Rapid and sensitive method for determining free amino acids in plant tissue by high-performance liquid chromatography with fluorescence detection

This paper describes a reliable and rapid method for the complete separation and quantitation of twenty-five amino acids typically found in plants, based on reversed phase high-performance liquid chromatography–linked fluorescence detector using a 150×4.6 mm Zorbax Eclipse AAA column. Plant tissue free amino acids(FAA)were extracted by ultrasonication with 5%(v/v) aqueous trifluoroacetic acid followed by ultrafiltration of extracts.The following analysis of amino acids was performed through programmed precolumn derivatization with orthophthalaldehyde and 9-fluorenylmethyl chloroformate reagents and efficient elution of derivatives within 26 min using binary gradient scheme. The method was validated over a concentration range of 4.5–450 μmol L^(-1)(μM).Separation analysis showed good selectivity(resolution>1.5) for most amino acids. The average repeatability(RSD%, relative standard deviation) of the analysis at seven calibration concentrations was below 4% and ranged from 1.13% to 12.04%. The intra-day mean coefficient of variation at two concentrations(22.5 and 90 μM) was within 2%, and the intermediate precision was less than 4%. The limits of detection were between 0.012 and 6.68 μM. The coefficients of determination(R2) of the linear calibration curves were from 0.9989 to 0.9999.When the method was applied to plant samples, the FAA recoveries at two spiked levels(25 and 100 μM) ranged from 67.0% to 108.9% with an average of 94.4%, and the precision was 0.26%–12.31% RSD. A specific application combining this method with optimized extraction and interference removal procedures was successfully used to determine the FAA pools in different plant tissues. Finally,a PLS-DA multivariate statistics model was validated for the classification of three plant species according to their FAA profiles.

Study on Amino Acid Composition of HSP70 and the Level of Plasma Free Amino Acids of Workers with Long-term Exposure to Harmful factors

In order to know the practical value of heat stress protein 70 (HSP70) and to know the changes of plasma free amino acids of workers with the induction of HSP70 by harmful factors, the amino acid composition of major HSP, HSP70 purified from the heated cultured human leukemia cancer cell line K562, rabbit liver, rat liver and heart, and mouse liver with two-step procedures of DE52-cel-lulose ion exchange chromatography and affinity chromatography on ATP-agarose was examined. The level of plasma free amino acids of workers with long-term exposure to heat, carbon monoxide and the combined effect of both heat and carbon monoxide was also investigated. The results showed that the three richest amino acids in HSP70 of all origins were Gly, Glu and Asp, except that of rat heart which was rich in Gly, Phe and Glu. Additionally, Lys, Val, Leu and Ala were also found very rich in HSP70 of all origins. Compared with controls, the most of plasma free amino acids tended to increase and free methionine and trypto-phan were increased significantly (P<0. 05) after a long-term exposure to heat, carbon monoxide, and both. These findings suggested that further studies need to be done to find the substances or drugs which induce the synthesis of HSP70 and reduce the inhibition of synthesis of normal proteins for the purpose of protecting people with exposure to harmful factors against the damage of the factors.

Floral gradient in flowering tobacco in relation to free amino acids

By employing TCLs (thin cell layers) culture, the floral gradient in flowering tobacco of different developmental stages was confirmed. The TCLs from early flowering tobacco regenerated more floral buds than those from the tobacco plants in full blooming or fruiting stages. Analysis of free amino acid levels revealed the acropetal gradient of Pro in flowering tobacco stem. L-Pro. L-Trp. D,L-Met and L-Arg were respectively added into the culture medium for testing their influence on floral bud formation from tobacco pedicel segments. Only L-Trp evidently enhanced the floral bud neoformation.

