Free Tryptophan Content Research Articles

Protease‐assisted process for tryptophan release from pumpkin ( Cucurbita maxima ) seed protein extracts

Squash pumpkin (Cucurbita maxima) seeds are currently underused food ingredient, despite being a high tryptophan-rich protein source. In this study, we obtained and characterized a protein hydrolysate from defatted C. maxima seed meal, focusing on the effect of the used protease on tryptophan release. Protein hydrolysates were prepared using five different proteases and compared regarding the degree of hydrolysis (DH), free tryptophan content, hydrolysis kinetics, and potential antioxidant activity over reaction time. Protein hydrolysis using Flavourzyme resulted in the highest DH (35.6%), and free tryptophan concentration (337.9 mg/100 g), whereas hydrolysates obtained using Chymotrypsin showed the highest in vitro antioxidant activity (oxygen radical absorbance capacity [ORAC]) (371.1 mmol TE/100 g DM of protein extract). Our results show that protein hydrolysates from C. maxima seeds are a promising source of free tryptophan, with high antioxidant activity, supporting its potential as a food ingredient or for nutraceutical applications. Practical application C. maxima seeds are a promising source of free tryptophan, and its functionalization through hydrolysis showed a significant effect increasing tryptophan release and antioxidant activity, increasing its potential for ingredient or nutraceutical applications.

Milk fermentation affects amino acid and fatty acid profile of mare milk from Polish Coldblood mares

The aim of this work was to demonstrate the influence of mare milk fermentation type on the amino acid profile, especially the content of tryptophan, water-soluble vitamins and magnesium. Additionally, the atherogenic, thrombogenic and hypercholesterolaemic fatty acid index was analysed. The dominant amino acids of mare milk were glutamic acid, leucine, lysine, and aspartic acid. Fermentation of mares' milk did not affect the riboflavin, cobalamin, and magnesium content. The type of lactic acid bacteria used had no effect on the free tryptophan content of fermented milk. The free tryptophan content of fermented mares' milk was 0.062 mg 100 g−1 (Δ22% compared with unfermented milk). The sum of saturated fatty acid concentration was lower in fermented than in the unfermented mares' milk. Atherogenic index was lower in mare milk than in fermented mares’ milk. The values of fatty acids with a hypocholesterolaemic effect and hypercholesterolaemic fatty acids decreased in fermented milk.

Dietary sources of melatonin and benefits from production of high melatonin pasteurized milk.

Melatonin is a neurohormone that regulates circadian rhythms in the human body. It can also be taken to alleviate insomnia and sleep disorders. Pasteurized milk is a good source of nutrients and some bioactive compounds. Recently, the growing trend of healthy foods has resulted in higher competition with regard to milk products. Functional milk has been developed with higher bioactive compounds to respond to consumer demand. High melatonin pasteurized milk was developed using selected edible grains and mulberry leaves to fortify melatonin in pasteurized milk. Melatonin and free tryptophan of fourteen edible grains and mulberry leaves were determined using HPLC-FD. Highest melatonin concentrations were observed in white sesame, sunflower and soybean (75.24, 67.45 and 56.49ng/g dry weight (dw), with highest concentrations of free tryptophan in soybean, red bean and mung bean (2617.83, 1527.23 and 845.27ng/gdw, respectively), while melatonin and free tryptophan contents in fresh mulberry leaves were 51.57ng/g and 210.53ng/gdw, respectively. Soymilk powder and mulberry leaf tea were supplemented to prepare high melatonin pasteurized milk. Results showed that chemical compositions, melatonin and free tryptophan contents significantly increased (P < 0.05) with increasing amounts of soymilk powder and mulberry leaf tea. Sensory evaluation gave highest overall liking score for the treatment consisting of mulberry leaf tea (4.00%), soymilk powder (4.00%) and milk (89.80%). Findings indicated that mulberry leaves and soybean are both good sources of melatonin and free tryptophan and can be applied to prepare high melatonin pasteurized milk.

