Laura Scheffler, PhD Thesis
Mohamed Mahmoud, PhD Thesis
Katja Lorber, PhD Thesis
GABAA and glycine receptors form pentameric arrangements of various subunit combinations and are involved in balancing excitation and inhibition in the central nervous system. Infusion and herbal products are being used over centuries as relaxants or sedatives. Binding of these substances to inhibitory receptors in the central nervous system has been suggested, however, distinct binding sites are not known, yet. Our group previously investigated Sideritis mountain tea, which is known for its sedative and relaxing effect in Mediterranean countries. Tea extracts enhanced the action of GABAA receptors. Linalool, myrtenol, and verbenol were among the single extracted compounds and showed enhancement of GABAergic currents when coapplied. Noteworthy, the analyzed α1β2γ2 GABAA receptors configuration is believed to be involved in mediating sedation. In this thesis, we took a closer look to the monoterpenes linalool, myrtenol and verbenol and explored effects of linalool and myrtenol derivatives on various GABAA receptor configurations. Using pharmacophore-based virtual screening and physiological readouts, novel inhibitors structurally related to cyclic monoterpenes were identified. In detail, our aim was to investigate linalool following metabolization. Although linalool being an acyclic monoterpene is a mild GABAA receptor modulator, its metabolic side products bear potential to enhance GABAergic activity. Moreover, linalool is highly abundant in food, perfumes and beverages and its metabolic products were well described in literature. In fact, metabolization of linalool can occur after inhalation or ingestion. The resulting side products are usually hydroxylated, oxidized or acetylated. Our published results showed that oxygenated linalool metabolites retained their modulatory character, while other modification did not enhance GABAergic currents significantly. Myrtenol and verbenol are strong positive modulators of phasic GABAA receptors. We further analyzed myrtenol and verbenol on α4β2δ GABAA receptors important for tonic inhibition. Experiments were performed in transfected HEK293 cells and brain stem slices. The resulting publication demonstrated a similar effect on ii endogenous GABAA receptors in the brain stem slices compared to transfected cells. Myrtenol and verbenol were further investigated on α2β3 receptors. Both monoterpenes were also analyzed on cultured primary olfactory bulb neurons. The olfactory bulb contains the first GABAA receptors that can be targeted following inhalation of these volatile odorants. Potentiation of GABAA receptors by both substances was verified in transfected cells and primary neurons. Together with pharmacophore-based studies, we screened for ‘myrtenol-like’ substances. Novel compounds were discovered and characterized that turned from the positive allosteric modulation of myrtenol into negative allosteric modulators. Hence, structural side chains were identified that are crucial for positive modulation or negative modulation. Another project dealt with the closely related inhibitory glycine receptor. The glycine receptor study described mutations that lead to a severe neuromotor phenotype in humans. Two mutations (G160R and T162M) have been found located in close proximity to the ligand-binding site. Five additional amino acid substitutions were introduced. The resulting mutants were analyzed for receptor trafficking and ligand potency alterations in vitro. Our findings showed that the mutations caused severe problems in cell trafficking and significantly reduced ligand binding.
In recent years a lot of research focused on the highly complex influence of nutrition on humans. Besides the impact of micronutrients, the effect of macronutrients, such as protein, carbohydrate and fat was investigated. However, the focus of these studies strongly differs regarding the nutriments used, timeframes and issues. Some studies considered the effects of macronutrients concerning psychophysical parameters or metabolism. In contrast, other studies investigated the effects on cognition or olfaction. In most of the studies food was applied via normal intake. Still, there is a need for elucidating the effects of different application forms on olfaction, cognition, psychophysical and metabolic function, e.g. regarding dietary research. The central point of the first part of this thesis is the analysis of the influence of orally and intravenously applied macronutrients concerning human cognition, olfaction, psychophysical and metabolic parameters, depending on different application forms (normal intake vs. slow intake). Therefore, three studies with healthy, young, normal weight, male subjects were conducted. The first study is a placebo-controlled study with slow intravenous application of 600 kcal nutrient solutions (protein, carbohydrates or fat) or placebo, i.e. bypassing the oro-pharyngeal-gastric pathway. None of the nutrient solutions or placebo applied, changed the perception of satiation, hunger or food craving. Further, olfaction and cognition were only affected in a minor manner. The second study demonstrated the influence of oral application form (normal intake vs. slow intervalled intake) of an isocaloric and isovolumetric nutrient solution (600 kcal). Normal intake significantly reduced hunger and food craving compared to slow intervalled intake. Furthermore, within slow intervalled application condition, identification scores of odors improved and subjectively rated hedonic values of odors impaired in satiated state compared to hunger state. Regarding cognition, there were no differences between both application forms concerning hunger and satiated state. The third study is a placebo-controlled study with normal oral application of three isocaloric nutrient solutions (protein, carbohydrate or fat 600 kcal) or
placebo. Intake of a placebo solution significantly reduced hunger and food craving directly after consumption. The isovolumetric condition showed that the effects on hunger and food craving are not only related to the distention of the stomach but also emphasizes the importance of the type of macronutrient applied. Moreover, hunger and food craving ratings and hunger hormone levels demonstrated that the hierarchical order that appears in satiating efficiencies of isovolumetric-isocaloric ingested macronutrients is protein > carbohydrate > fat. However, olfactory function was only affected by time. Besides, ingestion of a fat solution resulted in reduced cognitive tests error rates compared to protein intake. This implies that the different macronutrients affect psychophysical, metabolic and cognitive parameters in different ways. Taken together, the three studies show consistent effects during normal rate application compared to (1) low intervalled rate of oral food intake and to (2) intravenous low rate application.