Construction of Free Amino Acids Composition Data Base for Food

Significant amounts of free amino acids exist in commercially sold vegetables and fruits. Despite of the fact, only a little information is available about the free amino acid contents in foods. To utilize information of free amino acids in food, we have carried out the experiments to quantitate the free amino acids by derivatized with NBD-F （4-fluoro-7-nitrobenzofurazan） and analyzed on reversed-phase UHPLC （ultra-high pressure liquid chromatography） equipped with ultraviolet visible detector. Almost all of food extracts contained free amino acids including GABA （T-amino butyrate）. Contents of free amino acids vary considerably depending upon vegetables and fruits. Principal free amino acids found in vegetables and fruits were asparagine, glutamine, arginine and GABA, which are involved in important metabolic pathways in human. About 140 species of vegetables and fruits were subjected for the data base. All of the plants and fruits we examined exhibit significant amount of free amino acids, those are clearly distinct from data bases obtained after acid hydrolysis treated food samples. Since glutamate and GABA act as excitatory and inhibitory neurotransmitters in CNS, respectively; free amino acids in vegetables and fruits that we eat daily, should be an important source for the cellular metabolic activities.

Effect of different drying methods on the amino acids,α-dicarbonyls and volatile compounds of rape bee pollen

The significant demand for high quality food has motivated us to adopt appropriate processing methods to improve the food nutritional quality and flavors.In this study,the effects of five drying methods,namely,pulsed vacuum drying(PVD),freeze drying(FD),infrared drying(IRD),hot-air drying(HAD)and sun drying(SD)on free amino acids(FAAs),α-dicarbonyl compounds(α-DCs)and volatile compounds(VOCs)in rape bee pollen(RBP)were determined.The results showed that FD significantly released the essential amino acids(EAAs)compared with fresh samples while SD caused the highest loss.Glucosone was the dominantα-DCs in RBP and the highest loss was observed after PVD.Aldehydes were the dominant volatiles of RBP and SD samples contained more new volatile substances(especially aldehydes)than the other four drying methods.Comprehensively,FD and PVD would be potential methods to effectively reduce the quality deterioration of RBP in the drying process.

Metagenomic analysis revealing the metabolic role of microbial communities in the free amino acid biosynthesis of Monascus rice vinegar during fermentation

Free amino acid(FAA)is the important component of vinegar that infl uences quality perception and consumer acceptance.FAA is one of the major metabolites produced by microorganisms;however,the microbial metabolic network on FAA biosynthesis remains unclear.Through metagenomic analysis,this work aimed to elucidate the roles of microbes in FAA biosynthesis during Monascus rice vinegar fermentation.Taxonomic profiles from functional analyses showed 14 dominant genera with high contributions to the metabolism pathways.The metabolic network for FAA biosynthesis was then constructed,and the microbial distribution in different metabolic pathways was illuminated.The results revealed that 5 functional genera were closely involved in FAA biosynthesis.This study illuminated the metabolic roles of microorganisms in FAA biosynthesis and provided crucial insights into the functional attributes of microbiota in vinegar fermentation.

Rapid Determination of Free Amino Acids in Milk by Microchip Electrophoresis Coupled with Laser-induced Fluorescence Detection

A simple and sensitive method for determination of free amino acids in milk by microchip electrophoresis （MCE） coupled with laser-induced fluorescence （LIF） detection was developed. Seven kinds of standard amino ac- ids were derivated with sulfoindocyanine succinimidyl ester （Cy5） and then perfectly measured by MCE-LIF within 150 s. The parameters of MCE separation were carefully investigated to obtain the optimal conditions： 100 mmolo L^-1 sodium borate solution （pH 10.0） as running buffer solution, 0.8 kV as injection voltage, 2.2 kV as separation voltage etc. The linear range of the detection of amino acids was from 0.01 μmol·L^-1 to 1.0 μmol·L^-1 and the detection limit was as low as about 1.0 μmol·L^-1. This MCE-LIF method was applied to the measurements of free amino acids in actual milk samples and satisfactory experimental results were achieved.