Independent selection for seed free tryptophan content and vernalization response in chickpea domestication

AbstractChickpea shows a distinct domestication trajectory vis‐a‐vis pod dehiscence and growth cycle mediated by vernalization insensitivity compared with its companion Near Eastern legumes. Our objectives were: (i) to map the quantitative trait loci (QTLs) associated with vernalization response and seed free tryptophan in domesticated × wild chickpea progeny and (ii) estimate the genetic correlation between vernalization response and free tryptophan content. A domesticated × wild chickpea cross was used to document phenotypic segregation in both traits and to construct a skeletal genetic map for QTL detection. A number of vernalization response and seed free tryptophan content QTLs were documented in both F2 and F3 generations. No significant genetic correlation between these two traits was observed. Epistatic relationship between two free tryptophan loci was documented. It is evident that selection for high seed tryptophan is easier to accomplish relative to selection for vernalization insensitivity. This suggests that the two traits were selected independently in antiquity, thereby corroborating earlier claims for conscious selection processes associated with chickpea domestication.

Optimization of enzymatic hydrolysis of skipjack tuna by-product using protamex ®: a response surface approach

The goal of this study was to optimize enzymatic hydrolysis of the dark flesh of skipjack tuna (Katsuwonus pelamis). Protamex® was used as the hydrolytic enzyme at various concentrations (1%, 1.5%, 2%, 2.5% and 3% w/w) at pHs from 6.5 to 8.5, temperatures from 40 to 60 oC and times of 2 to 6 h. The experiment was performed following a five-level second-order central composite design with six replications at the centeer points. The high coefficients of determination for degree of hydrolysis (R2 = 0.9674) and free tryptophan content (R2 = 0.9426) indicate the suitability of the design for predicting the responses. The optimum hydrolytic conditions predicted by the response surface methodology were 3% Protamex® at pH 6.57 and 58 °C for 4 h. These conditions resulted in an experimental degree of hydrolysis of 18.48% and free tryptophan content of 72.10 mg kg-1.Keywords: Skipjack tuna; protein hydrolystate; response surface analysis; free trytophan

Development of high tryptophan GM rice and its transcriptome analysis

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of tryptophan (Trp), which is the precursor of bioactive metabolites like indole-3-acetic acid and other indole alkaloids. Alpha anthranilate synthase 2 (OsASA2) plays a critical role in the feedback inhibition of tryptophan biosynthesis. In this study, two vectors with single (F124V) and double (S126F/L530D) point mutations of the OsASA2 gene for feedback-insensitive subunit of rice anthranilate synthase were constructed and transformed into wildtype Dongjinbyeo by Agrobacterium-mediated transformation. Transgenic single and double mutant lines were selected as a single copy using TaqMan PCR utilized nos gene probe. To select intergenic lines, the flanking sequence of RB or LB was digested with a BfaI enzyme. Four intergenic lines were selected using a flanking sequence tagged (FST) analysis. Expression in rice (Oryza sativa L.) of the transgenes resulted in the accumulation of tryptophan (Trp), indole-3-acetonitrile (IAN), and indole-3-acetic acid (IAA) in leaves and tryptophan content as a free amino acid in seeds also increased up to 30 times relative to the wildtype. Two homozygous event lines, S-TG1 and D-TG1, were selected for characterization of agronomic traits and metabolite profiling of seeds. Differentially expressed genes (DEGs), related to ion transfer and nutrient supply, were upregulated and DEGs related to co-enzymes that work as functional genes were down regulated. These results suggest that two homozygous event lines may prove effective for the breeding of crops with an increased level of free tryptophan content.

Co-expression of the tobacco anthranilate synthase β subunit with its feedback-insensitive α subunit as a selectable marker that also markedly increases the free tryptophan content

Anthranilate synthase (AS), a tetramer consisting of two α subunits and two β subunits, is the key control enzyme in the tryptophan (Trp) biosynthesis pathway. A naturally occurring α subunit of AS called ASA2 that is insensitive to Trp feedback inhibition was isolated from a tobacco suspension cell culture and has been extensively studied and used for both nuclear and plastid transformation. However, the obligate β subunit of AS had not been studied in tobacco. Therefore, the tobacco AS β subunit-encoding cDNA was cloned and its encoded protein was verified. Agrobacterium-mediated transformation of tobacco plants was performed, under the selection of the toxic Trp analogs, 7-methyltryptophan or α-methyltryptophan, with a construct containing both ASA2 and AS β subunit genes. Many transgenic plants overexpressing both subunits were identified and examined. Compared to the wild-type plants, the transgenic plants had higher levels of enzymatic activities for both holoenzyme and α subunit. The transgenic plants had 9 to 68 times the amount of free Trp as the wild-type plants, which was more pronounced than plants overexpressing ASA2 alone. This study demonstrates the potential of co-expressing AS α and β subunits as a robust plant transformation system as well as overcoming feedback inhibition to obtain high levels of Trp biosynthesis.

