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.

Evolution of free amino acids, biogenic amines and volatile compounds in fermented sausages inoculated with Lactiplantibacillus plantarum and Staphylococcus simulans

Lactic acid bacteria and coagulase-negative staphylococci play an important role in the production of fermented sausages,such as inhibiting the growth of undesirable bacteria and antioxidant.In this study,the effects of inoculation with different starter cultures(Lactiplantibacillus plantarum HN108 and Staphylococcus simulans NJ209)on the free amino acids(FAAs),biogenic amines(BAs)and volatile compounds of fermented sausages were investigated using an amino acid analyzer,ultra performance liquid chromatography and gas chromatography-ion mobility spectrometry,respectively.The pH and carbonyl content of the inoculated group was significantly lower than those in the control group(P<0.05).L.plantarum HN108 significantly reduced the content of FAAs and BAs in fermented sausage production(P<0.05),while S.simulans NJ209 promoted the formation of FAAs(especially bitter amino acids)and exhibited slight BAs-reducing activity.In addition,L.plantarum HN108 promoted the formation of volatile compounds such as ketones,alcohols and alkenes in sausages.In conclusion,L.plantarum HN108 could contribute to reducing the content of putrescine and tyramine and forming the desirable flavor compounds in fermented sausages.Thus,L.plantarum HN108 is expected to be a starter culture that can improve the safety and flavor of fermented sausages.

USP19 Stabilizes TAK1 to Regulate High Glucose/Free Fatty Acid-induced Dysfunction in HK-2 Cells

Objective Obesity-induced kidney injury contributes to the development of diabetic nephropathy(DN).Here,we identified the functions of ubiquitin-specific peptidase 19(USP19)in HK-2 cells exposed to a combination of high glucose(HG)and free fatty acid(FFA)and determined its association with TGF-beta-activated kinase 1(TAK1).Methods HK-2 cells were exposed to a combination of HG and FFA.USP19 mRNA expression was detected by quantitative RT-PCR(qRT-PCR),and protein analysis was performed by immunoblotting(IB).Cell growth was assessed by Cell Counting Kit-8(CCK-8)viability and 5-ethynyl-2′-deoxyuridine(EdU)proliferation assays.Cell cycle distribution and apoptosis were detected by flow cytometry.The USP19/TAK1 interaction and ubiquitinated TAK1 levels were assayed by coimmunoprecipitation(Co-IP)assays and IB.Results In HG+FFA-challenged HK-2 cells,USP19 was highly expressed.USP19 knockdown attenuated HG+FFA-triggered growth inhibition and apoptosis promotion in HK-2 cells.Moreover,USP19 knockdown alleviated HG+FFA-mediated PTEN-induced putative kinase 1(PINK1)/Parkin pathway inactivation and increased mitochondrial reactive oxygen species(ROS)generation in HK-2 cells.Mechanistically,USP19 stabilized the TAK1 protein through deubiquitination.Importantly,increased TAK1 expression reversed the USP19 knockdown-mediated phenotypic changes and PINK1/Parkin pathway activation in HG+FFA-challenged HK-2 cells.Conclusion The findings revealed that USP19 plays a crucial role in promoting HK-2 cell dysfunction induced by combined stimulation with HG and FFAs by stabilizing TAK1,providing a potential therapeutic strategy for combating DN.

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.

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.

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.