The second part of this thesis deals with the development and validation of olfactory tests. The influence of nutrition on human olfaction has been analyzed by numerous scientists using nonexclusively food related olfactory tests. This is probably due to the fact that such tests are not commercially available. Thus, there is a need for the development of an exclusively food related olfactory test. Besides, the mainly in Europe used olfactory test, the Sniffin’ Sticks Test was not validated on a chemo-analytical basis until now. Certainly, the stability of the threshold tests is quite important because even small concentration fluctuations may have a huge impact on the reliability of the results. The first aim of this part of the thesis was the chemo-analytical validation of the n-butanol threshold test of the Sniffin’ Sticks Test. The second aim was the development, as well as sensory and chemo-analytical validation of an exclusively food related olfactory test that can be used for clinical and scientific issues. First, the chemo-analytical validation of the n-butanol threshold test demonstrated that the gas phase of new and six months old threshold pens are linear over the entire set. Additionally, the application-simulation test showed a valid performance of the set when used according to the manual. These results correspond to the expected performance, which was till then only assumed. Second, the newly developed exclusively food associated olfactory test (FAOT) is an identification test that consists of 16 different felt pens, filled with solutions of single odorants or aromas. The test contains pens representing the three macronutrients, protein, carbohydrate and fat. For identification each pen has a list of four items with one correct item. All items and odors are food associated and familiar to European population. Comparing the new test with the validated identification test of the Sniffin’ Sticks Test shows that both tests are similar concerning identification rate and intensity. Additionally, sensory and chemo-analytical validation of the new test demonstrated that the test is stable for at least 24 weeks. Thus, the innovative FAOT is a valid olfactory test that can be used for clinical as well as scientific issues.
Frauke Kirsch, PhD Thesis
Aroma is one of the main quality parameters of wine and off-odour taints can be highly detrimental to it, since they may overpower and obscure its natural bouquet, thereby diminishing consumer enjoyment of this highly valued product. As such, avoiding generation of off-odours is an issue of great relevance for winemakers worldwide. In order to deepen the knowledge about wine off-odours, four wines were investigated by gas chromatography-olfactometry (GC-O) with regard to the presence of earthy-smelling compounds. The samples had similar characteristics: made from Pinot Noir variety, cultivated in the Burgundy region (France) and from the 2006 vintage. However, it was found that they differed with regard to the intensity of an earthy odour defect, from clearly affected samples to a high quality wine. 2-Methylisoborneol (2-MIB) was detected in the two most affected wines, although the samples were seven years old. This finding contradicted previous studies that reported the instability of this substance in wine and must (La Guerche, Dauphin, Pons, Blancard, & Darriet, 2006). In order to investigate this seemingly paradoxical situation, 2-MIB stability was reinvestigated in wine and in model solution. Although the concentration of 2-MIB decreased in both media with time, rather than a complete degradation, only a decelerating degradation was observed, still yielding a final concentration at 90 days of storage that was well above its odour threshold. Based on this observation, it was concluded that 2-MIB can still be detected in wine and may be responsible for earthy off-odours, on condition that the start concentration is high enough.
The second part of this thesis dealt with the investigation of the effects of Botrytis cinerea and Erysiphe necator infections on wine aroma. These are among the most relevant fungi in viticulture and are responsible for bunch rot and powdery mildew diseases, respectively. The study characterized the impact on both grape must and final wine. With this aim, samples were produced from different grape varieties. The sample set included White Riesling, Red Riesling and Gewürztraminer to study the effects of bunch rot and the hybrid Gm 8622-3 in the case of powdery mildew. For each variety, a healthy sample and a sample that showed a pronounced case of infection were collected the same day in the same vineyard and were later processed identically in order to avoid the influence of additional factors. In each case, the main aroma compounds were determined by GC-O and ranked according to their relative intensities by means of aroma extract dilution analysis (AEDA). In case of the wine samples, these investigations were further supported by sensory analyses and quantitative determinations. In general, it was observed that fungus infections produced clear differences in the overall aroma composition of the samples, and these were predominantly related to quantitative shifts rather than qualitative changes in the odorant composition. In the must samples, bunch rot had a positive impact on fruity and floral notes while several earthy smelling compounds such as geosmin and 2-MIB were developed as result of the infection. Unlike in previous studies, however, no clear differences in the quantities of earthy, mushroom-like smelling substances as result of the powdery mildew infection were observed.
Wine analysis revealed that bunch rot infection induced a general increase in the Flavour Dilution (FD) factor of fruity smelling lactones, such as γ-decalactone, γ-undecalactone and (Z)-6-dodeceno-γ-lactone, and the curry-like smelling lactone sotolone together with an increase in vanillin. However, bunch rot led to inconsistent effects on the aroma composition in the three investigated varieties, demonstrating that both the type of fungal infection and the grape variety greatly influence the final product. Regarding the sensory tests, bunch rot affected samples were generally rated as more peach-like/fruity, floral and liquor-like/toasty than their corresponding healthy controls. Furthermore, in the hedonic evaluation, bunch rot affected samples were described as being more pleasant in all three varieties studied, which might be linked to the enhancement of positive aroma attributes already mentioned. On the contrary, the sample affected by powdery mildew was consistently evaluated as being more negative with regard to hedonics. This negative rating was, however, not related to any specific off-odour but was rather due to a lack of positive aromatic notes. In this sense, powdery mildew led to a decrease of the vanilla-like note in the affected sample corresponding to a decrease in the FD factor of vanillin, while the remaining sensory attributes were rated very similarly. In agreement with this, the FD factors of the acidic compounds, lactones and esters remained only slightly altered. Interestingly, no earthy smelling substances were detected in any wine sample. These compounds were perceived only with low FD factors in the musts, with the exception of 2-MIB, which was, most likely, degraded during fermentation. Lastly, the effects of the named fungal diseases were further characterized by quantification of representative aroma compounds by means of stable isotope dilution analysis (SIDA). Analyses revealed that both fungal diseases caused significant changes in the concentration of most target compounds. The greatest effects were an increase in the concentration of phenylacetic acid, acetic acid and γ-decalactone for both fungi and all grape varieties.
