SYNTHETIC FLAVORS: A NEW APPROACH TO EFFICIENCY AND SAFETY

More than 1000 volatile synthetic flavor compounds are GRASfood additives. Comprehensive toxicological testing to assuretheir safety would be prohibitive in cost. Instead, their numbermight be logically reduced to a few dozen by applying thesecriteria: (1) Measurement of normal odor detection threshold,to eliminate weakly odorous compounds. (2) Assessment with specificallyodor blind persons, to delete compounds having very similarodors. As an example, 12 lower fatty acids on the permittedlist range over 2700-fold in their thresholds, yet they belongto the same sweat-like primary odor. Hence they could be replacedby just one among their number, isovaleric acid, which has thestrongest and sweatiest odor. Analogous reasoning is appliedto three new primary odors: the spermous odor of 1-pyrroline,the fishy odor of trimethylamine, and the malty odor of isobutyraldehyde.In each case the number and quantity of flavor additives requiredin food could be substantially reduced by using the more effcientprimary odors. This should result in improved safety for theconsumer.     

Foot odor due to microbial metabolism and its control

To characterize foot odor, we analyzed its components by sensory tests, isolated microorganisms that produce it, and evaluated the mechanism of the occurrence of foot odor. As a result, foot odor was found to be derived from isovaleric acid, which is produced when Staphylococcus epidermidis, a resident species of the normal cutaneous microbial flora, degrades leucine present in sweat. In addition, Bacillus subtilis was detected in the plantar skin of subjects with strong foot odor, and this species was shown to be closely associated with increased foot odor. Therefore, we screened various naturally occurring substances and fragrant agents that inhibit microbial production of foot odor without disturbing the normal microbial flora of the human skin. As a result, we identified citral, citronellal, and geraniol as fragrant agents that inhibit the generation of isovaleric acid at low concentrations.     

Integrated separation process for isolation and purification of biosuccinic acid

Biotechnologically produced succinic acid has the potential to displace maleic acid and its uses. Therefore, it is of high interest for the chemical, pharmaceutical, and food industry.In addition to optimized production strains and fermentation processes, an efficient separation of succinic acid from the aqueous fermentation broth is indispensable to compete with the current petrochemical production of succinic acid. Isolation and purification of succinic acid from an Escherichia coli fermentation broth were studied with two amine-based reactive extraction systems: (i) trihexylamine in 1-octanol and (ii) diisooctylamine and dihexylamine in a mixture of 1-octanol and 1-hexanol. Back extraction of succinic acid from the organic phase was carried out using an aqueous trimethylamine solution. The trimethylammonium succinate generated after back extraction was split with an evaporation-based crystallization.The focus was on process integration, for example, reuse of the applied amines for extraction and back extraction. It was shown that the maximum trimethylamine concentration for back extraction should not exceed the stoichiometric amount (2 mol trimethylamine/mol the succinic acid in the organic phase) to ensure maximal extraction yields with the reused organic phase in subsequent extractions. Moreover, mixer-settler extraction and back extraction of succinic acid were scaled up from the milliliter- to the liter-scale making use of liquid–liquid centrifuges. The overall yield was 83.5% of the succinic acid from thefermentation supernatant. The final purity of the succinic acid crystals was 99.5%. Organic phase and amines can easily be recycled and reused.     

Individual differences in thresholds and quality reports of human subjects to various odors

Sixty-three subjects were selected on the basis of their relativeability (n = 30) or inability (n = 33) to detect the intenseurine–sweaty odor of the diastereoisomeric ketone, cis-4-(4'-t-butylcyclohexyl)-4-methyl-2-pentanone (pemenone). Absolute thresholds were determined,and quality reports and hedonic ratings were obtained for near-thresholdconcentrations of pemenone and five other odorous compounds.Several of these compounds were selected because large individualdifferences in sensitivity (specific anosmias) were known toexist. A principal compound analysis of threshold concentrationsindicated that three orthogonal factors well described the relationshipsbetween sensitivities to the compounds. Threshold concentrationsof pemenone and androstenone were highly correlated and thefirst factor was defined by relationships between those odorantsand isovaleric acid, all modally putrid-smelling compounds.Subjects relatively osmic for pemenone generally reported aputrid odor for pemenone, but anosmics reported mostly otherqualities. These relationships between relative sensitivityand the quality reports elicited by the different compoundsare consistent with a multiple-profile model of odor qualityperception. That model postulates that most odor molecules interactwith more than one perceptual channel (receptor process) andthat any individual alteration in the relative specificity ordeletion in the number of such receptor processes could alterthe pattern of interaction and thus should give rise to alterationsin the quality or intensity of the resulting odor perception.     