Optimizing the Enzymatic Hydrolysis of Skipjack Tuna (Katsuwonus pelamis) Dark Flesh Using Alcalase® Enzyme: A Response Surface Approach

The goal of this study was to optimize enzymatic hydrolysis of the dark flesh of skipjack tuna (Katsuwonus pelamis). Alcalase 2.4 L was used as the hydrolytic enzyme at various concentrations (1, 1.5, 2, 2.5 and 3% w/w) at pHs from 6 to 10, temperatures from 35 to 75?C and times of 2 to 6 h. The experiment was performed following a five-level second-order central composite design with six replications at the center points. The high coefficients of determination for degree of hydrolysis (R2 = 0.8909) and free tryptophan content (R2 = 0.9732) indicate the suitability of the design for predicting the responses. The optimum hydrolytic conditions predicted by the response surface methodology were 2% Alcalase at pH 8.86 and 65.4?C for 5.74 h. These conditions resulted in an experimental degree of hydrolysis of 20.74% and free tryptophan content of 102.23 mg kg-1.

Tryptophan over-producing cell suspensions of Catharanthus roseus (L) G. Don and their up-scaling in stirred tank bioreactor: detection of a phenolic compound with antioxidant potential

Five cell suspension lines of Catharanthus roseus resistant to 5-methyl tryptophan (5-MT; an analogue of tryptophan) were selected and characterized for growth, free tryptophan content and terpenoid indole alkaloid accumulation. These lines showed differential tolerance to analogue-induced growth inhibition by 30 to 70 mg/l 5-MT supplementation (LD(50) = 7-15 mg/l). Lines P40, D40, N30, D50 and P70 recorded growth indices (i.e. percent increment over the initial inoculum weight) of 840.9, 765.0, 643.9, 585.7 and 356.5 in the absence and, 656.7, 573.9, 705.8, 489.0 and 236.0 in the presence of 5-MT after 40 days of culture, respectively. A corresponding increment in the free tryptophan level ranging from 46.7 to 160.0 μg/g dry weight in the absence and 168.0 to 468.0 μg/g dry weight in the presence was noted in the variant lines. Higher tryptophan accumulation of 368.0 and 468.0 g/g dry weight in lines N30 and P40 in 5-MT presence also resulted in higher alkaloid accumulation (0.65 to 0.90 % dry weight) in them. High-performance liquid chromatography (HPLC) analysis of the crude alkaloid extracts of the selected lines did not show the presence of any pharmaceutically important monomeric or dimeric alkaloids except catharanthine in traces in the N30 line that was also unique in terms of a chlorophyllous green phenotype. The N30 line under optimized up-scaling conditions in a 7-l stirred tank bioreactor using Murashige and Skoog medium containing 2 mg/l α-naphthalene acetic acid and 0.2 mg/l kinetin attained 18-folds biomass accumulation within 8 weeks. Interestingly, the cell biomass yield was enhanced to 30-folds if 30 mg/l 5-MT was added in the bioreactor vessel one week prior to harvest. Crude alkaloid extract of the cells grown in shake flask and this bioreactor batch also showed the formation of yellow-coloured crystals which upon (1)HNMR and ESI-MS analysis indicated a phenolic identity. This crude alkaloid extract of bioreactor-harvested cells containing this compound at 50 μg/ml concentration registered 65.21, 17.75, 97.0, 100 % more total antioxidant capacity, reducing power, total phenolic content, and ferric-reducing antioxidant power, respectively, when compared with that of extracts of cells grown in shake flask cultures. The latter, however, showed 57.47 % better radical scavenging activity (DPPH) than the bioreactor-harvested cells.

