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• Reverse phase column for SEC and RP separation of hydrophilic and hydrophobic macro molecules. • Use of phosphate buffer for sensitive detection of polysaccharide at UV 200 nm. • SEC-RPLC/UV method is simple and generic for analysis of free polysaccharides. The level of free polysaccharide is a critical quality attribute of polysaccharide-protein conjugate vaccines. The work presented describes a simple and sensitive method for the determination of low level free polysaccharides in multiple polysaccharide-protein conjugates. The method utilizes a reverse phase (RP) column to perform a size exclusion chromatography (SEC) separation of free polysaccharide and a reverse phase liquid chromatography (RPLC) separation of free protein and protein-polysaccharide conjugate. The use of phosphate buffer in the mobile phase enables the universal and sensitive detection of low level free polysaccharides at UV 200 nm. The method has been validated to monitor low level free polysaccharide (<1 %) in multiple polysaccharide-protein conjugates. The limit of quantitation is 2 μg/ml or 0.3 % free polysaccharide in 0.6 mg/ml polysaccharide-protein conjugate. The accuracy is in the range of 94.1.0–108.5 %. [ABSTRACT FROM AUTHOR]

Small-aroma chicken with a high protein content, is widely distributed in the mountainous area of Yunnan-Guizhou Plateau, China. In this work, three novel ACE-inhibitory peptides, Leu-Thr-Glu-Lys-Val-Val-Phe (LTQKVVF), Leu-Asp-Asp-His-Phe-Leu (LDDHFL), and Val-Pro-Gly-Pro-Glu-Pro-Lys-Pro (VPGPEPKP), were prepared from the Small-Aroma Chicken breast. The purification and identification protocol employed comprised ion-exchange chromatography, gel filtration chromatography, reverse phase-high performance liquid chromatography (RP-HPLC), and nano liquid chromatography-electrospray ionization–tandem mass (Nano-LC-ESI–MS/MS). Compared with VPGPEPKP and VPGPEPKP, LTQKVVF displayed noticeable ACE inhibitory activity, with IC 50 values of 189.34 μM. What's more, the Lineweaver-Burk plots revealed peptides LTQKVVF and LDDHFL acted as the mixed-competitive inhibitor while VPGPEPKP inhibited ACE in a manner similar to uncompetitive inhibition. The binding free energies of LTQKVVF, LDDHFL, and VPGPEPKP for ACE were −8.1 kcal/mol, −8.6 kcal/mol, and −9.6 kcal/mol, respectively. The strong inhibition of ACE by VPGPEPKP may be attributed to the interaction of hydrogen bonds, alkyl, and pi-alkyl, which formed by crucial residues of ACE. [Display omitted] • Three novel peptides were characterized from small-aroma chicken. • The peptides exhibited inhibition activity on angiotensin converting enzyme (ACE). • Lineweaver Burk plots was used to identify the inhibition mode of peptides. • The interactions between peptides and ACE were simulated by molecular docking. [ABSTRACT FROM AUTHOR]

HPLC, also known as High Performance Liquid Chromatography is a technique used to separate individual components in a single mixture. There are various types of this technique. They are partition chromatography, Normal phase chromatography, Reverse phase chromatography, size exclusion chromatography, ion exchange chromatography, bioaffinity chromatography and aqueous chromatography. The most popular one is the reverse phase chromatography. The mechanism of high performance liquid chromatography relies on pumps in which the solvent which is the pressurized liquid is passed through a solid adsorbent material. In this review, around 50 articles from Pubmed, Google scholar and other article databases which are relevant to the topic are taken. The types, parameters, instrumentation, major and other applications of HPLC are discussed. The various applications of high performance liquid chromatography were discussed and compared with the current and existing articles. The opposing and limiting factors are also discussed. High performance liquid chromatography is a reliable method used as a confirmatory test accordingly. HPLC in combination with other methods produce a very accurate result without any flaws. Many studies have been going on to analyse and reduce the flaws to produce a better insight over the usage of this method. [ABSTRACT FROM AUTHOR]