The replacement of synthetic conventional compounds by natural ingredients; whether in medicine, food, or cosmetics; has been increasingly requested by consumers, especially since the last decade. Terpenes in general and monoterpenes in particular are secondary metabolites in plants, and they may be a promising natural alternative. Monoterpenes, the main constituents of plants’ essential oils, are odorous compounds that play a significant ecological role in plant evolution. They are primarily utilized by the flavour and fragrance industries due to their characteristic aroma. In addition, a series of representatives belonging to this substance class are antimicrobial, anti-inflammatory, antiseptic, and anticancer agents; or elicit other therapeutic effects. Thereby, acyclic monoterpene alcohols, mainly linalool, geraniol, nerol, and citronellol, are primarily in the focus of scientific research. Besides their aromatic character and their role in aromatherapy, they induce a series of pharmacological and physiological effects. In view of the latter, their metabolic pathways have been previously investigated in both plants and animals. Linalool and geraniol, for example, are metabolized giving 8-hydroxy and 8-carboxy derivatives; i.e. undergoing oxidation at C-8. However, these metabolites have not been tested in terms of odour or other physiological activities. Furthermore, no studies are at hand elucidating which structural features of these substances are responsible for specific odour qualities and potencies of these monoterpenes.
In the frame of this doctoral thesis, a comparison between chemical structure and odour character of selected monoterpenes relating to linalool, geraniol, nerol, and β-citronellol has been conducted, complemented by investigations on their acetate derivatives and previously identified oxygenated metabolites. To achieve this aim, a series of oxygenated derivatives, bearing an aldehyde, an alcohol, or an acid functional group at C-8, were synthesized from the aforementioned terpene alcohols and acetates yielding 24 compounds, yielding a comprehensive substance library for future elucidation of the substances’ presence in nature and evaluation of their further potential physiological properties. Within this study, however, the focus lies on a comprehensive characterization of the compounds’ olfactory properties. Accordingly, all compounds were tested in relation to their odour qualities and relative odour thresholds (OTs) in air, as well as potential inter-individual variations in sensory perception for each single substance. Overall, the results show that almost all investigated parent monoterpene alcohols and their acetates exhibited closely related odour characters; ranging between citrus-like, fresh, fruity, floral-sweet, and fatty.
Amongst others, linalool was demonstrated to be the most potent monoterpene of the group of investigated compounds, eliciting an OT of 3.2 ng/Lair. According to this study, the presence of an OH group at C-3 in the linalool basic structure is the main contributor to its characteristic odour quality and high potency. On the other hand, the occurrence of this OH at C-1 in geraniol, nerol, and citronellol does not alter their odour quality but increases their odour threshold levels, with values of 40, 60 and 10 ng/Lair, respectively. Esterification of this OH-group to the respective acetate barely affected the odour quality, but provoked a decline in odour potency. Substitution at C-8 of either the parent monoterpeneols or their acetates by another OH-group retained the smell of the parent compounds but led to a dramatic decrease in the potency. However, the smell potency was only retained when replacing the alcoholic group at C-8 by an aldehyde or an acid group. It is worth mentioning that among the acetate derivatives 8-oxolinalyl acetate elicits similar smell impressions as linalool, thus exhibiting a citrus-like, fresh odour with an OT of 5.9 ng/Lair. Apart from that, further oxidation of C-8 of linalool, geraniol, citronellol, and citronellyl acetate to their corresponding acids led to a total odour loss.
To summarize, the aim of this thesis was to evaluate if the target acyclic monoterpene alcohols and their acetates are the only odourous compounds in this substance class or if related derivatives also bear the potential of eliciting interesting olfactory effects. Finally, data of this substance library regarding smell properties was complemented by retention index data (RI values) as well as mass spectrometric and nuclear magnetic resonance data to aid researchers in future analytical studies when aiming at identifying the target molecules in nature.
Frankincense, the gum resins produced by tapping trees of the genus Boswellia, have a long and rich history as incense materials. In the course of this thesis, several unresolved aspects of volatiles in Boswellia gum resins were investigated:
First of all, a broad range volatile profiling study allowed to confirm and extend the knowledge about the general composition of the volatile fraction in Boswellia resins. Different species are identifiable by their volatile profile, and marker substances were identified for the commercially relevant species. To resolve open questions resulting from contradictory literature sources, a large sample set from nine different species was screened and the results were evaluated by multivariate statistical analysis.
Secondly, in order to identify odor active constituents in the resin of Boswellia sacra, an aroma extract dilution analysis was performed, resulting in the identification of 19 odorants in six different samples. Two odorants exhibited very high flavor dilution factors, but could not be sufficiently characterized or identified by two-dimensional GC-MS measurements. Consequently, a semipreparative enrichment procedure was established to isolate sufficient amounts of the respective trace constituents. Analytical data from the purified odorants allowed the identification of mustakone and rotundone, two sesquiterpene ketones with very low odor thresholds.
In a further part of the study, the use of frankincense in incense rituals was simulated in a newly developed pyrolysis-GC-MS/Olfactometry approach, which allowed the simultaneous tracking of MS and sensory data resulting from pyrolysis of the gum resin material. Through these experiments, several new odorants could be shown to be formed during the heating step. Fractions of the gum resin material produce specific odorants that were resolved within this study.
In the last step, a fast and easy quantification method using thermal desorption GC-MS in combination with stable isotope dilution assays was developed to investigate potential changes in the most volatile odorants’ profiles during storage of frankincense. It was observed that even long term storage for more than 10 years did not result in notable losses of monoterpenoid odorants. Overall, frankincense still continues to provide interesting research challenges, particulary in the field of odorant formation during pyrolysis.
Birth is the most challenging transition in the life cycle of mammals, requiring the mother-infant-dyad to adapt physiologically and behaviourally to postpartum life. In humans, the mother is around the infant most of the time, ensuring neonatal energy uptake through regular breastfeeding sessions. Successful breastfeeding is thought to be supported by olfactory cues related to lactation. However, neonatal olfactory perception and motivational state towards single odorants from lacteal fluids are still poorly understood, as are dynamic changes in the chemosensory properties of these fluids during the course of lactation. Does an odorant which occurs in both amniotic fluid and colostrum, viz. 5α-androst-16-en-3-one (AND), elicit specific responses in 3-day-old human neonates? Does the human neonate respond to odorants such as 3-methyl-3-sulfanylhexan-1-ol (MMH), 3-sulfanylhexan-1-ol (MH), 3-hydroxy-3-methylhexanoic acid (HMHA), and 3-methylhex-2-enoic acid (3M2H), which are assumed to be cleaved at moments of highly reinforcing conditions, that is delivery and breastfeeding? Does the newborn discriminate between the odours of lacteal stimuli of different lactation periods, viz. colostrum, transitional milk, and mature milk? Do colostrum and transitional milk differ in their odorant composition as determined by an adult nose? Ethological and aroma-analytical experiments were run to answer these questions. In the first part of this study, the odorants AND, MMH and MH, and HMHA and 3M2H were administered to 3-day-old neonates, and respiratory rate changes and changes in durations of oro-facio-cephalic movements were assessed. Aqueous solutions were administered at concentration ranges from 0.5 pg/L to 500 μg/L (AND), 1 to 50 ng/L (MMH), 1 and 10 ng/L (MH), 0.5 ng/L to 200 μg/L (HMHA), and 30 ng/L to 80 μg/L (3M2H)1, approaching physiologically-relevant concentrations. Reference stimuli were distilled water, vanillin, the familiar formula/breast milk (consumed since birth), and, in one of the experiments, also maternal sweat.