Food-borne amines and amides as potential precursors of endogenous carcinogens

This paper reviews the experimental results of our research in the past several years and other related papers that have been directed toward the occurrence, biotransformation and epidemiological significance of carcinogenic N-nitroso compounds in biosphere. Endogenous carcinogens are a group of cancer-causing compounds produced in vivo from harmless precursors. This category has been exemplified by the well-known carcinogens, N-nitroso compounds. The significance of naturally occurring amines and amides as precursors of carcinogenic N-nitroso compounds in vivo and their implication in the incidence of human cancer have been investigated and emphasized. Extremely high levels of trimethylamine-N-oxide and dimethylamine were detected in squids and other seafoods. More than 90% of trimethylamine-N-oxide were converted to dimethylamine and trimethylamine on pyrolysis. Low levels of dimethylamine and methylamine were also detected in the fermented soybean products, wines and sauces. Both dimethylamine and trimethylamine are excellent precursors of dimethylnitrosamine. Several naturally occurring aromatic amines especially 2-carboline derivatives such as harman, norharman, harmaline, harmalol, harmine and harmol are mutagenic and become more mutagenic to Salmonella typhimurium after nitrosation. Appreciable amounts of piperidine were detected in the popular spice white and black pepper powders. Under acidic condition, piperidine reacts readily with nitrite to form carcinogenic N-nitroso-piperidine. N-Nitrosophenacetin was formed from the reaction of nitrite with the amide drug phenacetin. This new compound showed strong mutagenicity to Salmonella typhimurium and Sarcina lutea and strong teratogenic activity to Leghorn chicken embryos. Studies have shown that the majority of N-nitroso compounds in the body come from in vivo conversion. Most investigators believe that this endogenous pool of N-nitroso compounds may prove to be a major exposure route in man. The presence of naturally occurring amines and amides in the diet then becomes one of the crucial limiting steps in the formation of endogenous N-nitroso compounds in vivo.     

Studies on the role of beta-cell metabolism in the insulinotropic effect of alpha-ketoisocaproic acid.

alpha-Ketoisocaproic acid has been shown to be a potent insulin secretagogue but the mechanism has not been elucidated. To define the role of beta-cell metabolism in the insulinotropic activity of alpha-ketoisocaproic acid the utilization of glucose and the oxidation of alpha-ketoisocaproic and isovaleric acid by incubated islets of obese hyperglycemic mice were measured. Glucose metabolism was never enhanced by alpha-ketoisocaproic acid. The same 14CO2 amounts were released from the non-secretagogue [1-14C]isovaleric acid (10 mM) or from alpha-keto[2-14C]isocaproic acid (5--20 mM). Pyruvate (20 mM) did not inhibit alpha-ketoisocaproic acid-induced insulin secretion in spite of reduction of decarboxylation of alpha-ketoisocaproic acid by more than 40%. The results indicate that stimulated insulin release in response to alpha-ketoisocaproic acid is not mediated by an indirect increase in glucose metabolism and further suggest that isovaleryl-CoA and following CoA-esters in alpha-ketoisocaproic acid degradation are not likely recognized as signals. The possibility, however, remains that enhanced intramitochondrial production of reducing equivalents elicits insulin secretion.     

Identification of new odoriferous compounds in human axillary sweat

3-Hydroxy-3-methylhexanoic acid (1) and the 3-sulfanylalkan-1-ols 2-5 were identified to contribute to the odor of human axillary sweat. Quantitative analyses of axillary sweat extracts from 50 healthy men showed an unambiguous correlation between the detected levels of 1 and the intensity of the axillary odor. Chiral-GC analyses revealed 1 to be a 72:28 mixture of the (S)/(R)-isomers. Optically pure (S)-1 (>97% ee) emanated a strong spicy note, which recalled typical axillary odors. 3-Methyl-3-sulfanylhexan-1-ol (2), the enantiomeric ratio of which equaled that of 1, was present in greater quantity than any of the other 3-sulfanylalkanols. Optically pure (S)-2 (>97% ee) had a strong meaty, fruity note, also reminiscent of axillary odor. The compounds identified, in particular (S)-1 and (S)-2, contribute significantly to the olfactory impression of human axillary odor.     