Vertical variations and composition of dissolved free amino acid in the seawater of the Yap Trench in the western Pacific Ocean

The composition and concentration of dissolved free amino acid(DFAA)of seawater samples collected in May 2016 from the surface to the hadal zone of the northern region of the Yap Trench were analyzed by pre-column derivatization of o-phthalaldehyde.Results show that the average concentration of DFAA in the study area was 0.47±0.36μmol/L.In different sampling stations,the concentrations of DFAA with water depth showed complex variation patterns.At the sediment-seawater interface,the concentrations of DFAA in the western side of the trench were obviously higher than that in its eastern side.In the study area,there were no significant correlations between the concentrations of DFAA and the environmental parameters such as concentrations of chlorophyll a(Chl a),dissolved oxygen(DO),pH,and dissolved inorganic nitrogen(DIN),indicating that the concentrations of DFAA in seawater of the trench are affected by many factors,such as photosynthesis,respiration,temperature,pressure,illumination,and circulation.The dominant DFAA are similar in different water layers of sampling stations,including aspartic acid(Asp),glutamic acid(Glu),glycine(Gly),and serine(Ser).The composition of different amino acids,and the relative abundance of acidic,basic,and neutral amino acids might be related to the sources and consumption of various amino acids.Nine pairs of amino acids in the DFAA showed significantly positive relationship by correlation matrix analysis,suggesting that they might share similar biogeochemical processes.The degradation index(DI)of the DFAA in seawater of the Yap Trench could reflect the degradation,source,and freshness of DFAA in the trench to some extents.This is a preliminary study of amino acids from sea surface to hadal zone in the ocean,more works shall be done in different trenches to reveal their biogeochemical characte ristics in extreme marine environme nts.

Influences of dietary protein sources and crude protein levels on intracellular free amino acid profile in the longissimus dorsi muscle of finishing gilts

Background： The current study was carried out to determine effects of dietary protein source and crude protein（CP）level on carcass characteristics, meat quality, and muscle amino acid（AA） profile in finishing gilts. The experiment was designed as a 2 × 2 factorial arrangement with two sources of dietary proteins（cottonseed meal, CSM vs. soybean meal, SBM） and two levels of CP（12 % vs. 14 %, as-fed basis）. Seventy-two crossbred gilts（89.5 ± 0.9 kg） were allotted to one of four dietary treatments in a randomized complete block design for a period of 28 d. All diets were formulated to be isoenergetic and similar concentrations of standardized ileal digestible essential AA covering the nutrient requirements of pigs.Results： Growth, carcass characteristics and meat quality were not affected by dietary protein source nor crude protein level（P ＆gt; 0.10） except that average daily feed intake was increased by CSM diets（P = 0.03）. Gilts offered reduced protein diets had lower muscle p H45min（P 〈 0.05）. Neither dietary protein source nor crude protein level influenced N deposition. However, reduced protein diets decreased N intake, N excretion, and serum urea nitrogen content, whilst improved N efficiency（P 〈 0.01）. CSM diets increased N intake（P = 0.04）,but did not depress N efficiency. The concentrations of phenylalanine, tryptophan, cysteine and tyrosine（P 〈 0.05） of the longissimus muscle were decreased when gilts offered CSM diets, while muscle intracellular free valine concentration was increased（P = 0.03）. The gilts offered reduced protein diets had greater intracellular concentrations of free methionine, lysine, and total AA in muscle（P 〈 0.05）.Conclusion： These results suggest that CSM could replace SBM as a primary protein source in finishing pig diets in terms of performance, N efficiency, carcass characteristics, and meat quality, but decrease the concentrations of muscle specific AA. Furthermore, the reduced protein diet played an important role in increasing muscle intracellular concentrations of specific free amino acids（FAA）, and in reducing the relative ratios of specific FAA to lysine in longissimus dorsi muscle of pig, whose biological meaning needs further studies.

