Identification and Quantitation of New Glutamic Acid Derivatives in Soy Sauce by UPLC/MS/MS

Glutamic acid is an abundant amino acid that lends a characteristic umami taste to foods. In fermented foods, glutamic acid can be found as a free amino acid formed by proteolysis or as a non‐proteolytic derivative formed by microorganisms. The aim of the present study was to identify different structures of glutamic acid derivatives in a typical fermented protein‐based food product, soy sauce. An acidic fraction was prepared with anion‐exchange solid‐phase extraction (SPE) and analyzed by UPLC/MS/MS and UPLC/TOF‐MS. α‐Glutamyl, γ‐glutamyl, and pyroglutamyl dipeptides, as well as lactoyl amino acids, were identified in the acidic fraction of soy sauce. They were chemically synthesized for confirmation of their occurrence and quantified in the selected reaction monitoring (SRM) mode. Pyroglutamyl dipeptides accounted for 770 mg/kg of soy sauce, followed by lactoyl amino acids (135 mg/kg) and γ‐glutamyl dipeptides (70 mg/kg). In addition, N‐succinoylglutamic acid was identified for the first time in food as a minor compound in soy sauce (5 mg/kg).     

Modelling the simultaneous processes occurring during the BOD5 test using synthetic waters as a pattern

Synthetic waters imitating BOD₅ test conditions have been studied to model the progress curves of cell growth, organic matter consumption and oxygen demand. Glucose and glutamic acid were used as organic matter and Bacillus subtilis, Escherichia coli and Pseudomonas putida as microorganisms. The concentrations of all the products were measured simultaneously over time and were fitted to the Logistic and Monod models. Following this, the goodness of fit was analysed. Discrimination between models allowed us to conclude that the Logistic model has acceptable accuracy to describe the kinetic behaviour inside a BOD₅ bottle, while the extra parameter in the Monod model would not be statistically justified. Thus, the logistic parameters were determined under different conditions by non-linear regression and their biological meaning was interpreted.     

Exudation-reabsorption in a mycorrhizal fungus, the dynamic interface for interaction with soil and soil microorganisms

 The mycelium of Suillus bovinus slowly absorbed [U-¹⁴C]glucose and other tracers from droplets placed on the cords, translocated them to the peripheral hyphae and exuded them into fluid drops on the hyphal tips. The exudate was characterized by ¹H NMR spectroscopy and by sugar and amino acid analysis. The exuded compounds were mainly carbohydrates and peptides. Acetic acid and oxalic acid were also present in the exudate along with a number of unidentified compounds. Released ions (K, Na, Cl, P, Mg and Ca) were identified by X-ray microanalysis. The mycelium was shown to reabsorb up to 65% of the exuded ¹⁴C compounds in 2 days. Glucose, mannitol, glutamic acid (pH 3.2), and Rb⁺ (as well as other mineral ions) were all readily absorbed by the mycelium, while oxalic acid at pH 4.2 and glutamic acid at pH 6.5 were not. Exudation of fluid droplets on the surface of the hydrophobic mycorrhizal fungus S. bovinus may represent an ecophysiologically important function of the extramatrical hyphae, which provides an interface for interaction with the immediate hyphal environment and its other microorganisms where the peripheral hyphae exchange their photosynthetically derived products for nutrients to be used later by the pine host. We hypothesize that actively absorbed carbohydrates from the root are translocated to the peripheral hyphae along a concentration gradient of sugars and polyols by means of active translocation and diffusion in cell elements and by acropetal water transport in the cord vessels. Accepted 27 April 1999     

Organic and inorganic nitrogen source effects on the metabolism of Clostridium acetobytulicum

Summary The effects of organic and inorganic nitrogen combinations on cell growth, solvent production and nitrogen utilization by Clostridium acetobutylicum ATCC 824 was studied in batch fermentations. Fermentations in media with 10 mM glutamic acid, as the organic nitrogen source, and 0 mM to 10 mM ammonium chloride, as the inorganic nitrogen source had a solvent yield of 0.8 to 1.08 mmol solvent/mmol glucose used, with a slow fermentation rate (2 mmol solvent/l h^-1). When media contained 20 mM or 30 mM glutamic acid as well as 2.5 to 7.5 mM ammonium chloride the fermentation rate increased (5.5 mmol/l h^-1) while the solvent yield remained constant (0.86 to 0.96 mmol solvent/mmol glucose used). Total solvent production was higher in media containing 20 mM or 30 mM glutamic acid than with 10 mM glutamic acid.     