Respiratory rate was measured via a respiratory belt and oro-facio-cephalic movements were coded from videos by a trained experimenter. Triangle tests were conducted with adult participants to estimate adult orthonasal olfactory detection thresholds of the target odorants. Respiratory rate changes were observed to be a poor indicator of neonatal odour detection. Oro-facio-cephalic responses, however, suggested neonatal odour detection and hedonic valence of odours.
Thus, it was concluded that the neonates detected the studied odorants at concentrations of 0.5 ng/L (AND), 10 ng/L (MH), 0.5 ng/L (HMHA), and 80 μg/L (3M2H). Likewise, results point to a detection of MMH at 50 ng/L, but only by male neonates. The sex of the newborns also modulated their responsiveness to AND. Feeding mode (bottle- vs. breast-fed) had no impact on the neonates’ responses. The aforementioned stimuli elicited longer negative facio-cephalic and/or shorter positive oro-facio-cephalic actions compared with water. Familiar milk and sweat were not discriminated from water in terms of neonatal facial responses, but vanillin was discriminated in some of the experiments. Adult women and men detected AND at 50 and 500 μg/L, MMH at 1 and 38 μg/L, MH at 0.7 μg/L, HMHA at 0.6 and 1.7 mg/L, and 3M2H at 120 and 360 mg/L, respectively.
In the second part of this study, the respiratory and behavioural responses of newborns to the odours of unfamiliar pools of lacteal fluids (donated from unrelated mothers) from three different lactational stages (colostrum, transitional milk, and mature milk) were assessed. Further, odour-active compounds were extracted from sample pairs of colostrum and transitional milk. The extracts and corresponding blanks were subjected to comparative aroma extract dilution assays (gas chromatography-olfactometry). The identification of odorants was based on a comparison of odour quality and retention indices with those of pure reference compounds. Neonates did not evince discriminative respiratory rate changes or facial activity towards the odours of colostrum, transitional milk, and mature milk. A multitude of odour impressions were detectable in aroma extracts of colostrum and transitional milk samples, with 32 odorants being identified. None of the detected odorants was found exclusively in colostrum extracts or in transitional milk extracts. However, some compounds yielded higher flavour dilution (FD) factors in a majority of colostrum samples compared with transitional milk samples. (E,Z,Z)-Trideca-2,4,7-trienal stands out of these odorants due to the fact that it was not found in blank samples but was detected in each sample pair. It was concluded from these results that 3-day-old neonates detect the investigated odorants at concentrations present in perinatal fluids. In contrast, adults required more elevated concentrations to detect these odorants in a triangle test. It was further concluded that the target odorants are negatively valenced for the newborns. Familiar milk, exhibiting an odour that is known to be detected by newborns, was not discriminated from water in the majority of the experiments. Hence, the detection of an odour did not always lead to a neonatal response that was distinctive to that of water. Thus, even lower concentrations of the studied odorants might have been detected by the newborns but elicited a neutral response.
Further, it was concluded that colostrum and transitional milk share many odorants, and together with mature milk are olfactorily and/or motivationally equivalent for the neonate under the present experimental conditions. Nevertheless, the present aroma-analytical data suggest that certain odorants are more concentrated in colostrum compared with transitional milk. In summary, the studies conducted during this doctoral thesis contribute to broadening the knowledge on early human odour perception and acquisition, as well as on the aroma composition of human colostrum and transitional milk.
1 1 g = 1000 mg = 106 μg = 109 ng = 1012 pg
Dairy products have been consumed by humans since the domestication of mammals, primarily due to their high nutritional values and palatable sensory attributes. Over the centuries, manifold dairy matrices have arisen that tend to prolong the rather brief edible period of pure, unprocessed milk. Nowadays, almost all dairy products are heat-treated at the beginning of production to decrease their native bacterial counts. But this treatment comes at a cost: the exposure of milk to high temperatures leads to heat-induced alterations of its organoleptic properties. In the past, extensive research was undertaken to understand and characterize such alterations. Most studies focused on bovine milk and on the standard formulations and technologies used in dairy product manufacture. Recently, however, with changing directions in consumer preferences, novel or even traditional raw materials and innovative processing technologies have emerged that offer a new dimension in production and treatment, for instance the increasing demand for milk of non-bovine origin, the supplementation of infant formulas with polyunsaturated fatty acids (PUFAs), or the search for alternative and gentle heating technologies.
In the present work, the aroma of goat milk was investigated via a systematic approach with regard to seasonal effects and heat treatment (Chapter 1). The most potent odor-active compounds were characterized in raw, pasteurized, ultra-high temperature (UHT) treated and sterilized goat milks by human sensory evaluations, gas chromatography/olfactometry (GC/O), and aroma extract dilution analyses (AEDA). Sensory differences were apparent in goat milks at altering stages of lactation, from different farms and after varying degrees of heat treatment. The most potent odorants in raw goat milk were identified as 4-ethyloctanoic acid, 3-methylindole (skatol) and one unknown compound with a canola-like, metallic odor quality. Heat treatment was found to induce increasing intensities in the compounds phenylacetic acid and 4-hydroxy-2,5-dimethyl-3(2H)-furanone (furaneol). Furthermore, some alkylated phenols were identified within the odor-active fraction of goat milk and presumably contribute to the typical goaty, waxy, animal-like flavor of the milk. In addition, 1-benzopyran-2-one (coumarin) was unequivocally identified in goat milk. Assuming a forage-based origin, the transfer of the latter compound into ruminant milk was confirmed for the first time in these studies.