Study on Free Amino Acid and Short Peptide Fertilizer Production by Solid State Fermented Castor Bean Meal

Using microbial fermentation to increase the content of free amino acids and short peptides in the organic fertilizer for castor bean meal can effectively promote plant growth and improve fruit quality.Using free amino acid and short peptide content as an indicator,through single factor and response surface optimization experiments,the process parameters(moisture content,fermentation time and inoculum quantity)of castor meal solid-state fermentation were optimized.The best process parameters for the solid-state fermentation were:the moisture content 62%,the fermentation time 20 d,and the inoculum quantity 0.23%.The moisture content had the greatest impact on the conversion rate of free amino acids and short peptides,and the protein conversion rate reached 65.6%.The scale-up experiment under the optimal conditions showed that the solid-state fermentation using the inoculum had a significant beneficial effect compared with other fermentation methods.The fermentation of castor cake fertilizer provides a theoretical and practical basis for production feasibility,and has important guiding significance for the effective utilization of castor bean meal.

Determination of 18 Kinds of Amino Acids in Fresh Tea Leaves by HPLC Coupled with Pre-column Derivatization

A rapid and accurate quantitative method of high performance liquid chromatography( HPLC) with fluorescence detector has been developed for the analysis of 18 kinds of amino acids in fresh tea leaves. The samples were minced and mixed,and extracted with ultra pure water at 90℃ for 20 min. The 6-aminoquinolyl N-hydroxy-succinimidyl carbamate( AQC) was used as pre-column derivatization reagent. Gradient HPLC separation was performed on a C_(18) column( Symmetry C_(18),3. 9 mm × 15 cm,4 μm). Good linearity between concentrations and peak areas was achieved in the concentration range of 5. 0-250 μmol/L for 18 kinds of amino acids. The method was validated by the analysis of five replicates. The 18 kinds of amino acid standards were spiked in fresh tea leaf samples and the average recovery rate was 86. 25%-109. 05% with relative standard deviations( n = 5) ranging from 6. 03% to 10. 56%. The limit of detection( LOD) for the analytes was0. 05-1. 27 μmol/L. The method was successfully applied to the analysis of the 18 kinds of amino acids in fresh tea leaves from east Dongting and west Dongting mountains in Suzhou. The results indicate that the method is simple,rapid,precise and reliable.

Free amino acid content in trunk,branches and branchlets of Araucaria angustifolia(Araucariaceae)

Araucaria angustifolia(Bertol.) O. Kuntze exhibits dimorphism in its stem structure, where the trunk is orthotropic and branches and branchlets(primary and secondary branches) are plagiotropic. These stems exhibit different behavior when used for vegetative propagation,and only segments of trunk can form a complete plant. The physiological and biochemical mechanisms that characterize these stems are still little known. The aim of this study was to describe the free amino acid profiles in trunks,branches, and branchlets of A. angustifolia. Segments of 5 cm in length were excised from young individuals below the stem apex. The needles were removed and samples were frozen and lyophilized. The determinations were made by high-performance liquid chromatography, and the results were expressed as lg/g fresh weight(FW). The trunks and branches had the highest content of total amino acids, which were 112.23 ± 20.57 lg/g FW and 111.97 ± 27.78 lg/g FW, respectively. The amino acids—glutamine, aspartate and c-aminobutyric acid and tyrosine—were noticeably higher in the three types of stems.In the trunk, a higher amount of asparagine and tryptophan,was also detected. Glutamic acid and glutamine were found in higher quantities in the branches. The branchlets had very low total amino acid content (30.79 ± 4.19 lg/g FW), wherein asparagine is the only amino acid not detected. Thus, it was observed that the profile of the free amino acid differs among trunks, branches, and branchlets in A. angustifolia, indicating that they perform different functions.

A Study of Bioactivity of Corn Peptides with Low Molecular Weight Ⅱ: Effect on Plasma Free Amino Acid Concentrations in Rats

The effects of the ingestion of corn peptides with a low molecular weight(LMCP) prepared from zein on some plasma free amino acid concentrations in rats that had taken ethanol were investigated. LMCP(1 0 g/kg body weight) in 15% ethanol(10 mL/kg body weight) was given to Wister rats by intragastrical administration. The amino acid analysis showed that the concentrations of alanine, leucine, and proline in the plasma reached their maximum levels at 30 min for the LMCP-intake group. They are 582.39, 99.60 and 272.51 μg/L , respectively. But in the control group, the plasma free amino acid levels were not changed obviously. Therefore, LMCP could cause an increase in concentration of some free amino acids such as alanine, leucine and proline etc . in plasma of the rats that have taken ethanol.