Biosynthesis and chemical reactions of poly(amino acid)s from microorganisms

The biosynthesis and chemical reactions of poly(amino acid)s produced by microorganisms are reviewed. A large amount of γ-poly(glutamic acid) (PGA) has been produced by Bacillus strains. ε-Polylysine (PL) has been produced by Streptomyces albulus. As a modification of PGA and PL, pH-sensitive hydrogels have been prepared by means of γ irradiation or the addition of a crosslinking agent to an aqueous solution of PGA and PL. Received: 4 September 1996 / Received revision: 27 January 1997 / Accepted: 28 January 1997     

Microbial production of poly-γ-glutamic acid

Poly-γ-glutamic acid (γ-PGA) is a natural, biodegradable and water-soluble biopolymer of glutamic acid. This review is focused on nonrecombinant microbial production of γ-PGA via fermentation processes. In view of its commercial importance, the emphasis is on l-glutamic acid independent producers (i.e. microorganisms that do not require feeding with the relatively expensive amino acid l-glutamic acid to produce γ-PGA), but glutamic acid dependent production is discussed for comparison. Strategies for improving production, reducing costs and using renewable feedstocks are discussed.     

Glutamic acid production byArthrobacter globiformis

Two hundred and fiftyArthrobacter strains were tested in a basal salts-glucose medium for their ability to produce glutamic acid; 50 strains produced small amounts of glutamic acid and alanine, as well as traces of other amino acids. Five biotin-dependent strains produced extraordinarily large amounts of glutamic acid. One of these, which was identified asA. globiformis, was selected for further study. Glutamic acid was only produced by this organism at biotin levels suboptimal for growth; maximal production (0.45 moles of glutamic acid per mole of glucose consumed) occurred at a biotin level of 10^–5 µg/ml. Other factors which markedly influenced glutamic acid production were temperature, (NH₄)₂SO₄ concentration, and pH of the growth medium.The taxonomy of glutamic acid-producing bacteria and the correlation between biotin deficiency and glutamic acid production are discussed.     

Solid state fermentation for L-glutamic acid production using Brevibacterium sp.

Summary Solid state fermentation system was used to cultivate Brevibacterium sp. on sugar cane bagasse impregnated with a medium containing glucose, urea, mineral salts and vitamins for producing L-glutamic acid. Maximum yields (80 mg glutamic acid per g dry bagasse with biomass and substrate - mg/gds) were obtained when bagasse of mixed particle size was moistened at 85–90 % mositure level with the medium containing 10 % glucose. This is the first report on the cultivation of Brevibacterium sp. in solid cultures for production of glutamic acid.     

Changes in cell volume,growth and respiration rate in response to hyperosmotic stress of NaCl,sucrose and glutamic acid in Brevibacterium lactofermentum and Corynebacterium glutamicum

Responses to hyperosmotic shock in the lysine-producing mutant Brevibacterium lactofermentum NRRL B-11470 and the wild-type Corynebacterium glutamicum ATCC 13032 were studied in batch and continuous culture. The strains were chosen because they are used in commercial production of lysine and glutamic acid. Both strains, as well as the wild type of B. lactofermentum, were able to grow at high osmotic stress, at least 3 osmol/kg, from NaCl, sucrose, glutamic acid or lysine. The specific growth rate decreased in a nearly linear fashion with increasing stress. However, low stress from glutamic acid stimulated growth, especially in the wild type of B. lactofermentum. Both cell and cytoplasmic volume decreased spontaneously after hyperosmotic shock and no plasmolysis was observed. Addition of betaine stimulated the subsequent increase in the volumes. The volumes decreased linearly with increasing stress, except at low glutamic acid stress, which caused a volume increase. The respiration rate, measured as CO₂ evolution, decreased immediately after shock, but increased again and stabilized within 2 h. Betaine stimulated the respiration recovery rate.     

The use of glutamic acid for the isolation and identification of Clostridium cochlearium and Cl. tetanomorphum

Summary A simple method has been developed to test the ability of anaerobes to ferment glutamic acid. This has been applied to a systematic study of seventeen strains of anaerobic spore-formers isolated from soil with the aid of enrichment cultures containing glutamic acid as the main source of carbon and energy. The twelve glutamic acid fermenting strains were found to be similar in their morphological and physiological characters except with respect to the utilization of glucose and pyruvate (with one strain also lactose, mannitol and salicin). These organisms are identified with two previously described species, Cl. cochlearium and Cl. tetanomorphum. The five glutamic acid negative strains differ distinctly from the positive strains also with respect to other characters. The ability to ferment glutamic acid therefore appears to be of taxonimic value.     