Effect of glucose,maltose, soluble starch,and CO<Subscript>2</Subscript> on the growth of the hyperthermophilic archaeon<Emphasis Type="Italic"> Pyrococcus furiosus</Emphasis>

The hyperthermophilic archaeon Pyrococcus furiosus was cultivated in batch and continuous fermentations on different carbon substrates. The cultivation of P furiosus on soluble starch as the only carbon source resulted in cell densities three times higher than in cultivations on maltose, 1.06 x 10(10) cells/ml compared to 3.4 x 10(9) cells/ml. The yield coefficient, Y(x/y) = 0.12 g/g, and the growth rate, mu = 0.33 h(-1), were almost equal on soluble starch and on maltose, but on glucose no growth could be detected. An inhibitory effect of glucose, when added to other carbon substrates, also could not be found. Isobutyric and isovaleric acid were detected as novel metabolites produced by P. furiosus. Inhibitory effects of these acids, as well as of the well-known products acetic acid, propionic acid, and alanine, could be precluded. Concentrations of 10% CO2 in the gas supply respective to the exhaust gas enhanced the growth of P furiosus significantly. The maximum cell number was two orders of magnitude higher than was observed with pure nitrogen. Further increase of the CO2 concentration up to 100% had no significant effect on the growth of P. furiosus.     

The role of learning in adult food location by the larval parasitoid,Microplitis croceipes (Hymenoptera: Braconidae)

Wind tunnel experiments were conducted to determine roles of odor learning in food foraging of the larval parasitoid,Microplitis croceipes (Hymenoptera: Braconidae). Females that had neither fed on sucrose water nor experienced any odor and females that had experienced an odor without feeding failed to respond to any odors in a wind tunnel. Most of the females that had fed without an odor also did not respond to odors. However, most of the females that had experienced an odor during feeding on sucrose water flew to the odor. These results indicate that when females experience an odor during feeding, they learn to associate the odor with food and subsequently respond to the odor. As age of females increased, their response to an experienced odor increased, peaked 2 to 5 days after emergence, and then decreased. With an increasing number of odor experiences while feeding, accuracy of females choosing the experienced odor increased. Females that experienced an odor while feeding three to five times chose the experienced odor 90% of the time. When females experienced an odor while feeding five times, the memory of food associated odor lasted at least 2 days. When they experienced food with two odors successively, they could memorize both odors, and multiple experiences did not cause memory interference. Even when females had learned a food-associated odor, their response to the learned odor ceased after several visits on patches containing the odor but no food. Such negative experience may cause switching of food searching to new odors by females.     

Synthesis and circular dichroism study of the human salivary proline-rich protein IB7.

The solid phase synthesis of a 59 amino acid human salivary protein IB7 has been accomplished using Fmoc strategy. Because the protein contains 25 proline, 13 glycine and 9 glutamine residues the coupling time, piperidine delivery and acetic anhydride reaction time were increased. Yield after HPLC purification was 35%. Circular dichroism studies revealed that about one third of IB7 residues adopted a type II helix secondary structure, as found in collagen helices. The rest of the sequence adopts a random coil secondary structure.     

Response of Cebus apella to Foods Flavored with Familiar or Novel Odor

What cues support social influences on food preference in tufted capuchins? Although vision is important for food discrimination, we hypothesized that olfactory cues might also be involved. In Experiment 1, we assessed whether semolina flavored with a novel odor and eaten by a demonstrator, elicits more interest than semolina flavored with a familiar odor and eaten by a demonstrator, and to what extent the observer's interest towards the demonstrator was elicited by the food itself or by the odor impregnating the demonstrator's oral area. In Experiment 2, we investigated whether having encountered a novel odor in a social context increases the observer's subsequent consumption of semolina carrying this odor versus semolina carrying another novel odor previously encountered in a non-social context. We tested 15 demonstrator-observer pairs. Eight observers were offspring of the demonstrator; 7 observers were not offspring of the demonstrator. Offspring (but not non-offspring) expressed interest towards the demonstrator's food significantly more when the odor was novel than when it was familiar. Offspring (but not non-offspring) were more interested when the demonstrator's food was present than when only its odor was available. Finally, having encountered the novel odor in the social context did not lead to greater consumption. Our findings demonstrate that in closely bonded pairs, foods carrying novel odors elicit interest, though it is prompted mainly by the food itself. In contrast with other macrosmatic mammalian species, capuchin consumption of a food whose odor was previously encountered in a social context was not greater than that of a food whose odor was previously encountered when alone.     