剩余污泥有机质资源回收利用原位驱动晚期垃圾渗滤液深度脱氮——一种具有显著能源优势的创新生物技术

The sustainable recovery and utilization of sludge bioenergy within a circular economy context has drawn increasing attention,but there is currently a shortage of reliable technology.This study presents an innovative biotechnology based on free nitrous acid(FNA)to realize sustainable organics recovery from waste activated sludge(WAS)in-situ,driving efficient nitrogen removal from ammonia rich mature landfill leachate by integrating partial nitrification,fermentation,and denitrification process(PN/DN-F/DN).First,ammonia((1708.5±142.9)mg·L^(-1))in mature landfill leachate is oxidized to nitrite in the aerobic stage of a partial nitrification coupling denitrification(PN/DN)sequencing batch reactor(SBR),with nitrite accumulation ratio of 95.4%±2.5%.Then,intermediate effluent(NO_(2)^(-)-N=(1196.9±184.2)mg·L^(-1))of the PN/DN-SBR,along with concentrated WAS(volatile solids(VSs)=(15119.8±2484.2)mg·L^(-1)),is fed into an anoxic reactor for fermentation coupling denitrification process(F/DN).FNA,the protonated form of nitrite,degrades organics in the WAS to the soluble fraction by the biocidal effect,and the released organics are utilized by denitrifiers to drive NOx-reduction.An ultra-fast sludge reduction rate of 4.89 kg·m^(-3)·d^(-1) and nitrogen removal rate of 0.46 kg·m^(-3)·d^(-1) were realized in the process.Finally,F/DN-SBR effluent containing organics is refluxed to PN/DN-SBR for secondary denitrification in the post anoxic stage.After 175 d operation,an average of 19350.6 mg chemical oxygen demand organics were recovered per operational cycle,with 95.2%nitrogen removal and 53.4%sludge reduction.PN/DN-F/DN is of great significance for promoting a paradigm transformation from energy consumption to energy neutral,specifically,the total benefit in equivalent terms of energy was 291.8 kW·h·t^(-1) total solid.

Berberine reverses free-fatty-acid-induced insulin resistance in 3T3-L1 adipocytes through targeting IKKβ

AIM: To investigate the effects and molecular mechanisms of berberine on improving insulin resistance induced by free fatty acids (FFAs) in 3T3-L1 adipocytes. METHODS: The model of insulin resistance in 3T3-L1 adipocytes was established by adding palmic acid (0.5 mmol/L) to the culture medium. Berberine treatment was performed at the same time. Glucose uptake rate was determined by the 2-deoxy-[3H]-D-glucose method. The levels of IkB kinase beta (IKKβ) Ser181 phosphorylation, insulin receptor substrate-1(IRS-1) Ser307 phosphorylation, expression of IKKβ, IRS-1, nuclear transcription factor kappaB p65 (NF-κB p65), phosphatidylinositol-3-kinase p85 (PI-3K p85) and glucose transporter 4 (GLUT4) proteins were detected by Western blotting. The distribution of NF-κB p65 proteins inside the adipocytes was observed through confocal laser scanning microscopy (CLSM). RESULTS: After the intervention of palmic acid for 24 h, the insulin-stimulated glucose transport in 3T3-L1 adipocytes was inhibited by 67%. Meanwhile, the expression of IRS-1 and PI-3K p85 protein was reduced, while the levels of IKKβ Ser181 and IRS-1 Ser307 phosphorylation, and nuclear translocation of NF-κB p65 protein were increased. However, the above indexes, which indicated the existence of insulin resistance, were reversed by berberine although the expression of GLUT4, IKKβ and total NF-κB p65 protein were not changed during this study. CONCLUSION: Insulin resistance induced by FFAs in 3T3-L1 adipocytes can be improved by berberine. Berberine reversed free-fatty-acid-induced insulin resistance in 3T3-L1 adipocytes through targeting IKKβ.

Correlation between serum free fatty acids levels and Gensini score in elderly patients with coronary heart disease

Objectives To investigate the relationship between serum fxee fatty acids （FFAs） levels and the severity of coronary artery lesions in elderly patients with coronary heart disease （CAD）. Methods A total of 172 elderly patients who underwent coronary angiography were divided into CAD group （n = 128） and non-CAD group （n = 44） according to the results of coronary angiography. Serum FFAs and lipid levels were measured and the Gensini score were calculated. Results No matter the differences between age, gender and the usage of stat- ins or not, there was no statistical significance in FFAs levels （P 〉 0.05）. In terms of the Gensini score, it was higher in patients aged 70-79 years than in patients 60-69 years old [15.00 （5.00, 34.00） vs. 10.00 （2.00, 24.00）, P 〈 0.05], higher in men than women [14.00 （4.00, 34.00） vs. 7.00 （2.50, 19.75）, P 〈 0.05], and higher in patients on statins [13.50 （4.25, 33.50）vs. 6.50 （2.00, 18.00）, P 〈 0.05]. The serum FFAs lev- els [449.50 （299.00, 624.75） mEq/L vs. 388.00 （258.50, 495.25） mEq/L, P 〈 0.05J and Gensini score [17.50 （8.00, 41.75） vs. 1.00 （0, 5.00）, P 〈 0.05] were higher in the CAD group than in the non-CAD group. In the CAD group, there was no statistical significance in FFAs levels among patients with different numbers of diseased coronary vessels （P 〉 0.05）. Furthermore, the FFAs levels were positively correlated with the Gensini score （r = 0.394, P = 0.005）. Regression analysis showed that the FFAs levels were related to the Gensini score independently after adjusting for the other risk factors. Conclusions The serum FFAs levels were associated with the Gensini score in elderly patients with CAD. It might indicate FFAs as a biomarker predicting the severity of coronary artery lesions.