Optimization of cell growth and poly(glutamic acid) production in batch fermentation by Bacillus subtilis

Poly(glutamic acid) was produced maximally by Bacillus subtilis in batch fermentations at pH 7 and using glycerol at 20 g l^–1 in a glutamic acid/citric acid medium. Poly(glutamic acid) reached 23 g l^–1 after 30 h.     

La destinée des groupements aminés alpha et epsilon de la lysine dans le foie de rat in vitro

The fate of alpha and epsilon lysine amino groups has been explored in rat liver homogenate by means of l-lysine labelled selectively in the two positions. α-¹⁵NH₂ and ?-¹⁵NH₂ are rapidly incorporated into the amino group of glutamic acid and it seems at first that both transaminations occur simultaneously. But the reversible transfer of the amino group between α-aminoadipic acid and glutamic acid, determined by means of labelled α-aminoadipic acid, proceeds swiftly, and the incorporation of α-¹⁵NH₂ from the corresponding labelled lysine in glutamic acid may be easily explained by ?-transamination and saccharopine formation. The direct transamination of the α-amino group of l-lysine is most improbable and might be limited to some microorganisms and to ?-N-substituted lysine derivatives.     

Enzymatic synthesis of theanine from glutamic acid γ-methyl ester and ethylamine by immobilized Escherichia coli cells with γ-glutamyltranspeptidase activity

Theanine (γ-glutamylethylamide) is the main amino acid component in green tea. The demand for theanine in the food and pharmaceutical industries continues to increase because of its special flavour and multiple physiological effects. In this research, an improved method for enzymatic theanine synthesis is reported. An economical substrate, glutamic acid γ-methyl ester, was used in the synthesis catalyzed by immobilized Escherichia coli cells with γ-glutamyltranspeptidase (GGT) activity. The results show that GGT activity with glutamic acid γ-methyl ester as substrate was about 1.2-folds higher than that with glutamine as substrate. Reaction conditions were optimized by using 300 mmol/l glutamic acid γ-methyl ester, 3,000 mmol/l ethylamine, and 0.1 g/ml of immobilized GGT cells at pH 10 and 50°C. Under these conditions, the immobilized cells were continuously used ten times, yielding an average glutamic acid γ-methyl ester to theanine conversion rate of 69.3%. Bead activity did not change significantly the first six times they were used, and the average conversion rate during the first six instances was 87.2%. The immobilized cells exhibited favourable operational stability.     

On the Manner of Cyclization of <Emphasis Type="Italic">N</Emphasis>-Acylated Aspartic and Glutamic Acid Derivatives

Abstract  

When synthesizing arylpiperazine library modified with N-acylated amino acid derivatives (e.g., cyclized aspartic acid, cyclized glutamic acid, proline) we wished to rapidly determine the way of cyclization of N-acylated glutamic acid derivatives. During concomitant cleavage and cyclization two alternative routes were possible—either formation of six-member imide (glutarimide) or five-member lactam. Application of MS/MS and ¹H NMR method allowed us to establish that cyclization of N-acylated glutamic acid derivatives preceded to lactams—N-acylated pyroglutamic acid derivatives.     

Synthesis of Amino Acids from Hydrocarbons

Isolation of microorganisms capable of synthesising amino acids, utilizing hydrocarbons, has been reported. These microorganisms were isolated from soil samples by selective culture techniques. 91 strains were found capable of producing amino acids in the broth. Different amino acids and their maximum yield obtained were glutamic acid 160 mg/1; leucine 90.0 mg/1; isoleucine 40.0 mg/1; valine 105.0 mg/1; methionine 25.0 mg/1; tryptophan 2.5 mg/1; arginine 70.0 mg/1; and histidine 10.0 mg/1.     

