Study of aroma formation and transformation during the manufacturing process of Biluochun green tea in Yunnan Province by HS-SPME and GC-MS.

BACKGROUND: Biluochun is a typical non-fermented tea and is also famous for its unique aroma in China. Few studies have been performed to evaluate the effect of the manufacturing process on the formation and content of its aroma. RESULTS: The volatile components were extracted at different manufacturing process steps of Biluochun green tea using fully automated headspace solid-phase microextraction (HS-SPME) and further characterised by gas chromatography-mass spectrometry (GC-MS). Among 67 volatile components collected, the fractions of linalool oxides, ß-ionone, phenylacetaldehyde, aldehydes, ketones, and nitrogen compounds were increased while alcohols and hydrocarbons declined during the manufacturing process. The aroma compounds decreased the most during the drying steps. CONCLUSION: We identified a number of significantly changed components that can be used as markers and quality control during the producing process of Biluochun. The drying step played a major role in the aroma formation of green tea products and should be the most important step for quality control. © 2016 Society of Chemical Industry.

Different expression profiles of bioactive peptides in Pelophylax nigromaculatus from distinct regions.

Amphibian skin is an abundant repository of bioactive peptides, important components of the defensive system. The variability of the bioactive peptide repertoires of individual species remains unclear. In this study, dark-spotted frogs were collected from Kunming in Yunnan Province, China and Guiyang in Guizhou Province, China to determine whether the bioactive peptides in amphibian skin differ between the two regions. Eight antimicrobial peptides and an antioxidant peptide were identified by screening of cDNA library. Among the identified peptides, three antimicrobial peptides (pelophylaxin-2GY, temporin-1GY, and temporin-1KM) and an antioxidant peptide (antioxidin-PN) are reported here for the first time. Nigrocin-1, nigrocin-2, and pelophylaxin-2 were expressed by frogs in both regions. Pelophylaxin-2GY and temporin-1GY were found only in the frogs from Guiyang, whereas antioxidin-PN, esculetin-1, esculetin-2, and temporin-1KM were found only in those from Kunming. This difference was confirmed by allele-specific RT-PCR. The bioactive peptides expressed clearly varied between these populations of the same species.

[Screening and breeding high 1,3-propanediol producing strains by genome shuffling].

OBJECTIVE: To improve the tolerance of main metabolites, we used genome shuffling to achieve high 1,3-propanediol producing mutants. METHODS: Based on 96 deep-well palates containing prepared ended fed-batch broth as an efficient selection method, genome shuffling has been applied in strain improvement. RESULTS: Five high producers were obtained after genome shuffling (LSG1, LSG2, LSG4, LSG5 and LSG6). During batch fermentation (3 L), the 1, 3-propanediol production of the five mutants were improved 17.0%, 19.0%, 12.9%, 23.9% and 18.0% , compared with the parent strain; the conservations from glycerol were improved 17.7%, 20.0%, 13.3%, 24.4% and 17.7%. CONCLUSION: Genome shuffling was an efficient approach for strain improvement, and 96 deep-well palates containing fed-batch broth has been demonstrated as an efficient selection approach.

An anticoagulant serine protease from the wasp venom of Vespa magnifica.

Wasp is an important venomous animal that can induce human fatalities. Coagulopathy is a clinical symptom after massive wasp stings, but the reason leading to the envenomation manifestation is still not known. In this paper, a toxin protein is purified and characterized by Sephadex G-75 gel filtration, CM-Sephadex C-25 cationic exchange and fast protein liquid chromatography (FPLC) from the venom of the wasp, Vespa magnifica (Smith). This protein, named magnvesin, contains serine protease-like activity and inhibits blood coagulation. The cDNA encoding magnvesin is cloned from the venom sac cDNA library of the wasp. The deduced protein from the cDNA is composed of 305 amino acid residues. Magnvesin shares 52% identity with allergen serine protease from the wasp Polistes dominulus. Magnvesin exerted its anti-coagulant function by hydrolyzing coagulant factors TF, VII, VIII, IX and X.

Significance of the extra C-terminal tail of CaLP, a novel calmodulin-like protein involved in oyster calcium metabolism.

Oyster (Pinctada fucata) calmodulin-like protein (CaLP), containing a C-terminally extra hydrophilic tail (150D-161K), is a novel protein involved in the regulation of oyster calcium metabolism. To investigate the importance of the extra fragment to the Ca(2+)/Mg(2+)-dependent conformational changes in the intact CaLP molecule and the interactions between CaLP and its target proteins, a truncated CaLP mutant (M-CaLP) devoid of the extended C-terminus was constructed and overexpressed in Escherichia coli. The conformational characteristics of M-CaLP were studied by CD and fluorescence spectroscopy and compared with those of the oyster CaM and CaLP. The far-UV CD results reveal that the extra tail has a strong effect on the Ca(2+)-induced, but a relatively weak effect on the Mg(2+)-induced conformational changes in CaLP. However, upon Ca2+ or Mg2+ binding, only slight changes for intrinsic phenylalanine and tyrosine fluorescence spectra between M-CaLP and CaLP are observed. Our results also indicate that the extra tail can significantly decrease the exposure of the hydrophobic patches in CaLP. Additionally, affinity chromatography demonstrates that the target binding of CaLP is greatly influenced by its additional tail. All our results implicate that the extra tail may play some important roles in the interactions between CaLP and its targets in vivo.