Effect of sodium salicylate on oxidative stress and insulin resistance induced by free fatty acids

BACKGROUND: It has been reported that high-dose salicylates improve free fatty acids (FFAs)-induced insulin resistance and beta-cell dysfunction in vitro, but the mechanism remains uncertain. In insulin-resistant rats, we found that the supplementation of sodium salicylate is associated with a reduction of plasma malondialdehyde (MDA), a marker of oxidative stress. Few studies have investigated the effects of salicylates on oxidative stress levels in insulin-resistant animal models. This study aimed to assess the effect of sodium salicylate on insulin sensitivity and to explore the potential mechanism by which it improves hepatic and peripheral insulin resistance. METHODS: Intralipid+heparin (IH), saline (SAL), or intralipid+heparin+sodium salicylate (IHS) were separately infused for 7 hours in normal Wistar rats. During the last 2 hours of the infusion, hyperinsulinemic-euglycemic clamping was 3 performed with [6-(3)H] glucose tracer. Plasma glucose was measured using the glucose oxygenase method. Plasma insulin and C-peptide were determined by radioimmunoassay. MDA levels and glutathione peroxidase (GSH-PX) activity in the liver and skeletal muscle were measured with colorimetric kits. RESULTS: Compared with infusion of SAL, IH infusion increased hepatic glucose production (HGP), and decreased glucose utilization (GU) (P<0.05). The elevation of plasma free fatty acids increased the MDA levels and decreased the GSH-PX activity in the liver and muscle (P<0.01). Sodium salicylate treatment decreased HGP, elevated GU (P<0.05), reduced MDA content by 60% (P<0.01), and increased the GSH-PX activity by 35% (P<0.05). CONCLUSIONS: Short-term elevation of fatty acids induces insulin resistance by enhancing oxidative stress levels in the liver and muscle. The administration of the anti-inflammatory drug sodium salicylate reduces the degree of oxidative stress, therefore improving hepatic and peripheral insulin resistance. IKK-beta and NF-kappa B provide potential pathogenic links to oxidative stress.

The Kinetics of the Esterification of Free Fatty Acids in Waste Cook- ing Oil Using Fe2（SO4）3/C Catalyst

The esterification of free fatty acids(FFA) in waste cooking oil with methanol in the presence of Fe2(SO4)3/C(ferric sulfate/active carbon) catalyst was studied.The effects of different temperature,methanol/FFA mole ratio and amount of catalyst on the conversion of FFA were investigated.The results demonstrated that under optimal esterification conditions the final acid value of the resultant system can be reduced to ～1(mg KOH)·g-1,which met fully the requirements in post-treatment for efficient separation of glycerin and biodiesel.The kinetics of the esterification were also investigated under different temperatures.The results indicated that the rate-control step could be attributed to the surface reaction and the esterification processes can be well-depicted by the as-calculated kinetic formula in the range of the experimental conditions.