Human safety controversies surrounding nitrate and nitrite in the diet

Nitrate and nitrite are part of the human diet as nutrients in many vegetables and part of food preservation systems. In the 1950s and 1960s the potential for formation of nitrosamines in food was discovered and it ignited a debate about the safety of ingested nitrite which ultimately focused on cured meats. Nitrate impurities in salt used in the drying of meat in ancient times resulted in improved protection from spoilage during storage. This evolved into their deliberate modern use as curing ingredient responsible for 'fixing' the characteristic color associated with cured meats, creating a unique flavor profile, controlling the oxidation of lipids, and serving as an effective antimicrobial. Several critical reports and comprehensive reviews reporting weak associations and equivocal evidence of nitrite human health safety have fostered concerns and debate among scientists, regulators, press, consumer groups, and consumers. Despite periodic controversy regarding human health concerns from nitrite consumption, a building base of scientific evidence about nitrate, nitrite, heme chemistry, and the overall metabolism of nitrogen oxides in humans has and continues to affirm the general safety of nitrate/nitrite in human health. As nitrite based therapeutics emerge, it is important to consider the past controversies and also understand the beneficial role in the human diet.     

Timberol? Inhibits TAAR5-Mediated Responses to Trimethylamine and Influences the Olfactory Threshold in Humans

In mice, trace amine-associated receptors (TAARs) are interspersed in the olfactory epithelium and constitute a chemosensory subsystem that is highly specific for detecting volatile amines. Humans possess six putative functional TAAR genes. Human TAAR5 (hTAAR5) is highly expressed in the olfactory mucosa and was shown to be specifically activated by trimethylamine. In this study, we were challenged to uncover an effective blocker substance for trimethylamine-induced hTAAR5 activation. To monitor blocking effects, we recombinantly expressed hTAAR5 and employed a commonly used Cre-luciferase reporter gene assay. Among all tested potential blocker substances, Timberol®, an amber-woody fragrance, is able to inhibit the trimethylamine-induced hTAAR5 activation up to 96%. Moreover, human psychophysical data showed that the presence of Timberol® increases the olfactory detection threshold for the characteristic fishy odor of trimethylamine by almost one order of magnitude. In conclusion, our results show that among tested receptors Timberol® is a specific and potent antagonist for the hTAAR5-mediated response to trimethylamine in a heterologous system. Furthermore, our data concerning the observed shift of the olfactory detection threshold in vivo implicate that hTAAR5 or other receptors that may be inhibited by Timberol® could be involved in the high affinity olfactory perception of trimethylamine in humans.     

Isolation and identification of an alkyldiacylglycerol containing isovaleric acid

We have isolated and identified a unique subclass of alkyldiacylglycerols from the pink portion of the harderian gland of the New Zealand white rabbit. Using chemical, enzymic, chromatographic, and physical procedures, the lipid class has been identified as 1-alkyldiacylglycerol containing 1 mole of isovaleric acid. More than 50% of the O-alkyl moieties consist of 16:0 and 18:0 carbon chains, whereas the other major O-alkyl moieties are 15:0 and 17:0 branched chains ( approximately 30%). The long-chain acyl groups of the alkyldiacylglycerol subclass consist primarily of saturated fatty acids (60% 16:0 and 30% 18:0) and a small amount of branched-chain fatty acids ( approximately 5%), whereas the 3-position appears to be occupied by isovaleric acid.     

From musk to body odor: Decoding olfaction through genetic variation

The olfactory system combines input from multiple receptor types to represent odor information, but there are few explicit examples relating olfactory receptor (OR) activity patterns to odor perception. To uncover these relationships, we performed genome-wide scans on odor-perception phenotypes for ten odors in 1000 Han Chinese and validated results for six of these odors in an ethnically diverse population (n = 364). In both populations, consistent with previous studies, we replicated three previously reported associations (β-ionone/OR5A1, androstenone/OR7D4, cis-3-hexen-1-ol/OR2J3 LD-band), but not for odors containing aldehydes, suggesting that olfactory phenotype/genotype studies are robust across populations. Two novel associations between an OR and odor perception contribute to our understanding of olfactory coding. First, we found a SNP in OR51B2 that associated with trans-3-methyl-2-hexenoic acid, a key component of human underarm odor. Second, we found two linked SNPs associated with the musk Galaxolide in a novel musk receptor, OR4D6, which is also the first human OR shown to drive specific anosmia to a musk compound. We noticed that SNPs detected for odor intensity were enriched with amino acid substitutions, implying functional changes of odor receptors. Furthermore, we also found that the derived alleles of the SNPs tend to be associated with reduced odor intensity, supporting the hypothesis that the primate olfactory gene repertoire has degenerated over time. This study provides information about coding for human body odor, and gives us insight into broader mechanisms of olfactory coding, such as how differential OR activation can converge on a similar percept.     