Infant formula was the subject matter of the next set of investigations. Studies were carried out on spray-dried infant formulas in terms of improving the sensory quality of PUFA-enriched formulas. The overall aim of this research was to identify a sensitive method for the early detection of fatty acid oxidation via a comparative evaluation of different primary and secondary oxidation markers (Chapter 2). More specifically, lipid hydroperoxides (LPOs), conjugated dienes and several odor-active volatiles were comparatively quantified via photometric assays and gas chromatography-mass spectrometry (GC-MS). The latter technique allowed oxidative variances in the infant formulas to be screened down to low ppbV-levels and within eight weeks of production. The photometric method for analyzing the primary oxidation markers was found to lack sufficient sensitivity for the early indication of the development of sensory defects. In addition to these analyses and based on their outcome, predictions were made on improving PUFA-enriched infant formulas by varying their antioxidant and mineral composition. Statistical analysis using an experimental design approach revealed a high impact of copper on promoting fatty acid oxidation. Hence, the generation of rancid, metallic and/or fishy off-flavors in infant formulas could be delayed by adding copper to the product in an encapsulated form.
Yoghurt was the focus of the final investigations, with studies relating to the sensory and textural changes of yoghurt products upon heat treatment. More specifically, a second (post-pasteurization) gentle heat treatment was applied to a fermented yoghurt matrix. The aim of this research was to improve the shelf-life of yoghurt without impairing its very susceptible organoleptic properties. Gentle heating via radio frequency (RF) treatment was applied at mild temperatures between 58°C and 72°C and compared to convectional (CV) heating. An initial study focused on the general applicability of a second, post-pasteurization heating step for yoghurt, as well as on the differences between RF and CV heating with respect to microbial numbers and sensory quality (Chapter 3). A second aspect of this research was to overcome the technological challenge of preserving the fine and homogeneous microstructure and texture of yoghurt during the second heating (Chapter 4). Overall, RF heating was found to be effective in rapidly attaining the required homogenous temperatures of between 58°C and 65°C. For CV heating, heat transfer limitations that imposed a mandatory prolonged heating period became apparent. Microbial investigations demonstrated the efficacy of the gentler mode of the RF treatment, whereby lactic acid bacteria (LAB) partially survived the heat treatment, as required by food law. In particular, a reduction in LAB highlighted the potential of this method for prolonging the yoghurt shelf-life, not only by inactivating yeasts and molds, but also by preventing strong post-acidification. Although no significant changes in the overall sensory quality were observed, slight color changes occurred after heat treatment, possibly due to the onset of caramelization processes. Furthermore, microstructural changes after post-pasteurization heat treatments were observed by means of cryo-scanning electron microscopy (cryo-SEM). Additionally, the texture changed with increasing thermal stress, for instance via the formation of white flakes or a decrease in inner gel strength, although such textural changes are presumably reversible. Overall, these studies demonstrated that RF heat treatment is a viable procedure for further product developments.
In conclusion, the research conducted during this doctoral thesis provides new perspectives on the sensory and/or textural properties of selected dairy products following heat treatment, by use of novel or traditional raw materials and after the application of electro-thermal heating technologies. In addition, odor compounds and previously unknown structural changes in dairy matrices were observed for the first time. Further, a sensitive method for monitoring lipid oxidation in its early stages was shown to be suitable for use on dairy matrices. Hence, the results of these investigations provide important insights into the quality aspects of milk products and offer opportunities for the industry to further develop safe dairy products with high nutritional values and palatable sensory properties.
The volatile and odorous fraction of human urine contains a wealth of information. Analysing this fraction can increase our knowledge on the metabolism and excretion processes of both endogenous and exogenous compounds. Changes in the profile of urine volatile constituents can arise from disease or hormonal variations, and can equally be influenced by diet. Although a wide range of non-volatile urinary compounds has been comprehensively characterised to date, the volatile and odorous fraction of human urine has received only little attention. Nevertheless, this compound fraction is important and informative in proving insights into metabolic and physiological processes, possibly with diagnostic relevance.
In the investigations reported herein, commonly-occurring odorants in human urine were identified. A preliminary screening of compounds was accomplished using odour extract dilution analysis and identification was achieved by one and two-dimensional gas chromatography-olfactometry/mass spectrometry (GC-MS and GC-GC-MS, respectively). Overall, a wide range of individual samples was analysed due to initial observations of pronounced inter-individual variations in the constituent compounds of different urine samples. Only two compounds, namely 2-acetyl-1-pyrroline and 3(methylthio)-propanal,
were commonly detected in all samples and number of compounds were detected in only a minority of samples. In total, 20 odorants were identified as common constituents in the urine samples analysed, ten of which are reported here for the first time as being urine constituents.
Glucuronide conjugates of odorants, which are important phase-II metabolites, were analysed in human urine in addition to the free odorants. This was achieved by the enzymatic cleavage of the glucuronides by β-glucuronidase-assays, with a subsequent analysis of the odorants released using the methods described above for the untreated urine. A number of additional odorants that were not found in the untreated samples were detected in the glucuronidase-treated urine samples. This included several phenolic substances, as well as other hydroxylated compounds and acids, to name just a few. Furthermore, a number of compounds that were identified in the untreated urine samples were similiarly detected in the hydrolysed samples, albeit with considerably higher flavour dilution (FD) factors. This observation provides evidence that the respective odorants are partly present as glucuronide conjugates in urine. In total, 34 odorants were identified in enzymatically hydrolysed urine, 15 of which are reported here for the first time.