Monthly Changes of Glycogen, Lipid and Free Amino Acid of Oyster

Monthly difference of the chemical composition of oyster cultured along the eastern coast of Shandong Province was analyzed. The components analyzed included glycogen, fatty acid and free amino acid （FAA）. The content of glycogen was high in January and March （2.89 and 2.82 g（100 g） ^-1 on average, respectively） and low in October （2.07g（100g）^- 1 on average）. The low content of neutral lipids in October reflected a relatively poor nutritional value of oyster （1.42 g（ 100 g）^- 1 on average）. The main fatty acids of oyster were palmitic acid （16：0）, oleic acid （18： 1）, eicosapentaenoic acid （EPA, 20： 5（0 3） and docosahexaenoic acid （DHA, 22：6（0 3）. The major FAAs of oyster were Taurine, Glutamicacid, Glycin, Alanine, Arginine and Proline. Taurine was the most abundant FAA with its content ranging from 603 mg（100 g） ^-1 to 1 139 mg（ 100 g） ^-1. The high contents of glycogen, polyunsaturated fatty acid and FAA showed that oyster cultured along the eastern coast of Shandong Province was nutritionally good in January and March.

Effect of Allelic Variation of β-conglycinin α-subunit Locus cgy-2 on Soybean(Glycine max) Quality Ⅰ. Evaluation of Free Amino Acid Content

The effects of the deficiency of the allergenicα-subunit on soybean amino acid(AA)composition were studied using the cultivar Dongnong 47(DN47)as a genetic background.The near-isogenic line(NIL)NIL-DN47-Δα of DN47,with an introgression of theα-null trait allele from the high protein donor parent RiB,was created by marker assisted background selection and used to investigate the AA content and nutritional quality.The contents of crude protein,the total AAs,the total essential amino acids(EAAs)and sulfur-containing(Met and Cys)AAs increased by 4.11%,4.16%,5.20% and 11.96%,respectively in NIL-DN47-Δα compared with DN47.Analyses of the total EAAs(TEAAs)and the EAA index(EAAI)revealed that both parameters in NIL-DN47-Δα were higher than those in DN47.The null-allele of theα-subunit positively affected the AA scores.The quantitative changes in free AAs(FAAs)in the developing seeds of NIL-DN47-Δα and DN47 were compared as of 15 days after flowering(DAF)until maturity.The results showed that the total FAA content in NIL-DN47-Δα was significantly higher than that in the DN47 throughout the late maturation stage(40-60 DAF)of seeds.The high concentration of the FAAs in cgy-2 mutant seeds was a consequence of the high rates of synthesis and/or accumulation of individual FAAs during seed maturation where 25 DAF was an important turning point in the accumulation of the FAAs.The FAA contents of single soybeanα-null,double(α+α′)and triple(α+α′+group I)-null mutant combination lines were investigated.In all of these combinations,introduction of the cgy-2 gene invariably raised the FAA content of mature seeds above that of the DN47.In summary,the enhanced protein quality in cgy-2 mutants resulted from several factors.(1)There was a general increase in the contents of most AAs and FAAs in NIL-DN47-Δα.(2)The induced synthesis of free Arg contributed effectively to the high FAAs of various storage-protein-deficiency mutants.For example,in the S2(null α,group I),the free Arg content was seven times as much as that of DN47,accounting for more than half of the total FAA content in the seed.(3)The increase of sulfur-containing AAs in theα-null type NIL mainly resulted from elevated Met content.These data suggested that the cgy-2 mutation might improve the protein quality of soybean seeds and that lacked of the allergenicα-subunit resulted in increased the FAA content.