Effects of different free fatty acids on insulin resistance in rats

BACKGROUND: Much evidence demonstrates that elevated free fatty acids (FFAs) are associated with insulin resistance. However, it is not clear whether different FFAs can cause different degrees of peripheral insulin resistance. This study aimed to investigate the effects of short-term elevation of FFAs on hepatic and peripheral insulin action, and determine whether FFAs with different degrees of saturation have differential effects on hepatic insulin resistance. METHODS: Intralipid+heparin (IH, polyunsaturated fatty acids), oleate (OLE), lard oil+heparin (LOH), and saline (SAL) were separately infused intravenously for 7 hours in normal Wistar rats. During the last 2 hours of the fat/saline infusion, a hyperinsulinemic-euglycemic clamping was performed with [6-H-3] glucose tracer. Plasma glucose was measured using the glucose oxygenase method. Plasma insulin and C-peptide were determined by radioimmunoassays. Plasma FFAs were measured using a colorimetric method. RESULTS: Compared with infusion of SAL, plasma FFA levels were significantly elevated by infusions of IH, OLE, and LOH (P<0.001). All three fat infusions caused remarkably higher hepatic glucose production (HGP) than SAL (P<0.001). OLE and LOH infusions induced much higher HGP than IH (P<0.01). Glucose utilization (GU) was decreased with all three fat infusions relative to SAL (P<0.001), but GU did not differ among the three types of fat infusions. CONCLUSIONS: Short-term elevation of FFAs can induce hepatic and peripheral insulin resistance. Polyunsaturated fatty acids induced less hepatic insulin resistance than monounsaturated or saturated fatty acids. However, IH, OLE, and LOH infusions induced similar peripheral insulin resistance.

Inhibitory effect of schisandrin B on free fatty acid-induced steatosis in L-02 cells

AIM:To investigate the effects of schisandrin B (Sch B) on free fatty acid (FFA)-induced steatosis in L-02 cells.METHODS:Cellular steatosis was induced by incubating L-02 cells with a FFA mixture (oleate and palmitate at the ratio of 2:1) for 24 h.Cytotoxicity and apoptosis were evaluated by 3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and Annexin V/propidium iodide staining,respectively.Cellular total lipid was determined using a photocolorimetric method after Nile red staining,and triglyceride content was measured using an enzymatic kit.To study the effects of Sch B on steatosis,L-02 cells were treated with Sch B (1-100 μmol/L) in the absence or presence of 1 mmol/L FFA for 24 h,and cellular total lipid and triglyceride levels were measured.To explore the mechanisms of action of Sch B in the steatotic L-02 cells,mRNA levels of several regulators of hepatic lipid metabolism including adipose differentiation related protein (ADRP),sterol regulatory element binding protein 1 (SREBP-1),peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ were measured by quantitative real-time polymerase chain reaction (PCR),and protein levels of ADRP and SREBP-1 were measured by immunoblotting.RESULTS:Treatment with 1 mmol/L FFA for 24 h induced intracellular lipid accumulation in L-02 cells comparable to that in human steatotic livers without causing apparent apoptosis and cytotoxicity.Sch B mitigated cellular total lipid and triglyceride accumulations in the steatotic L-02 cells in a dose-dependent manner.Quantitative real-time PCR and Western blot analyses revealed that treatment of L-02 cells with 100 μmol/L Sch B reverted the FFA-stimulated up-regulation of ADRP and SREBP-1.CONCLUSION:Sch B inhibits FFA-induced steatosis in L-02 cells by,at least in part,reversing the up-regulation of ADRP and SREBP-1.

Genistein Reverses Free Fatty Acid-induced Insulin Resistance in HepG2 Hepatocytes through Targeting JNK

This study investigated the effects and molecular mechanisms of genistein in improving insulin resistance induced by free fatty acids （FFAs） in HepG2 hepatocytes. A model of insulin resistance in HepG2 cells was established by adding palmitic acid （0.5 mmol/L） to the culture medium and the cells were treated by genistein. Glucose consumption of HepG2 cells was determined by glucose oxidase method. The levels of c-jun N-terminal kinase （JNK） phosphorylation, insulin receptor substrate-1 （IRS-1） Ser307 phosphorylation, JNK, IRS-1, phosphatidylinositol-3-kinase p85 （PI-3K p85） and glucose transporter 1 （GLUT1） proteins were detected by Western blotting. The results showed that after the treatment with palmitic acid for 24 h, the insulin-stimulated glucose transport in HepG2 cells was inhibited, and the glucose consumption was substantially reduced. Meanwhile, the expressions of IRS-1, PI-3K p85 protein and GLUT1 were obviously reduced, while the levels of JNK phosphorylation and IRS-1 Ser307 phosphorylation and the expression of JNK protein were significantly increased, as compared with cells of normal control. However, the aforementioned indices, which indicated the existence of insulin resistance, were reversed by genistein at 1-4 μmol/L in a dose-dependent manner. It was concluded that insulin resistance induced by FFAs in HepG2 hepatocytes could be improved by genistein. Genistein might reverse FFAs-induced insulin resistance in HepG2 cells by targeting JNK.