Most odorants were present over a wide range of FD factors, both in the native as well as in the glucuronidase-treated individual urine samples. This further indicates that there are huge inter-individual variations in the concentrations of these odorants in urine. Following compound identification, quantification of both the free as well as glucuronidated compounds was made via stable isotope dilution assays (SIDA) in combination with GC-MS and GC-GC-MS. Furthermore, the concentration of creatinine was determined in the urine samples in order to present concentrations of the quantified odorants not only as absolute concentrations (in [μg/L]), but also normalised to the creatinine concentration (in [μg/mol creatinine]). Quantification of substances is essential to gain insights into the degree of glucuronide formation and the inter-individual differences therein, thereby enhancing our knowledge on the metabolism and excretion of odorants. This knowledge is essential for detecting potential variations and abnormalities resulting from peripheral influences, e.g. diet, environment, or disease.
In this study, odorants that were detected in untreated urine were found to be present over a broad range of concentrations, with median concentrations of 2-510 μg/mol creatinine. Moreover, the large inter-individual variations, as indicated from the odour extract dilution analyses, could be proven quantitatively. The most pronounced inter-individual variations in the untreated urine were observed for the substances (E)-β-damascenone and 4-ethylguaiacol, whose maximum concentrations exceeded their respective minimum concentrations by a factor of 90. Furthermore, a sex-related difference in concentration was observed for most compounds. This difference was most distinct for the compounds vanillin, indole and 4-vinylguaiacol in the untreated urine. Odorants in the glucuronidase-treated urine were found to be present at median concentrations of ~50-174,000 μg/mol creatinine. As observed for the untreated urine, there were also marked inter-individual differences in the odorant concentrations of the hydrolysed urine samples. The inter-individual concentration differences in the hydrolysed urine were most pronounced for the compounds 3-methylbutanoic acid and 4-vinylguaiacol, whose respective maximum concentrations were a factor 50 and 90 higher than their minimum concentrations. Differences between the hydrolysed urine of male and female subjects were not nearly as marked as in the untreated samples.
Comparing the concentrations of the substances in the untreated and in the glucuronidase-treated samples, the creatinine-normalised median concentrations were between a factor 3 and 650 higher in the hydrolysed samples compared to the untreated samples, depending on the compound in question. The greatest increase here was observed for the compounds guaiacol and indole. Little is known about the influence of diet on the excretion of volatile and odorous compounds and their metabolites in human urine. Thus, dietary-related changes in concentrations of selected volatile and odorous substances and their glucuronide conjugates in human urine were studied, as illustrated with coffee. Coffee is a highly popular beverage that is consumed in vast quantities worldwide. Although positive and negative effects of coffee on human health are frequently discussed, little is known about the metabolism and excretion of volatile and odorant constituents of coffee. As such, urine samples prior to and after the consumption of coffee were analysed and compared using the quantitation methods described above. Prior to investigating the metabolism and excretion of those substances, the volatile and odorant constituents of coffee were analysed. On the one hand, the analyses focussed on the volatiles (odorants) that are essential for the aroma of coffee, which were determined by aroma extract dilution analysis (AEDA) of the coffee beverage. On the other hand, the quantitatively dominating volatiles were determined, which was achieved via GC-MS analysis of coffee samples. Subsequently, a selection of these qualitatively and quantitatively relevant odorants and volatiles was quantified in urine. Again, quantification was accomplished in both untreated and glucuronidase-treated urine by SIDA. Here, concentrations prior to and after the consumption of coffee were compared. A series of compounds was found to be present as free substances or as glucuronide conjugates in urine at elevated concentrations after the intake of coffee. These results were consistent with sensory analyses, whereby orthonasal descriptions of the respective smells of the urine samples were used as indicators for specific urinary odorants. It was thereby observed that the majority of urine samples developed a coffee-like or roasty odour within only relatively short time intervals after the consumption of coffee, while none of the control samples that were obtained prior to coffee consumption were described with these attributes. As mentioned above, the concentrations of the selected odorants in urine increased soon after coffee intake. The same was true in untreated urine samples for all of the substances quantified based on the creatinine-normalised data. This increase was most distinct for the compound furfuryl alcohol, whose median concentration increased by a factor six after coffee consumption. The median creatinine-normalised concentrations also increased after coffee intake for all substances quantified in the hydrolysed samples compared to the untreated samples. The most pronounced increases in the hydrolysed samples were observed for the three methoxyphenols, namely 4-ethylguaiacol, 4-vinylguaiacol and guaiacol, whose median creatinine-normalised concentrations increased by factors 57, 37 and seven, respectively. In order to provide a measure of the proportion of the respective odorants and volatiles in the coffee beverage being excreted in urine relative to the coffee itself, the theoretical percentage of their excretion into urine was calculated. The sum of free compounds and their respective glucuronide conjugates was thereby taken into account and it was shown that a huge percentage of some of the consumed odorants were excreted within relatively short periods. One particularly striking example for this was the excretion of the compounds guaiacol and 4-ethylguaiacol, whereby on average about 60 % were excreted into urine as free as well glucuronidated compounds within 1.5 hours after coffee consumption. Again, inter-individual variations were quite pronounced.
In conclusion, the results of this work offer a valuable insight into physiological processes occurring in humans in terms of the metabolism and excretion of both endogenous and exogenous compounds. Furthermore, the present findings highlight the tremendous impact of the diet on the qualitative and quantitative composition of urinary volatiles. The results of the present work form the basis for further studies targeting deviations from the norm and elucidating variations caused by diet, environmental influences, diseases and other endogenous and exogenous factors.
The odorant composition of human body fluids, with the exception of axillary secretions, remains largely unexplored. This might be due to the fact that most studies dealing with body fluids have a medical, forensic or toxicological background and only a limited number of studies to date have focused exclusively on the identification of odorants. Odorants in body fluids might play an important role in inter-individual communication, especially in the mother-infant relationship, and also in the inter-cell communication, e.g. between sperm and egg cells. The overall aim of this research was therefore to explore the odorant composition of selected body fluids, namely amniotic fluid, human milk and follicular fluid. A small number of studies have investigated the volatile fraction of human milk. However, most of the substances identified so far are common dairy constituents and are therefore not unique in humans. One of the identified substances which might be potentially specific to milk of human origin was the ketosteroid androstenone (5α-androst-16-en-3-one). However, previous studies were not successful in unequivocally identifying or quantifying this steroid in human milk due to its presence at only trace concentrations in this medium. The present work therefore sought to develop a method to determine the concentration of this substance in human breast milk. This method included the purification of milk samples via gel permeation and column chromatography, followed by the analysis of these extracts by gas chromatography-mass spectrometry (GC-MS). The concentrations of androstenone, which were determined by means of a stable isotope dilution assay, were found to be in the range of ng/kg human milk.