Variations in the amino acid composition of cyanophycin in the cyanobacterium Synechocystis sp. PCC 6308 as a function of growth conditions

Gas chromatography-mass spectrometry studies of the nitrogen isotopic composition of the N-trifluoroacetyl n-butyl ester derivatives of the amino acids from isolated hydrolyzed cyanophycin from ¹⁵N-enriched cells led to two major findings: (1) the amino acid composition of this granular polypeptide, isolated using procedures optimized for extracting and purifying cyanophycin from cells in the stationary growth phase, varied with the culture growth condition; (2) the rate of incorporation of exogenous nitrate differed for each nitrogen atom of the amino acid constituents of cyanophycin or cyanophycin-like polypeptide. Arginine and aspartic acid were the principle components of cyanophycin isolated from exponentially growing cells and from light-limited stationary phase cells, with glutamic acid as an additional minor component. The cyanophycin-like polypeptide from nitrogen-limited cells contained only aspartic and glutamic acids, but no arginine. The glutamic acid content decreased and arginine content increased as nitrate was provided to nitrogen-limited cells. These cells rapidly incorporated nitrate at different rates at each cyanophycin nitrogen site: guanidino nitrogens of arginine>aspartic acid >-amino nitrogen of arginine>glutamic acid. Little media-derived nitrogen was incorporated into cyanophycin of exponentially growing cells during one cellular doubling time.Abbreviations asp-TAB, glu-TAB, arg-TAB N-Trifluoroacetyl n-butyl ester derivatives of aspartic acid, glutamic acid and arginine, respectively - CAP chloramphenicol - CF correction factor - TFAA Trifluoroacetic anhydride - MBTFA N-Methyl-bis-trifluoroacetamide     

Immobilization of Brevibacterium flavum cells on collagen for the production of glutamic acid in a recycle reactor

In this study, live cells of Brevibacterium flavum were immobilized for the production of glutamic acid. The reason for such a choice was that glutamic acid fermentation is an extensively studied fermentation and one which requires the viability of entire cellular faculties for the acid production. Brevibacterium flavum was chosen because it is an industrially used bacterium, and is very potent via a vis glutamic acid production. Studies were performed to find aeration and agitation conditions for optimal growth and glutamic acid productivity. Experiments were also done to find the optimum harvesting time. The cell activity peaks during the run of fermentation, and the time at which the peak occurs, was found. Conventional methods for immobilizing the cells on collagen were found to be lacking. The pH and drying were the two main reasons for loss of viability of the cells; the latter being more important. A modified immobilization procedure has been devised, which can immobilize live cells at any given pH and ionic strength, in contrast to the conventional method which requires the pH to be above 11 or below 3. This new method involves dialysis of collagen in suitable dialysis bags against water at pH7 (or buffer at any desired pH). The dialysed collagen blended at 20,000 rpm, resulted in a very smooth dispersion, unnoticeably different from collagen dispersion prepared at pH 11. The dispersed collagen was then cast and dried at an elevated temperature, and high air flow rate over the cast membrane, decreasing the time of drying from 6–8 hr ( in the conventional method) to 1.5–2 hr. The membrane has been tested for glutamic acid producing capabilities in a column reactor with the membrane spirally wound. The reactor has been operated under continuous conditions for 5–10 days with stable activities.     

NMR Spectra of Glutamic Acid-containing Dipeptides in Relation to Sequence Determination

It is possible to determine the sequence of a dipeptide containing glutamic acid as a constituent and also to decide whether the glutamyl bond is α or γ when glutamic acid is the N-terminal component by measuring the NMR spectra of the peptide in acidic, aqueous and basic solutions.     

Chemically Defined Media for the Cultivation of Naegleria: Pathogenic and High Temperature Tolerant Species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCCr?30100, ATCCr?30863, and ATCCr?30896) and two strains of N. lovaniensis (ATCCr?30467 and ATCCr?30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCCr?30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysine for ATCCr?30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCCr?30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCCr?30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B₁₂, nicotinamide, and Ca pantothenate) was required.     

Cycling treatment of anaerobic and aerobic incubation increases the content of γ-aminobutyric acid in tea shoots

Summary. γ-Aminobutyric acid (GABA), a hypotensive compound, is formed from glutamic acid under anaerobic condition in tea shoots. Glutamic acid was exhausted in the first three hours of anaerobic incubation and the increase of GABA stopped. After that, when tea shoots were released under aerobic condition, glutamic acid reproduced rapidly. After one hour of aerobic incubation, tea shoots were given three hours of anaerobic incubation again and then accumulated glutamic acid changed to GABA. The content of GABA increased much more than usual anaerobic incubation. GABA was more in the tea stem than in the leaf. Received January 4, 2000 Accepted March 1, 2000