Junrui Wu

Isolation and preliminary probiotic selection of lactobacilli from koumiss in Inner Mongolia.

From 16 samples of traditional fermented koumiss collected in Inner Mongolia Autonomous Region of China, forty‐eight lactobacilli strains were isolated and phenotypically characterized by their abilities to ferment different carbohydrates and by additional biochemical tests. The dominant lactobacilli species were identified as L. casei (17 strains), L. helveticus (10 strains) and L. plantarum (8 strains), with a lower frequency of isolation for L. coryniformis subsp. coryniformis (5 strains), L. paracasei (3 strains), L. kefiranofaciens (2 strains), L. curvatus (1 strain), L. fermentum (1 strain) and W. kandleri (1 strain). The pH values of all these samples were ranging from 3.37 to 3.94. In isolates, L. casei Zhang, L. helveticus ZL12‐1, and L. plantarum BX6‐6 were selected as potentially probiotic strains through the preliminary tests including resistance to low acid, abilities to grow in MRS with bile salts, antimicrobial activities and the viabilities during prolonged cold storage in fermented milk. Moreover 16S rDNA was conducted to confirm the identification. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Proteomics Analysis of Lactobacillus casei Zhang, a New Probiotic Bacterium Isolated from Traditional Home-made Koumiss in Inner Mongolia of China

Lactobacillus casei Zhang, isolated from traditional home-made koumiss in Inner Mongolia of China, was considered as a new probiotic bacterium by probiotic selection tests. We carried out a proteomics study to identify and characterize proteins expressed by L. casei Zhang in the exponential phase and stationary phase. Cytosolic proteins of the strain cultivated in de Man, Rogosa, and Sharpe broth were resolved by two-dimensional gel electrophoresis using pH 4–7 linear gradients. The number of protein spots quantified from the gels was 487 ± 21 (exponential phase) and 494 ± 13 (stationary phase) among which a total of 131 spots were identified by MALDI-TOF/MS and/or MALDI-TOF/TOF according to significant growth phase-related differences or high expression intensity proteins. Accompanied by the cluster of orthologous groups (COG), codon adaptation index (CAI), and GRAVY value analysis, the study provided a very first insight into the profile of protein expression as a reference map of L. casei. Forty-seven spots were also found in the study that showed statistically significant differences between exponential phase and stationary phase. Thirty-three of the spots increased at least 2.5-fold in the stationary phase in comparison with the exponential phase, including 19 protein spots (e.g. Hsp20, DnaK, GroEL, LuxS, pyruvate kinase, and GalU) whose intensity up-shifted above 3.0-fold. Transcriptional profiles were conducted to confirm several important differentially expressed proteins by using real time quantitative PCR. The analysis suggests that the differentially expressed proteins were mainly categorized as stress response proteins and key components of central and intermediary metabolism, indicating that these proteins might play a potential important role for the adaptation to the surroundings, especially the accumulation of lactic acid in the course of growth, and the physiological processes in bacteria cell.

Proteomic analysis of responses of a new probiotic bacterium Lactobacillus casei Zhang to low acid stress

Tolerance to acid is an important feature for probiotic bacteria during transition through the gastrointestinal tract. Proteomics analysis of a new probiotic bacterium, Lactobacillus casei Zhang, was performed upon 30-min exposure to low acid stress (pH 2.5 vs. pH 6.4) using two-dimensional electrophoresis. Out of 33 protein spots that showed changes of expression between the two pHs, 22 showed 1.5-fold higher expression at pH 2.5 than at pH 6.4, whereas five spots had expression decreased by 1.5-fold at pH 2.5. There were also six protein spots that were exclusively present on different pH maps. Further analysis showed that eight of the enhanced proteins, NagA, NagB, PGM, GlmM, LacC, TDP, GALM and PtsI, were involved in carbohydrate catabolism. Moreover, quantitative RT-PCR showed that the mRNA expression levels of dnaK, nagB, galm, estC, tuf and luxS were consistent with changes in protein expression. We postulate that there might be some relationship between differentially expressed proteins and acid tolerance in L. casei Zhang.