Free amino acid (FAA) analysis of phloem sap in hybrid rice Shanyou 63 and its parents

Hyhrid rice has greatly contributed to rice production inChina. But concurrently a dramatic upsurge of rice plant-hopper occurred, particularly for the whitebacked plant-hopper (WBPH), Sogatella furcifera. It has become animportant economic insect pest of hybrid rice, although ithad only been a minor herbivore of rice before nation-wideexploitation of hybrid rice.

Dynamic changes in physicochemical property,biogenic amines content and microbial diversity during the fermentation of Sanchuan ham

Sanchuan ham is appreciated in Yunnan Province,China,for its characteristic flavor and taste,while the microbial community structure and biogenic amines content remain unclear during fermentation processes.In this study,we explored the physicochemical property,biogenic amines concentration and microbial diversity of external and internal Sanchuan ham by high-throughput sequencing during the processing of Sanchuan ham.Results showed that the nitrite remained at a stable level of 0.15 mg/kg which was significantly lower than the national health standard safety level of 20 mg/kg.In addition,compared with fresh hams,the content of total free amino acids in ripe Sanchuan ham has grown 14 folds;sour and bitter were the main tastes of Sanchuan ham.Notably,the concentration of cadaverine was the highest of all biogenic amines during the entire fermentation period.At the bacterial phyla level,Firmicutes and Actinobacteria were the two main phyla,while at the genus level,Staphylococcus was a significant strain throughout the whole fermentation.Moreover,the dry stage has a great impact on the succession change of microbial community structure.Simultaneously,the change trends and composition of bacteria in the interior have slight discrepancies with those of the exterior of Sanchuan ham.

Changes of Amino Acid Content in Leaves of Agropyron cristatum Under Grazing Pressure

By measuring and comparing the contents of amino acids in Agropyron cristatum under grazing pressure, this study showed that the contents of total and each of the amino acids in A. cristatum changed little under grazing pressure. How- ever, the contents of free glycine （Gly） and proline （Pro） were very sensitive to grazing. Glycine content in grazing population was higher than that in non-grazing population at flowering stage; the content of free proline in non-grazing population was higher than that in grazing population at tillering, heading and especially flower- ing stage; the content of free proline under grazing pressure at maturity stage was higher than that of non-grazing population.

Amino Acid Analysis of Astragalus Membranaceus (Fisch.)Bge

Amino acid composition of Astragalus Membranaceus Bge(AMB)was analysed by automatic analyzer.The results showed that the amount of combined amino acids was 8.05%of total weight,the free amino acids was 0.38%and the whole amino acids was 8.43%.AMB contains all of the essential amino acids except Trp xysshhant.Besides the therapy effect,the AMB has significant nutrient effect.

The dual-function of bioactive peptides derived from oyster (Crassostrea gigas) proteins hydrolysates

Oysters(Crassostrea gigas)have a wide range of functionality due to their nutritional and bioactive components. However, the bioactive peptides of oyster proteins are rarely reported, particularly their antidiabetes effects and antioxidants. Oyster proteins were extracted from fresh oysters using phosphatebuffered saline and simulated gastrointestinal digestion was performed. The degree of hydrolysis(DH), structural characterization, molecular weight(Mw)distribution, free amino acid, anti-diabetic activity, and antioxidant activity were studied during in vitro simulated gastrointestinal digestion. The results showed that the α-glucosidase inhibitory activity, α-amylase inhibitory activity, DPPH radical scavenging activity, and ABTS radical scavenging activity of the oyster protein gastrointestinal digest were increased(P < 0.05)from 0 to 33.96%, from 9.17% to 44.22%, from 9.01 μg trolox/mg protein to 18.48 μg trolox/mg protein, and from 21.44 μg trolox/mg protein to 56.21 μg trolox/mg protein, respectively. Additionally, the DH, β-turn structure, fluorescence intensity, free amino acid, and short peptide content(Mw < 1 000 Da)increased in the simulated gastrointestinal digestion. These results indicate that the digestive hydrolysates obtained from oyster proteins could be used as natural anti-diabetic and antioxidant agents.