Umami‐ and kokumi‐taste‐active peptides in Maillard reaction products of enzymatic hydrolysate of bovine bone marrow extract (BBME) were purified and identified by ultrafiltration, gel filtration chromatography, reverse‐phase high‐performance liquid chromatography, liquid chromatography/quadrupole‐time of flight mass spectrometry (LC‐Q‐TOF‐MS/MS) and sensory evaluation. Eight umami and kokumi taste oligopeptides were found in the enzymatic hydrolysate of BBME. After Maillard reaction, the numbers of oligopeptides increased to twelve, such as Ala‐His, Ala‐Val‐His, Gly‐Pro, His‐Gly, Pro‐Ala‐His, Pro‐Val‐Asn‐His, Phe‐Glu‐Ala, Ala‐Ala‐Cys‐Arg, Leu‐Met, Leu‐Thr‐His, Pro‐Gly‐His, Ser‐Thr‐Gly‐Arg, which are linked to a variety of functional properties, such as ACE inhibitor, prolyl endopeptidase inhibitor and dipeptidyl peptidase IV inhibitor, etc. Eight peptides, including Cys‐Pro‐Arg, Ser‐Gly‐Val‐Glu, Pro‐Cys, Cys‐Glu, Cys‐Met‐Thr, Phe‐Glu‐Ala, Ala‐Gln and Leu‐Met, were synthesised and then subjected to sensory evaluation. The evaluation showed that among the eight peptides, Cys‐Pro‐Arg, Pro‐Cys and Leu‐Met led to the distinctly enhanced kokumi taste in blank beef soup. The influence of enzymatic hydrolysis and Maillard reaction to taste‐active components in bovine bone extract including amino acids, nucleotide and peptides. [ABSTRACT FROM AUTHOR]

The mechanism of action of food-derived angiotensin-I-converting enzyme (ACE) inhibitory peptides has not been completely elucidated. In the present study, ion-exchange chromatography, gel filtration chromatography, reverse phase-high performance liquid chromatography, and liquid chromatography-electrospray ionization–tandem mass (LC-ESI-MS/MS) were employed for purifying and identifying the ACE inhibitory peptides from hazelnut. To understand the mode of action of these peptides, ACE inhibition kinetics, in vitro and in vivo bioavailability assays, active site analysis, and interaction between the inhibitory peptides and ACE were investigated. The results identified novel ACE inhibitory peptides Ala-Val-Lys-Val-Leu (AVKVL), Tyr-Leu-Val-Arg (YLVR), and Thr-Leu-Val-Gly-Arg (TLVGR) with IC 50 values of 73.06, 15.42, and 249.3 μM, respectively. All peptides inhibited the ACE activity via a non-competitive mode. The binding free energies of AVKVL, YLVR, and TLVGR for ACE were −3.46, −6.48, and −7.37 kcal/mol, respectively. The strong inhibition of ACE by YLVR may be attributed to the formation of cation–pi interactions. [ABSTRACT FROM AUTHOR]

Our knowledge of the biological and environmental reactivity of siderophores is limited by the difficulty and cost of obtaining reasonable quantities by purification or synthesis. In this note, we describe a modified procedure for the low-cost, mg-scale purification of pyoverdin-type siderophores using a dual-flash chromatography (reverse-phase absorption and size exclusion) approach. [ABSTRACT FROM AUTHOR]

Abstract: The proteome of the venom of Naja haje legionis, the only medically important elapid species in Morocco, has been elucidated by using a combination of proteomic techniques that includes size exclusion chromatography, reverse-phase HPLC, Tricine/SDS-Page, tryptic digestion, Q-TOF tandem mass spectrometry and database search. The sequence analysis of venom fractions revealed a highly complex venom proteome which counts a total of 76 proteins identified from database that can be assigned into 9 proteins families. We report the identification of: cobra venom factor (CVF), l-amino-acid oxidases (LAAO), acetylcholinesterase (AChE), snake venom metalloproteinases (SVMP), cysteine rich secretory proteins (CRISP), venom nerve growth factor (vNGF), phospholipases A2 (PLA2), vespryns, kunitz-type inhibitor, short neurotoxins, long neurotoxins, weak neurotoxins, neurotoxin like proteins, muscarinic toxins, cardiotoxins and cytotoxins. Comparison of these proteins showed high sequence homology with proteins from other African and Asian cobras. Further works are needed to assess the contribution of individual toxins in venom toxicity. Biological significance: Naja haje legionis is one of the medically important snakes implicated in the pathogenesis of snake bite in Morocco. The absence of information about venom composition and clinical manifestations of envenomation by this cobra represents an obstacle for the management of this environmental disease in the country. The elucidation of Moroccan cobra venom composition will provide a reasonable guidance for clinician to understand the pathophysiological conditions associated with cobra envenomation and the elaboration of better management strategies. [Copyright &y& Elsevier]

A soy protein hydrolysate was found to inhibit rat platelet aggregation induced by ADP, an aggregating agent. To find out its principal antiplatelet peptide(s), the soy protein hydrolysate was separated successively by gel filtration chromatography, reverse-phase HPLC, and cation exchange HPLC. During the course of separation, we observed that most fractions had antiplatelet effects, which suggests that most peptides have some degree of antiplatelet effect. Following the inhibitory fractions, we purified and identified two new peptides, SSGE and DEE, by LC–electrospray ionization MS and peptide sequencing. Both peptides were highly hydrophilic. The concentrations to obtain 50% inhibition (IC50) of the aggregation intensity were approximately 480 and 460 μM, respectively, for SSGE and DEE. [Copyright &y& Elsevier]