Effect of bile salts stress on protein synthesis of Lactobacillus casei Zhang revealed by 2-dimensional gel electrophoresis

Lactobacillus casei Zhang, isolated from koumiss in Inner Mongolia of China, is known from previous findings to be tolerant to bile salts. Bile salts secreted by mammals act as a natural antibacterial barrier and may serve as a component of innate immunity, as they have limited antagonistic effect against resident microflora. In this work, we compared the growth and protein expression patterns of L. casei Zhang with and without bile salts. Twenty-six proteins were found to be differentially expressed using 2-dimensional gel electrophoresis. Peptide mass fingerprinting was used to identify these proteins. Further verification by using real-time, quantitative reverse transcription-PCR and bioinformatics analysis showed that the implicated pathways are involved with a complex physiological response under bile salts stress, particularly including cell protection (DnaK and GroEL), modifications in cell membranes (NagA, GalU, and PyrD), and key components of central metabolism (PFK, PGM, CysK, LuxS, PepC, and EF-Tu). These results provide insight on the protein expression pattern of L. casei under bile salts stress and offer a new perspective for the molecular mechanisms involved in stress tolerance and adaptation of bacteria.

Changes in flavour and microbial diversity during natural fermentation of suan-cai, a traditional food made in Northeast China.

We measured changes in the main physical and chemical properties, flavour compounds and microbial diversity in suan-cai during natural fermentation. The results showed that the pH and concentration of soluble protein initially decreased but were then maintained at a stable level; the concentration of nitrite increased in the initial fermentation stage and after reaching a peak it decreased significantly to a low level by the end of fermentation. Suan-cai was rich in 17 free amino acids. All of the free amino acids increased in concentration to different degrees, except histidine. Total free amino acids reached their highest levels in the mid-fermentation stage. The 17 volatile flavour components identified at the start of fermentation increased to 57 by the mid-fermentation stage; esters and aldehydes were in the greatest diversity and abundance, contributing most to the aroma of suan-cai. Bacteria were more abundant and diverse than fungi in suan-cai; 14 bacterial species were identified from the genera Leuconostoc, Bacillus, Pseudomonas and Lactobacillus. The predominant fungal species identified were Debaryomyces hansenii, Candida tropicalis and Penicillium expansum.

Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS

Abstract Whey, an essential source of dietary nutrients, is widely used in dairy foods for infants. A total of 584 whey proteins in human and bovine colostrum and mature milk were identified and quantified by the isobaric tag for relative and absolute quantification (iTRAQ) proteomic method. The 424 differentially expressed whey proteins were identified and analyzed according to gene ontology (GO) annotation, Kyoto encyclopedia of genes and genomes (KEGG) pathway, and multivariate statistical analysis. Biological processes principally involved biological regulation and response to stimulus. Major cellular components were extracellular region part and extracellular space. The most prevalent molecular function was protein binding. Twenty immune-related proteins and 13 proteins related to enzyme regulatory activity were differentially expressed in human and bovine milk. Differentially expressed whey proteins participated in many KEGG pathways, including major complement and coagulation cascades and in phagosomes. Whey proteins show obvious differences in expression in human and bovine colostrum and mature milk, with consequences for biological function. The results here increase our understanding of different whey proteomes, which could provide useful information for the development and manufacture of dairy products and nutrient food for infants. The advanced iTRAQ proteomic approach was used to analyze differentially expressed whey proteins in human and bovine colostrum and mature milk. Graphical Abstract

Comparative proteomic analysis of milk-derived exosomes in human and bovine colostrum and mature milk samples by iTRAQ-coupled LC-MS/MS

