analyses of samples A – J found the protein samples did not exert DNA protective effects (except hydrolysate H) or antioxidant effects by the comet and SOD assays, respectively. Samples D, E, F and J selectively reduced IFN-γ production (P < 0.05) in Jurkat T cells, measured using enzyme linked immunosorbent assay (ELISA). Further testing of hydrolysates K – W, including fractionated hydrolysates with molecular weight < 3, < 5 and > 5 kDa, found that higher molecular weight (> 5 kDa) and unfractionated hydrolysates demonstrate greatest anti-inflammatory effects, while fractionated hydrolysates were also shown to have antioxidant activity, by the SOD activity assay. A commercially available yogurt drink (Actimel) and snack-bar and chocolate-drink formulations were fortified with the most bioactive phenolic and protein samples – P2, B2, W, W < 3 kDa, W < 5 kDa, W > 5 kDa. All fortified foods were subjected to a simulated gastrointestinal in vitro digestion procedure and bioactivity retention in the digestates was determined using the comet and ELISA assays. Yogurt fortified with B2 digestate significantly (P < 0.05) protected against H2O2-induced DNA damage in Caco-2 cells. Greatest immunomodulatory activity was demonstrated by the snack-bar formulation, significantly (P < 0.05) reducing IFN-γ production in con-A stimulated Jurkat T cells. Hydrolysate W significantly (P < 0.05) increased the IFN-γ reducing capacity of the snack-bar. Addition of fractionated hydrolysate W < 3 kDa and W < 5 kDa to yogurt also reduced IL-2 production to a greater extent than the unfortified yogurt (P < 0.05).

Comparative genomics of Bifidobacteria

Chapter 2 of this thesis describes the sequence analysis of 14 bifidobacterial genomes from various species of the genus Bifidobacterium, and the determination of their open pan-genome trend. This analysis first determined the total number of genes to be considered as the reservoir of functions available to representatives of this genus. Many identified genes are still uncharacterized, but may be involved in the adaptation to the gut environment. This comparative genomic analysis also determined a pool of ortholog functions used to infer their phylogenetic relationship, thereby providing a more robust approach compared to that based solely on the16S rRNA-encoding gene. The genome sequence of an isolate from the insect hindgut Bifidobacterium asteroides PRL2011 was fully characterized in Chapter 3, surprisingly revealing a putative respiratory metabolism, which was also found to be present in other insect isolates, suggesting that respiration was an ancient feature of this genus, but also an adaptative trait to different atmosferic oxygen levels. Chapter 4 of this thesis outlines a comparative study which focused on the analysis of representatives of the Bifidobacterium breve species, revealing that the genetic variability among members of this species principally consists of genes with a role in adaptation to host environment and gut colonization. Finally, Chapter 5 describes the analysis of the genome sequence of Bifidobacterium animalis subsp. lactis BLC1, a probiotic bacterium widely used in food industries as an ingredient of functional foods, providing information that will allow future investigations of this species.

Novel structured emulsions for delivering of food flavours

Flavour release from food is determined by the binding of flavours to other food ingredients and the partition of flavour molecules among different phases. Food emulsions are used as delivery systems for food flavours, and tailored structuring in emulsions provides novel means to better control flavour release. The current study investigated four structured oil-in-water emulsions with structuring in the oil phase, oil-water interface, and water phase. Oil phase structuring was achieved by the formation of monoglyceride (MG) liquid crystals in the oil droplets (MG structured emulsions). Structured interface was created by the adsorption of a whey protein isolate (WPI)-pectin double layer at the interface (multilayer emulsion). Water phase structured emulsions referred to emulsion filled protein gels (EFP gels), where emulsion droplets were embedded in WPI gel network, and emulsions with maltodextrins (MDs) of different dextrose-equivalent (DE) values. Flavour compounds with different physicochemical properties were added into the emulsions, and flavour release (release rate, headspace concentration and air-emulsion partition coefficient) was described by GC headspace analysis. Emulsion structures, including crystalline structure, particle size, emulsion stability, rheology, texture, and microstructures, were characterized using differential scanning calorimetry and X-ray diffraction, light scattering, multisample analytical centrifuge, rheometry, texture analysis, and confocal laser scanning microscopy, respectively. In MG structured emulsions, MG self-assembled into liquid crystalline structures and stable β-form crystals were formed after 3 days of storage at 25 °C. The inclusion of MG crystals allowed tween 20 stabilized emulsions to present viscoelastic properties, and it made WPI stabilized emulsions more sensitive to the change of pH and NaCl concentrations. Flavour compounds in MG structured emulsions had lower initial headspace concentration and air-emulsion partition coefficients than those in unstructured emulsions. Flavour release can be modulated by changing MG content, oil content and oil type. WPI-pectin multilayer emulsions were stable at pH 5.0, 4.0, and 3.0, but they presented extensive creaming when subjected to salt solutions with NaCl ≥ 150 mM and mixed with artificial salivas. Increase of pH from 5.0 to 7.0 resulted in higher headspace concentration but unchanged release rate, and increase of NaCl concentration led to increased headspace concentration and release rate. The study also showed that salivas could trigger higher release of hydrophobic flavours and lower release of hydrophilic flavours. In EFP gels, increases in protein content and oil content contributed to gels with higher storage modulus and force at breaking. Flavour compounds had significantly reduced release rates and air-emulsion partition coefficients in the gels than the corresponding ungelled emulsions, and the reduction was in line with the increase of protein content. Gels with stronger gel network but lower oil content were prepared, and lower or unaffected release rates of the flavours were observed. In emulsions containing maltodextrins, water was frozen at a much lower temperature, and emulsion stability was greatly improved when subjected to freeze-thawing. Among different MDs, MD DE 6 offered the emulsion the highest stability. Flavours had lower air-emulsion partition coefficients in the emulsions with MDs than those in the emulsion without MD. Moreover, the involvement of MDs in the emulsions allowed most flavours had similar release profiles before and after freeze-thaw treatment. The present study provided information about different structured emulsions as delivery systems for flavour compounds, and on how food structure can be designed to modulate flavour release, which could be helpful in the development of functional foods with improved flavour profile.

Polycyclic aromatic hydrocarbons (PAHS) (I): Measures of prevention and control

Measures of prevention and control against polycyclic aromatic hydrocarbons (PAHs) focus on an official food control, a code of best practice to reduce PAHs levels by controlling industry and in the development of a chemopreventive strategy. Regulation (EU) 835/2011 establishes maximum levels of PAHs for each food group. In addition, Regulations (EU) 333/2007 and 836/2011 set up the methods of sampling and analysis for its official control. Scientific studies prove that the chemopreventive strategy is effective against these genotoxic compounds effects. Most chemopreventive compounds studied with proven protective effects against PAHs are found in fruit and vegetables.