Comparative analysis of volatiles difference of Yunnan sun-dried Pu-erh green tea from different tea mountains: Jingmai and Wuliang mountain by chemical fingerprint similarity combined with principal component analysis and cluster analysis.

BACKGROUND: Modern instrumental analysis technology can provide various chemical data and information on tea samples. Unfortunately, it remains difficult to extract the useful information. We describe the use of chemical fingerprint similarities, combined with principal component and cluster analyses, to distinguish and recognize Pu-erh green teas, which from two tea mountains, Wuliang and Jingmai, in the Pu-erh district of Yunnan province. The volatile components of all 20 Pu-erh green teas (10 Wuliang and 10 Jingmai teas) were extracted and identified by headspace solid-phase micro extraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS). RESULTS: Sixty-three volatiles (including alcohols, hydrocarbons, ketones, and aldehydes) were identified in the 20 Pu-erh green teas, and differences in compound compositions between them were also observed. Through fingerprint similarity, combined with principal component and cluster analyses, the 20 Pu-erh green teas were differentiated successfully based on their volatile characteristics. CONCLUSIONS: This study demonstrates that the GC-MS combined with chemical fingerprint and unsupervised pattern recognition method is suitable for the investigation of the volatile profiling and evaluating the quality and authenticity of teas related to the different origins.Graphical abstractDifferentiate Pu-erh green teas from different tea mountains by using chemical fingerprint similarity and multivariate statistical methods.

Oolong tea made from tea plants from different locations in Yunnan and Fujian, China showed similar aroma but different taste characteristics.

Consistent aroma characteristics are important for tea products. However, understanding the formation of tea aroma flavor and correspondingly proposing applicable protocols to control tea quality and consistency remain major challenges. Oolong tea is one of the most popular teas with a distinct flavor. Generally, oolong tea is processed with the leaves of tea trees belonging to different subspecies and grown in significantly different regions. In this study, Yunnan and Fujian oolong teas, green tea, black tea, and Pu-erh tea were collected from major tea estates across China. Their sensory evaluation, main water-soluble and volatile compounds were identified and measured. The sensory evaluation, total polysaccharide, caffeine, and catechin content of Yunnan oolong tea was found to be different from that of Fujian oolong tea, a result suggesting that the kinds of tea leaves used in Yunnan and Fujian oolong teas were naturally different. However, according to their aroma compounds, principal component analysis (PCA) and cluster analysis (CA) of the volatile compounds showed that the two types of oolong teas were similar and cannot be clearly distinguished from each other; they are also different from green, black, and Pu-erh teas, a result indicating that the same oolong tea processing technology applied to different tea leaves results in consistent aroma characteristics. The PCA analysis results also indicated that benzylalcohol, indole, safranal, linalool oxides, ß-ionone, and hexadecanoic acid methyl ester highly contributed to the distinct aroma of oolong tea compared with the other three types of teas. This study proved that the use of the same processing technology on two kinds of tea leaves resulted in a highly consistent tea aroma.

Structure-function relationship of three neurotoxins from the venom of Naja kaouthia: a comparison between the NMR-derived structure of NT2 with its homologues, NT1 and NT3.

Three homologous short-chain neurotoxins, named NT1, NT2 and NT3, were purified from the venom of Naja kaouthia. NT1 has an identical amino acid sequence to cobrotoxin from Naja naja atra [Biochemistry 32 (1993) 2131]. NT3 shares the same sequence with cobrotoxin b [J. Biochem. (Tokyo) 122 (1997) 1252], whereas NT2 is a novel 61-residue neurotoxin. Tests of their physiological functions indicate that NT1 shows a greater inhibition of muscle contraction induced by electrical stimulation of the nerve than do NT2 and NT3. Homonuclear proton two-dimensional NMR methods were utilized to study the solution tertiary structure of NT2. A homology model-building method was employed to predict the structure of NT3. Comparison of the structures of these three toxins shows that the surface conformation of NT1 facilitates the substituted base residues, Arg28, Arg30, and Arg36, to occupy the favorable spatial location in the central region of loop II, and the cation groups of all three arginines face out of the molecular surface of NT1. This may contribute greatly to the higher binding of NT1 with AchR compared to NT2 and NT3.