Exosomes are membranous vesicles found in biological fluids with important functions. However, milk-derived exosome proteins from humans and bovines have not been studied in detail. The advanced iTRAQ proteomic approach was used to analyze milk-derived exosomes in human and bovine colostrum and mature milk samples. A total of 920 milk exosome proteins were identified and quantified. Among these, 575 differentially expressed exosome proteins (P<0.05) were found. Multivariate analysis, gene ontology (GO) annotation and the KEGG pathway were used to interpret the identified proteins. The major biological processes involved were: response to stimulus (22%), localization (16%), establishment of localization (14%), and cellular component organization (14%). Cellular components engaged in intracellular (31%) and intracellular part (31%). The most prevalent molecular function mainly touched upon binding (52%). Milk exosome proteins participated in several KEGG pathways containing ribosome, regulation of actin cytoskeleton, glycolysis/gluconeogenesis, leukocyte transendothelial migration, aminoacyl-tRNA biosynthesis, pentose phosphate pathway, galactose metabolism and fatty acid biosynthesis. These results provide important information on human and bovine milk exosomes, and increase knowledge on the proteomes of these exosomes across different lactation stages, which could provide potential directions for newborn milk powder, biological markers and functional foods.

Gene expression of Lactobacillus plantarum FS5-5 in response to salt stress

Lactobacillus plantarum FS5-5—a new isolate from agricultural soybean paste—shows good growth in an environment with 12xa0% (w/v) NaCl. To understand the potential relationship between regulatory systems and its growth under different salt concentrations, the expression of genes encoding enzymes involved in five regulatory systems was determined by quantitative real-time polymerase chain reaction (qPCR). The results showed that the optimal growth of this strain relied on the cooperative action of different regulatory systems, with each system playing an important role in L. plantarum FS5-5 in adapting to salt stress. Interestingly, most of the genes showed higher expression under higher concentrations of NaCl, suggesting that L. plantarum FS5-5 uses its regulatory systems preferentially to sustain its growth. In addition, the expression change of a gene encoding glyceraldehyde-3-phosphate dehydrogenase (gapB) was not significant under different salt concentrations, indicating that it may be used as a housekeeping gene in the qPCR experiment on L. plantarum after exposure to salt stress. This study provided a global view of the effect of NaCl on the ability of L. plantarum FS5-5 to resist salt stress and may help us to develop strategies to improve the robustness of industrial production.

Characterization and comparison of whey N-glycoproteomes from human and bovine colostrum and mature milk

Milk glycoproteins are crucial nutrients with a variety of functions. However, whey N-glycoproteomes in human and bovine milks have not been characterized during lactation. Herein, using lectin enrichment and liquid chromatography tandem mass spectrometry, 68, 58, 100, and 98 N-glycoproteins were identified in human colostrum and mature milk as well as bovine colostrum and mature milk whey. Gene Ontology and KEGG pathway analyses were used to elucidate the biological functions of whey N-glycoproteins in human and bovine colostrum and mature milks. Whey N-glycoproteomes differed dramatically between human and bovine milks and across lactation stages. The conserved and specific whey N-glycoproteins in all four sample types were also determined. Our results improve understanding of the properties and biological functions of whey N-glycoproteins in human and bovine milk and colostra, and provide insight into the potential application of some N-glycoproteins in infant formulae at different stages of development.

The dynamic changes of chemical components and microbiota during the natural fermentation process in Da-Jiang, a Chinese popular traditional fermented condiment

There is limited information on the chemical components and microbiota changes involved in da-jiang, a Chinese popular traditional fermented soybean condiment. Therefore, dynamic changes both in physicochemical quality and microbial community diversity of 20 samples of naturally fermented da-jiang collected over 95u202fdays were measured, and we classified the samples into 5 groups (each containing 4 time points) according to the results. Based on OTUs analysis, Group 2 and Group 3 might represent the shift-stage between bacteria and fungi. Also, our results demonstrated that the formation of flavour, nutrient and quality of the traditional da-jiang were actually related to all of microbial community, especially the increased bacteria, including Staphylococcus and Leuconostoc. Correspondingly, Membrane Transport, Carbohydrate Metabolism, Amino Acid Metabolism and Replication and Repair were the top 4 KEGG pathways based on bacterial genes. The findings are expected to be helpful for us not only to understand the formation of flavour, taste and quality of traditional fermented da-jiang, but also to improve the industrialised fermented da-jiang.