Direct determination of free tryptophan contents in soy sauces and its application as an index of soy sauce adulteration

A simple and rapid HPLC method using UV detector for determination of tryptophan (Trp) contents in pure fermented soy sauces, acid-HVP, and commercial soy sauces was developed. The method requires only simple sample pretreatment. The limit of detection (LOD) is estimated at 1 mg/L (signal-to-noise ratio of 3), and the recovery yields ranged from 92% to 108%. The analysis showed that Trp in all of the analysed acid-HVP samples was not detected and there was obvious detection of Trp (ranged from 136.4 to 261.8 mg/L) in all of the analysed fermented soy sauce samples. The observations suggest that Trp is a practical index of pure fermented soy sauce. The absence of Trp contents or lower level of Trp contents than that in pure fermented soy sauces suggests the soy sauces are not pure fermented soy sauces or presence of soy sauce adulteration. Simultaneous determination of levulinic acid (LV) in samples may be a good help in making assessment of soy sauce adulteration with acid-HVP.

Nonproteic Contents Interfere with the Analysis of Added Tryptophan in Feeds - Test of a Correction Model

The nonproteic, free tryptophan contents in 23 important feed ingredients were analysed. Wheat, its bran and middlings, soybean meal, sunflower meal and other vegetable feed ingredients contain significant fractions of free trypto- phan, up to 38% of total tryptophan. These natural contents considerably interfere with the accurate determination of sup- plemented L-tryptophan in mixed feeds. Two correction methods were successfully tested in four feeds, which were each supplemented at two levels. They allow a distinct improvement in the analysis of tryptophan addition.

Structure-Based in Vitro Engineering of the Anthranilate Synthase, a Metabolic Key Enzyme in the Plant Tryptophan Pathway

Rice (Oryza sativa) anthranilate synthase alpha-subunit, OASA2, was modified by in vitro mutagenesis based on structural information from bacterial homologs. Twenty-four amino acid residues, predicted as putative tryptophan binding sites or their proximal regions in the OASA2 sequence, were selected and 36 mutant OASA2 genes were constructed by PCR-based site-directed mutagenesis. Corresponding mutant proteins were synthesized in a combination of two in vitro systems, transcription with a bacteriophage SP6 RNA polymerase and translation with a wheat-embryo cell-free system. Enzymatic functions of the mutant proteins were simultaneously examined, and we found six mutants with elevated catalytic activity and five mutants with enhanced tolerance to feedback inhibition by tryptophan. Moreover, we observed that some sets of specific combinations of the novel mutations additively conferred both characteristics to the mutant enzymes. The functions of the mutant enzymes were confirmed in vivo. The free tryptophan content of mutant rice calli expressing OASA2 enzyme with a double mutation was 30-fold of that of untransformed calli. Thus, our in vitro approach utilizing structural information of bacterial homologs is a potent technique to generate designer enzymes with predefined functions.

国内産小麦粉のくすみと遊離トリプトファン含量

国内産小麦粉のくすみと遊離トリプトファン含量

Chemical and microbial analyses of squid muscle ( Loligo plei) during storage in ice

Chemical and microbial changes in squid muscle ( Loligo plei) and the efficiency of chemical indices for freshness evaluation were investigated during storage in contact ice (CI) and non-contact ice (NC). Psychrotrophic bacterial counts and non-protein nitrogen, free amino acids, trimethylamine, volatile basic nitrogen (VBN), urea and free tryptophan contents were periodically determined in squid muscle. No difference ( P < 0.05) was detected between treatments regarding the increase of psychrotrophic bacterial counts in squid muscle during storage. The leaching of soluble compounds in CI treatment drastically affected the efficiency of the chemical indices for freshness. Trimethylamine and VBN contents in squid muscle increased only after day 9 and no increase was observed in free tryptophan and urea contents in CI treatment. In the NC treatment the contents of VBN and trimethylamine slowly increased during the first days of storage whereas free tryptophan and urea contents markedly increased during the whole storage. Free tryptophan and urea can be useful freshness indices for L. plei in storage conditions wherein leaching have been minimized.

Comparison between the Metabolic Effects of Tryptophan and Histidine Deficiencies in the Rat