Studies on the role of β-cell metabolism in the insulinotropic effect of α-ketoisocaproic acid

α-Ketoisocaproic acid has been shown to be apotent insulin secretagogue but the mechanism has not been elucidated. To define the role of β-cell metabolism in the insulinotropic activity of α-ketoisocaproic acid the utilization of glucose and the oxidation of α-ketoisocaproic and isovaleric acid by incubated islets of obese hyperglycemic mice were measured.Glucose metabolism was never enhanced by α-ketoisocaproic acid. The same ¹⁴CO₂ amounts were released from the non-secretagogue [1-¹⁴C]isovaleric acid (10 mM) or from α-keto [2-¹⁴C]isocaproic acid (5–20 mM). Pyruvate (20 mM) did not inhibit α-ketoisocaproic acid-induced insulin secretion in spite of reduction of decarboxylation of α-ketoisocaproic acid by more than 40%.The results indicate that stimulated insulin release in response to α-ketoisocaproic acid is not mediated by an indirect increase in glucose metabolism and further suggest that isovaleryl-CoA and following CoA-esters in α-ketoisocaproic acid degradation are not likely recognized as signals. The possibility, however, remains that enhanced intramitochondrial production of reducing equivalents elicits insulin secretion.     

Transforming growth factor-beta down-regulates apolipoprotein M in HepG2 cells

Apolipoprotein M (apoM) is a novel apolipoprotein presented mostly in high-density lipoprotein (HDL) in human plasma, and is exclusively expressed in liver and in kidney. The pathophysiological function of apoM has not yet been elucidated. Apolipoprotein B (apoB), the characteristic apolipoprotein of low-density lipoprotein (LDL), is like apoM, a very hydrophobic protein, and thereafter they both must co-circulate with lipoprotein particles in plasma. The cytokine, transforming growth factor-beta (TGF-beta), has been shown to decreased apoB secretion in HepG2 cells, and we hypothesized that TGF-beta may have the same effects on apoM expression in HepG2 cells. In the present study, we used real-time RT-PCR to analyze apoM and apoB mRNA levels during administration of TGF-beta, as well as TGF-alpha, epidermal growth factor (EGF) and hepatic growth factor (HGF). TGF-beta significantly inhibited both apoM and apoB mRNA expression in HepG2 cells. The inhibitory effects of TGF-beta were dose-dependent, i.e. 1 ng/ml of TGF-beta decreased apoM mRNA levels by 30%, and 10 or 100 ng/ml of TGF-beta decreased apoM mRNA levels more than 65%. The effect of TGF-beta on apoB mRNA expression was slightly weaker than that of apoM, with a maximum effect at 10 or 100 ng/ml TGF-beta where apoB mRNA levels decreased about 55%. The inhibitory effects of TGF-beta on apoM and apoB mRNA levels also increased with increasing incubation time, where the maximum effect was obtained at 24 h. Moreover TGF-alpha, EGF and HGF all decreased both apoM and apoB mRNA levels, but to a less extent than TGF-beta. Further, all four cytokines had more pronounced effects on apoM mRNA expression than apoB mRNA expression. The present study suggested that apoM, like apoB, may be involved in the hepatic lipoprotein assembly in vivo.     

Amino acid and lactate catabolism in trimethylamine oxide respiration of Alteromonas putrefaciens NCMB 1735

The nonfermentative Alteromonas putrefaciens NCMB 1735 grew anaerobically in defined media with trimethylamine oxide as external electron acceptor. All amino acids tested, except taurine and those with a cyclic or aromatic side chain, were utilized during trimethylamine oxide-dependent anaerobic growth. Lactate, serine, and cysteine (which are easily converted to pyruvate) and glutamate and aspartate (which are easily converted to tricarboxylic acid cycle intermediates) were metabolized at the fastest rate. Growth with lactate as growth-limiting substrate gave rise to the formation of 40 mol% acetate, whereas serine and cysteine were nearly completely oxidized to CO2. Molar growth yields with the latter substrates were the same and were 50% higher than with lactate. This showed that more ATP was formed when acetyl coenzyme A entered the tricarboxylic acid cycle than when it was converted via acetyl phosphate to acetate. Also, growth with formate as substrate indicated that the reduction of trimethylamine oxide to trimethylamine was coupled with energy conservation by a respiratory mechanism.