Investigations were additionally carried out on human amniotic fluid. The most potent odorants in individual human amniotic fluid samples were determined via gas chromatography-olfactometry (GC-O) in combination with aroma extract dilution analysis (AEDA). Amongst the odorous compounds identified were the two ketosteroids androstenone and 4,16-androstadien-3-one, which are both characterised by a sweaty, urine-like odour, the compound sotolone, which has a savoury-like note, methional, which smells like cooked potatoes, 4-ethyloctanoic acid, with a goat-like odour, and the metallic-smelling tr-4, 5-epoxy-(E)-2-decenal. These substances were identified in nearly all individual amniotic fluid samples, albeit at varying concentrations, as was indicated by their different flavour dilution (FD) factors in different samples that were determined by AEDA.
Further investigations were carried out to determine whether the human-specific odorant precursors that have been previously identified in human axillary secretions, namely the glutamine conjugates of (E/Z)-3-methyl-2-hexenoic and 3-hydroxy-3-methylhexanoic acid, as well as the cysteinylglycine-S-conjugates of (R)/(S)-3-sulfanylhexan-1-ol and (R)/(S)-3-methyl-3-sulfanylhexan-1-ol, are also present in human milk, amniotic fluid and colostrum. Since these compounds were expected to be present at only trace concentrations, an enrichment and purification procedure for these compounds in human milk and colostrum was developed using ultra-performance liquid chromatography-mass spectrometry (UHPLCMS). This allowed for their quantification even in low-volume samples of the mentioned media. The developed method included fat extraction using hexane followed by a column chromatographic treatment of the resulting extracts. The following substances could be thereby identified in the majority of the human milk and colostrum samples: N-α-3-Hydroxy-3-methylhexanoyl-(L)-glutamine, N-α-3-methylhex-2-enoyl-(L)-glutamine, S-[1-(2-hydroxyethyl)-1-methylbutyl])-(L)-cysteinylglycine and S-[1-(2-hydroxyethyl)-1-butyl]-(L)-cysteinylglycine.
By contrast, only two of the glutamine conjugates were found to be present in amniotic fluid. The concentrations of both glutamine conjugates in human milk, colostrum and amniotic fluid were in the μg/kg range, while the concentrations of the cysteinylglycineconjugates quantified in human milk and colostrum were generally lower and in the ng/kg range. The absolute quantity, as well as their relative amounts, varied considerably between the individual body fluid samples. Supporting data was sought by analysing bovine milk for the presence of these compounds. However, neither glutamine nor cysteinylglycine-Sconjugates were detected in this medium, at least not via the approach employed here.
Finally, follicular fluid was investigated for its constituent odorants using GC-O. The presence of odorous compounds in this medium could have a potential chemotaxis effect on spermatozoa. Individual follicular fluid samples were found to contain up to 25 different odour-active substances, amongst which were a series of short, medium and long-chained fatty acids that were primarily characterised by sweaty, cheesy or wax-like odours and a variety of fatty-smelling aldehydes. Additionally, the two urine-like smelling ketosteroids androstenone and androstadienone, as well as the two furanones sotolone and furaneol, with savoury and caramel-like odour notes, were detected. To confirm the identity of these substances detected by GC-O, the chemical ionisation (CI) or electron ionisation (EI) mass spectra of most of these compounds after one or two-dimensional gas chromatographic separation (i.e. GC-MS or GC-GC-MS) were recorded and compared with those of reference compounds. This lead in most cases to unequivocal identification of the target substances.
In conclusion, the data of the present study demonstrate that there exists a vast variety of odour-active substances and odour precursors in human body fluids, some of which are potentially human-specific while others are more ubiquitous. Further, it could be shown that the majority of the substances identified in a single sample were commonly present in nearly all individual samples of the respective body fluid, thus representing a common profile of odour constituents, despite concentrations of these substances varying considerably in most cases between individuals. The results reported in this thesis ultimately provide a basis for future behavioural tests, as well as for cell assays, to investigate possible influences of odouractive substances in chemosensory communication processes.
Seeds of sweet lupins are a valuable source for the production of lupin protein concentrates and isolates due to their high protein content and their high nutritive value. Besides, lupin proteins exhibit excellent functional properties regarding protein solubility and emulsifying characteristics. However, the sensory properties of the lupin protein isolates and changes of these characteristics during storage impede their commercial availability. Therefore, the aim of the present work was to characterise impact factors on the functional properties of protein isolates during processing. Additionally, the odour-active compounds most likely responsible for the characteristic flavour of lupin flours and protein isolates were identified using high resolution gas chromatography-olfactometry and two-dimensional high resolution gas chromatography-mass spectrometry. Furthermore, de-oiling with organic solvents and supercritical CO2 were investigated as possibilities to improve the flavour of the isolates.