Rats fed a tryptophan-free diet for 2 days showed less weight loss but more nitrogen loss than those fed a histidine-free diet. The metabolic nature of the body weight loss and of urinary nitrogen loss in rats fed the tryptophan-free diet was investigated in comparison with those fed the histidine-free diet. More body water was retained in rats fed the tryptophan-free diet than in those fed the histidine-free diet. This suggests that the lesser weight loss associated with tryptophan deficiency than with histidine deficiency may be due to the greater water content in rats fed the tryptophan-free diet. The tryptophan contents of the muscle, plasma, and liver decreased rapidly in rats fed the tryptophan-free diet, whereas the histidine contents of these tissues did not decrease as much as in rats fed the histidine-free diet. The conversion of radioactivity of (U-14-C)phenylalanine into respiratory CO-2 and liver and muscle proteins of rats fed the tryptophan- and histidine-free diets were compared. The relative recovery ratio of 14-C into respiratory CO-2 was found to be higher in tryptophan deficiency than in histidine deficiency, and the recovery ratio in muscle was lower in tryptophan deficiency than in histidine deficiency. These results suggest that the greater nitrogen loss seen in tryptophan deficiency than in histidine deficiency may be due to reduced synthesis of muscle protein, presumably due to the low free-tryptophan content in muscle resulting from the tryptophan-free diet.

Indoleacetic Acid Biosynthesis in Avena Coleoptile Tips and Excised Bean Shoots

Avena coleoptiles did not elongate when incubated with tryptophan under sterile conditions. Indole, anthranilic acid, and tryptamine promoted elongation. Under the same conditions, the tissue converted tryptophan-(14)C to IAA-(14)C. More IAA-(14)C was produced from indole-(14)C than from tryptophan-(14)C; however, the free tryptophan content of the tissue was also greatly increased by the indole treatment. Tryptophan-(14)C was readily taken up by the tissue but was mainly incorporated into protein and did not increase the free tryptophan level. When bean shoots were labeled with tryptophan-(14)C or indole-(14)C, the label incorporation into IAA-(14)C was very nearly the same. In this tissue the free tryptophan level in the tryptophan-(14)C and indole-(14)C treatments was also about equal. These results suggest that failure of exogenously supplied tryptophan to promote the elongation of Avena coleoptiles is a result of its predominant incorporation into protein and consequent unavailability for conversion to IAA.

Accumulation of free tryptophan and tryptairiine in zinc deficient maize seedlings

Maize seedlings were grown in culture solutions containing various levels of zinc. The changes in free tryptophan content due to the different zinc nutritions were estimated by using an amino acid analyzer. Free tryptophan was detected in normal leaves, but the amounts were very small relative to those in zinc deficient plants. The maximum accumulation coincided with the development of severe symptoms associated with advanced zinc deficiency. Changes in tryptamine content due to different zinc nutritions were estimated by paper chromatography and thin layer chromatography. The content of tryptamine was highest in maize leaves, which developed symptoms of zinc deficiency. Under conditions of adequate zinc nutrition, tryptamine was also found in maize leaves, but the amounts were very small in contrast to those in zinc deficient plants.

Total, bound and free tryptophan content of food; contribution to formulation of the amino acid balance

Die bei der Ermittlung des Aminosauregehaltes von Lebensmitteln und Proteinkonzentraten wesentlichen Gesichtspunkte wurden erortert und im Anschlus daran in einer Reihe von Lebensmitteln, Proteinkonzentraten und Proteinen der Gehalt an gesamtem, freiem und gebundenem Tryptophan ermittelt und mit den entsprechenden Stickstoff-Fraktionen [Gesamt-Stickstoff, Nichteiweis- (Rest.) Stickstoff und Eiweis-Stickstoff] in Vergleich gesetzt. Dabei zeigte sich, das in der Mehrzahl der Falle die prozentuale Verteilung des Tryptophans im Eiweisund Nichteiweisanteil der Stickstoffsubstanz nicht mit dem Gehalt an diesen Stickstoff-Fraktionen korrespondiert. Infolge eines betrachtlichen Anteils an freiem Tryptophan in manchen Lebensmitteln kann die Beschrankung der Aminosaurebestimmung auf isoliertes Reinprotein zu wesentlichen Irrtumern hinsichtlich der Aminosaurezusammensetzung und der biologischen Bewertung eines Lebensmittels fuhren. Da ernahrungsphysiologische Untersuchungen auf dem Gebiet der Proteine in erster Linie den Zweck verfolgen, die Aminosaurezusammensetzung von Lebensmitteln festzulegen und nicht Betrachtungen uber den Aufbau einzelner Proteine anzustellen, sind klare Einblicke in die fur die Ernahrung verfugbaren Aminosauremengen nur dann zu gewinnen, wenn die Aminosaurebestimmungen am Gesamtmaterial durchgefuhrt und die erhaltenen Werte auf den Gesamtstickstoffgehalt der Lebensmittel berechnet werden.