Free fatty acids receptors 2 and 3 control cell proliferation by regulating cellular glucose uptake

BACKGROUND Colorectal cancer(CRC)is a worldwide problem,which has been associated with changes in diet and lifestyle pattern.As a result of colonic fermentation of dietary fibres,short chain free fatty acids are generated which activate free fatty acid receptors(FFAR)2 and 3.FFAR2 and FFAR3 genes are abundantly expressed in colonic epithelium and play an important role in the metabolic homeostasis of colonic epithelial cells.Earlier studies point to the involvement of FFAR2 in colorectal carcinogenesis.AIM To understand the role of short chain FFARs in CRC.METHODS Transcriptome analysis console software was used to analyse microarray data from CRC patients and cell lines.We employed short-hairpin RNA mediated down regulation of FFAR2 and FFAR3 genes,which was validated using quantitative real time polymerase chain reaction.Assays for glucose uptake and cyclic adenosine monophosphate(cAMP)generation was done along with immunofluorescence studies to study the effects of FFAR2/FFAR3 knockdown.For measuring cell proliferation,we employed real time electrical impedancebased assay available from xCELLigence.RESULTS Microarray data analysis of CRC patient samples showed a significant down regulation of FFAR2 gene expression.This prompted us to study the FFAR2 in CRC.Since,FFAR3 shares significant structural and functional homology with FFAR2,we knocked down both these receptors in CRC cell line HCT 116.These modified cell lines exhibited higher proliferation rate and were found to have increased glucose uptake as well as increased level of glucose transporter 1.Since,FFAR2 and FFAR3 signal through G protein subunit(Gαi),knockdown of these receptors was associated with increased cAMP.Inhibition of protein kinase A(PKA)did not alter the growth and proliferation of these cells indicating a mechanism independent of cAMP/PKA pathway.CONCLUSION Our results suggest role of FFAR2/FFAR3 genes in increased proliferation of colon cancer cells via enhanced glucose uptake and exclude the role of PKA mediated cAMP signalling.Alternate pathways could be involved that would ultimately result in increased cell proliferation as a result of down regulated FFAR2/FFAR3 genes.This study paves the way to understand the mechanism of action of short chain FFARs in CRC.

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.

Development of a Derivatization-free GC-FID Method for Evaluation of Free Fatty Acid Levels in Plasma of Diabetic Nephropathy Patients

Metabolism of free fatty acids（FFAs） is related to several important physiological events and therefore their quantitaion in biological samples arouses extensive interest and efforts.Existing gas chromatography with flame ionization detector（GC-FID） methods for the analysis of FFAs normally require derivatization of them in order to lower boiling points.But this extra procedure tends to induce additional error and it is laborious and time-consuming.A derivatization-free method was therefore established in the present investigation to determine FFAs in human plasma by capillary（GC-FID）.After extraction of FFAs from plasma,a highly polar FFAP（free fatty acid in plasma） column was employed to directly quantitate FFAs concentration,free from derivatization reaction.All sample pretreatments were carried out at room temperature,improving recovery of short-chain FFAs.Heptadecanoic acid（C17：0） was employed as internal standard,and the proposed method was validated for recovery,precision,sensitivity,stabi-lity,and linearity.Validation data show that it is suitable for clinical study that has been applied to the evaluation of FFAs levels in plasma of diabetic nephropathy（DN） patients during the course of treatment.Forty-seven patients diagnosed with DN were admitted to the double-blind experiment.Control group（n=17） underwent solely basic treatment and the patients did not show significant change in FFAs concentration during six months of treatment.Experiment group（n=30） was supplied with traditional Chinese medicine besides basic treatment.After six months of medication,their plasma concentration of palmitic acid（C16：0）,stearic acid（C18：0） and oleic acid（C18：1n-9） decreased while linolenic acid（C18：3n-3） increased significantly（P〈0.05）.These four compounds could be served as biomar-kers in the evaluation of drug efficacy,and their quantitation in plasma may provide additional information for disease progression in DN patients.

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.

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.

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.