In a first experimental series, the influences of different numbers of acidic preextractions and protein extractions on protein recoveries were investigated. The results indicated that the protein recoveries were not only influenced by processing conditions (i.e. temperature, time, solid-to-liquid ratio, pH), but also by the particle size (flour, flakes), the protein content of the raw materials and the equipment used for protein isolation. Furthermore, the effects of different lupin species (L. albus cv. TypTop, L. luteus cv. Bornal) and lupin varieties of L. angustifolius L. on the chemical composition of flours and isolates as well as on the functional characteristics of the produced isolates were investigated. Diverging protein functionalities were obtained for protein isolates derived from different lupin species. Generally, the protein isolates of L. angustifolius L. revealed excellent emulsifying properties, whereas only moderate emulsifying characteristics were observed for the protein isolates derived from other species. In comparison to the LPI of the other species, the proteins of L. albus cv. TypTop formed viscous gels at a concentration of 15% (w/w). Thus, the lupin protein isolates with different functional profiles are suitable for various food applications, e.g. as emulsifiers in case of L. angustifolius L. and as a gelling agent in case of L. albus cv. TypTop. The diverging functionalities seem to be caused by the presence of different protein fractions with varying molecular weights as shown by one-dimensional polyacrylamide gel electrophoresis. However, a correlation between particular molecular weight protein fractions and specific functional properties was not possible in the present work. Altogether, greater variations were obtained between lupin species than between lupin varieties, but also environmental conditions during growth influenced dry matter recoveries during protein isolation. Due to its availability and the highest protein recovery of the investigated narrowleafed lupin varieties, L. angustifolius cv. Boregine was chosen for further sensory investigations. Thereby, the present thesis focussed on the identification of odouractive compounds in lupin flour and potential changes during storage and isolate production. The odour-active compounds, which were identified for the first time in lupin flour and protein isolates, comprised compounds of various chemical classes including aldehydes, ketones, carboxylic acids, 3-alkyl-2-methoxypyrazines, lactones and terpenes. According to the different chemical properties and the specific structural features of the identified aroma compounds different metabolic and reaction pathways leading to these substances can be assumed. These formation pathways most likely include lipoxygenase-mediated reactions, oxidation of fatty acids, degradation of amino acids as well as secondary plant metabolism.
Besides, the aroma profile of the protein isolate changed significantly to higher intensities of fatty, hay-like, green and oat flakes-like odour impressions in relation to the lupin flour samples. Consequently, higher FD-factors were obtained for saturated and unsaturated aldehydes in the isolate compared to lupin flour representing oxidation of fatty acids, which is most likely related to the activity of lipoxygenase. In order to improve the aroma of the LPI, lipid oxidation should be avoided, which might be accomplished by either enzyme inactivation or by de-oiling of lupin flakes. In the present thesis the effect of de-oiling by the application of various organic solvents as well as supercritical CO2 on the flavour and the functional properties of the isolates were investigated. Only de-oiling with ethanol and 2-propanol resulted in slightly decreased protein solubilities of the flakes, which subsequently resulted in lower protein recoveries. Furthermore, independent of the de-oiling process all isolates revealed excellent functional properties. The overall acceptance of the lupin protein isolates produced from supercritical CO2-extracted flakes was rated higher (5.2 to 5.5) than that of the protein isolates derived from organic solvent de-oiled (3.3 to 4.6) and from full-fat lupin flakes (2.9). Therefore, de-oiling with supercritical CO2 is a preferable alternative to de-oiling with organic solvents considering the protein recoveries, the functional properties of the isolates and the sensory properties.
The present thesis characterised the potential of narrow-leafed lupin varieties, in particular L. angustifolius cv. Boregine, as a valuable source for the efficient production of highly functional protein isolates. Additionally, the present work is a basis for the production of lupin protein isolates with improved flavour due to deoiling with supercritical CO2. However, the inactivation of enzymes to improve the flavour was not part of the present work and should be addressed in future.
The modern diet nowadays invariably includes a wide range of supplements that support special nutritional needs and optimise the daily intake of essential and/or scarcely present ingredients of our daily nutrition. The D-A-CH foundation of the German, Austrian, Liechtensteinian and Swiss Associations for Nutrition periodically publishes reference data for adequate nutrient intake. These data provide recommendations for daily energy intake, including requirements for optimal supply of vitamins, trace elements and minerals. Furthermore, particular requirements for different ages and specific situations such as pregnancy and lactation are considered. In terms of so-called lifestyle diseases such as cardiovascular diseases and certain cancers, the public foundations refer to a series of studies that have demonstrated close correlations between disease occurrence and common dietary lapses. Discoveries in the last 20 years especially have laid great stress upon polyunsaturated fatty acids (PUFAs) such as
eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) as being particularly advantageous for good health and longevity. These are synthesised from the nutritionally essential fatty acids (FA) α-linolenic acid of the n-3 FA series and linoleic acid of the n-6 FA series. A series of studies demonstrated the protective effects of PUFAs with regard to coronary heart diseases in adults and reported that a diet supplemented with PUFAs may decrease mortality from myocardial reinfarction and sudden death (Bucher, Hengstler, Schindler & Meier, 2002; Marckmann & Grønbæk, 1999). Apart from that, PUFAs have been shown to be beneficial for diverse aspects of foetal and neonatal development such as vision, cognition, and several others (Colombo, 2001; Fleith & Clandinin, 2005; Koletzko et al., 2001). These effects have been attributed in particular to n-3 FAs. With this in mind, the currently recommended balance of n-6 to n-3 FAs of 7 : 1 in the modern diet should be reevaluated to increase the n-3 proportion to a 5 : 1 ratio (DGE, 2004). Health benefits are routinely associated with consumption of fish, with fish oil being one of the major sources of n-3 long-chain polyunsaturated fatty acids (LC-PUFAs). The German Association of Nutrition (DGE; Deutsche Gesellschaft für Ernährung) recommends eating fish twice a week to increase the n-3 FA intake (DGE, 2004). The current recommendations in the United Kingdom are at least two portions a week of oily fish like salmon or mackerel to double the average consumption of n-3 LC-PUFA from about 0.1 g per day to about 0.2 g per day (SACN & COT, 2004).
Apart from regular fish consumption, the enrichment of common food products with the respective unsaturated FAs seems to be an applicable way to reach the recommended intake without radical changes of eating habits. In recent studies, possible formulations of functional foods have been proposed and introduced to the market, like fish oil enriched salad oils, mayonnaise, poultry meat, and cow’s and sheep’s milk, and n-3 FA supplemented infant formula (Jacobsen et al., 2001; Jimenez-Alvarez et al., 2008; Kitessa et al., 2004; Kitessa, Peake, Bencini & Williams, 2003; Kolanowski, 1999; Rymer & Ian Givens, 2010). However, despite the physiological impact of supplementation having been investigated in many studies, the potential deterioration of the sensory properties associated with the degradation of primary oxidation products of supplemented FAs poses a major problem. This represents a significant recent challenge for the food industry in order to maintain optimum food quality and to minimise consumer complaints.
Sabine Otto, PhD Thesis