Molecular cloning and expression of a pearl oyster (Pinctada fucata) homologue of mammalian putative tumor suppressor QM.

The QM gene was originally identified as a putative tumor suppressor gene from a Wilms' tumor cell line by subtractive hybridization assay. Later studies showed that the QM protein is multifunctional, involved in cell growth and differentiation, energy metabolism, respiration, and cytoskeletal function. In this report a full-length complementary DNA encoding a QM counterpart in pearl oyster (Pinctada fucata) was isolated. Phylogenetic analysis shows that oyster QM is more closely related to its insect homologues than to the mammalian homologues. Analysis of the tissue expression pattern of the oyster QM gene showed that oyster QM messenger RNA is expressed in all tissues tested, with highest levels in the digestive gland and mantle. Furthermore, we expressed the QM protein in Escherichia coli; Western blotting showed that the antibody of human QM is immunoreactive to the expressed oyster QM protein. Incubation of the oyster QM with Zn2+ resulted in the reduction of intrinsic emission fluorescence and a red-shift in the lambda(max) emission, indicating the occurrence of Zn(2+)-induced conformational changes. This evidence presents a possible mechanism for the critical function of zinc ion in the interaction of QM with Jun.

A novel ferritin subunit involved in shell formation from the pearl oyster (Pinctada fucata).

Iron is one of the most important minor elements in the shell of bivalves. This study was designed to investigate the involvement of ferritin, the principal protein for iron storage, in shell formation. A novel ferritin cDNA from the pearl oyster (Pinctada fucata) was isolated and characterized. The ferritin cDNA encodes a 206 amino acid polypeptide, which shares high similarity with snail soma ferritin and the H-chains of mammalian ferritins. Oyster ferritin mRNA shows the highest level of expression in the mantle, the organ for shell formation. In situ hybridization analysis revealed that oyster ferritin mRNA is expressed at the highest level at the mantle fold, a region essential for metal accumulation and contributes to metal incorporation into the shell. Taken together, these results suggest that ferritin is involved in shell formation by iron storage. The identification and characterization of oyster ferritin also helps to further understand the structural and functional properties of molluscan ferritins.

A novel matrix protein participating in the nacre framework formation of pearl oyster, Pinctada fucata.

Understanding the molecular composition is of great interest for both nacre formation mechanism and biomineralization in mollusk shell. A cDNA clone encoding an MSI31 relative, termed MSI7 because of its estimated molecular mass of 7.3 kDa, was isolated from the pearl oyster, Pinctada fucata. This novel protein shares similarity with MSI31, a prismatic framework protein of P. fucata. It is peculiar that MSI7 is much shorter in size, harboring only the Gly-rich sequence that has been proposed to be critical for Ca(2+) binding. In situ hybridization result showed that MSI7 mRNA was expressed specifically at the folds and outer epithelia of the mantle, indicating that MSI7 participates in the framework formation of both the nacreous layer and prismatic layer. In vitro experiment on the function of MSI7 suggested that it accelerates the nucleation and precipitation of CaCO(3). Taken together, we have identified a novel matrix protein of the pearl oyster, which may play an important role in determining the texture of nacre.

A Novel Myotoxin from the Venom of Trimeresurus mucrosquamatus.

A novel myotoxin, designated TMPB, was purified from the venom of Trimeresurus mucrosquamatus by Sephadex G-100 superfine gel chromatography and fast protein liquid chromatography (FPLC). The N-terminal sequence of 24 amino acid residues was determined by protein sequencer. The sequence similarities between TMPB and other two phospholipase A(2) (PLA2s) previously purified from the same venom were 41.7% and 54.2%, respectively, but TMPB showed no detectable PLA(2) hydrolytic activity. Its molecular weight was estimated to be 16 000 by reducing SDS-PAGE and isoelectric point was determined to be 9.2 by isoelectric focusing electrophoresis. TMPB exhibited strong myotoxicity and platelet aggregation inhibiting activity, and the two activities could all be inhibited by heparin.

Comparative analysis of Pu-erh and Fuzhuan teas by fully automatic headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry and chemometric methods.

Thirteen Pu-erh teas and 13 Fuzhuan teas obtained from two different production areas in China were profiled using fully automatic headspace solid-phase microextraction (HS-SPME)/gas chromatography-mass spectrometry (GC-MS) coupled with chemometric methods. A total of 93 aroma components were identified in 26 dark teas; among them, methoxyphenolic compounds (31.77%) were the most abundant components in Pu-erh teas, whereas ketone compounds were the most abundant components (25.42%) in Fuzhuan teas. Cluster analysis (CA) and principal component analysis (PCA) showed that these two types of dark teas could be clearly distinguished according to their chemical characteristics. This study suggested that the proposed strategy could provide a feasible and rapid technique to differentiate dark teas with similar morphological characteristics from different production areas by their volatile composition and relative content.

The study of fingerprint characteristics of Dayi Pu-Erh tea using a fully automatic HS-SPME/GC-MS and combined chemometrics method.

The quality of tea is presently evaluated by the sensory assessment of professional tea tasters, however, this approach is both inconsistent and inaccurate. A more standardized and efficient method is urgently needed to objectively evaluate tea quality. In this study, the chemical fingerprint of 7 different Dayi Pu-erh tea brands and 3 different Ya'an tea brands on the market were analyzed using fully automatic headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS). A total of 78 volatiles were separated, among 75 volatiles were identified by GC-MS in seven Dayi Pu-erh teas, and the major chemical components included methoxyphenolic compounds, hydrocarbons, and alcohol compounds, such as 1,2,3-trimethoxybenzene, 1,2,4-trimethoxybenzene, 2,6,10,14-tetramethyl-pentadecane, linalool and its oxides, α-terpineol, and phytol. The overlapping ratio of peaks (ORP) of the chromatogram in the seven Dayi Pu-erh tea samples was greater than 89.55%, whereas the ORP of Ya'an tea samples was less than 79.10%. The similarity and differences of the Dayi Pu-erh tea samples were also characterized using correlation coefficient similarity and principal component analysis (PCA). The results showed that the correlation coefficient of similarity of the seven Dayi Pu-erh tea samples was greater than 0.820 and was gathered in a specific area, which showed that samples from different brands were basically the same, despite have some slightly differences of chemical indexes was found. These results showed that the GC-MS fingerprint combined with the PCA approach can be used as an effective tool for the quality assessment and control of Pu-erh tea.

[Separation strategy of affinity chromatography for mixed antibodies].

A mammary gland bioreactor can efficiently express human recombinant monoclonal antibody. However, the target products are similar to the bovine antibody in the raw emulsion material in properties and structures. Thus it is difficult to achieve effective separation of the target products. In this work, the species differences between bovine antibody and recombinant human antibody were analyzed and a new separation strategy was raised based on it. We employed two kinds of affinity chromatography to separate these two antibodies from each other and studied the effect of elution mode upon separation. The results demonstrated that Protein A affinity chromatography could get hybrid antibodies using gradient elution mode, but hardly separate the recombinant human antibody and bovine antibody from each other. In contrast, the combination of Protein A affinity chromatography and displacement chromatography could separate the hybrid antibodies effectively and finally give recombinant human IgG (rHGG) product with the purity of 95% and the yield of more than 95%. Immuno-affinity chromatography could also effectively purify recombinant monoclonal antibodies and owned better generality, which could be used in purification of recombinant antibody expressed by any animal mammary gland. -

A weak neurotoxin exhibits synergic effect with cardiotoxins when co-applied to two non-neural cell lines.

A basic peptide with mass weight of 7.597 kDa was isolated and purified from the Naja atra venom by using the combination of ion exchange chromatography and reverse phase high performance liquid chromatography. N-terminal protein sequence determination revealed that this peptide was a weak neurotoxin. Neurotoxicity and cytotoxicity assay were performed. It was noticed that although the analysis of protein sequence did not show it was much more basic, this neurotoxin was eluted out after a cardiotoxin-like basic protein (CLBP). It was also found that, despite of low neurotoxicity, when applied to two non-neural cell lines including K562 cells and K1735-M2 cells, this weak neurotoxin exhibits synergic effects with cardiotoxins, which is firstly reported. It was presumed that the synergic effect might be due to the presence of their common characteristic tertiary structure, three-finger structure. This fact might bring us some new sights about the functions of the un-lethal components in the complex venom system and may help us to understand how the venom really works as an integrative system.

A novel short neurotoxin, cobrotoxin c, from monocellate cobra (Naja kaouthia) venom: isolation and purification, primary and secondary structure determination, and tertiary structure modeling.

A novel short neurotoxin, cobrotoxin c (CBT C) was isolated from the venom of monocellate cobra (Naja kaouthia) using a combination of ion-exchange chromatography and FPLC. Its primary structure was determined by Edman degradation. CBT C is composed of 61 amino acid residues. It differs from cobrotoxin b (CBT B) by only two amino acid substitutions, Thr/Ala11 and Arg/Thr56, which are not located on the functionally important regions by sequence similarity. However, the LD50 is 0.08 mg/g to mice, i.e. approximately five-fold higher than for CBT B. Strikingly, a structure-function relationship analysis suggests the existence of a functionally important domain on the outside of Loop III of CBT C. The functionally important basic residues on the outside of Loop III might have a pairwise interaction with alpha subunit, instead of gamma or delta subunits of the nicotinic acetylcholine receptor (nAChR).