Heat, cold, acid, and bile salt induced differential proteomic responses of a novel potential probiotic Lactococcus garvieae C47 isolated from camel milk.

Probiotics encounter various stresses during food processing and digestion. This study evaluated the differential proteomic responses of a newly identified potential probiotic lactic acid bacteria, Lactococcus garvieae, isolated from camel milk. Lc. garvieae C47 was exposed to heat, cold, acid, and bile conditions, and stress-responsive proteins were identified. The proteomic analysis was done using 2D-IEF SDS PAGE and nano-LC-MS/MS. Out of 91 differentially expressed proteins, 20 upregulated and 27 downregulated proteins were shared among the stresses. The multivariate data analysis revealed abundance of elongation factor Ts (spot C42), uridine phosphorylase, fructose-bisphosphate aldolase, peptidase T, cobalt ECF transporter T component CbiQ, UDP-N-acetylmuramate-l-alanine ligase, uncharacterized protein, aspartokinase, chaperone protein DnaK, IGP synthase cyclase subunit, probable nicotinate-nucleotide adenylyltransferase, NADH-quinone oxidoreductase, holo-[acyl-carrier-protein] synthase, l-lactate dehydrogenase, and uncharacterized protein. The maximum number of differentially expressed proteins belonged to carbohydrate and protein metabolism, which indicates Lc. garvieae shifts towards growth and energy metabolism for resistance against stress conditions.

Potential Probiotic Pediococcus pentosaceus M41 Modulates Its Proteome Differentially for Tolerances Against Heat, Cold, Acid, and Bile Stresses.

Probiotics containing functional food confer health benefits in addition to their nutritional properties. In this study, we have evaluated the differential proteomic responses of a potential novel probiotic Pediococcus pentosaceus M41 under heat, cold, acid, and bile stress conditions. We identified stress response proteins that could provide tolerances against these stresses and could be used as probiotic markers for evaluating stress tolerance. Pediococcus pentosaceus M41 was exposed for 2 h to each condition: 50°C (heat stress), 4°C (cold stress), pH 3.0 (acid stress) and 0.05% bile (bile stress). Proteomic analysis was carried out using 2D-IEF SDS PAGE and LC-MS/MS. Out of 60 identified proteins, 14 upregulated and 6 downregulated proteins were common among all the stress conditions. These proteins were involved in different biological functions such as translation-related proteins, carbohydrate metabolism (phosphoenolpyruvate phosphotransferase), histidine biosynthesis (imidazole glycerol phosphate synthase) and cell wall synthesis (tyrosine-protein kinase CapB). Proteins such as polysaccharide deacetylase, lactate oxidase, transcription repressor NrdR, dihydroxyacetone kinase were upregulated under three out of the four stress conditions. The differential expression of these proteins might be responsible for tolerance and protection of P. pentosaceus M41 against different stress conditions.

Biological activities of the bioaccessible compounds after in vitro digestion of low-fat Akawi cheese made from blends of bovine and camel milk.

The objective of this study was to assess protein degradation and biological activities of the water-soluble extract (WSE) and the 10 kDa permeable and nonpermeable fractions of in vitro digesta of low-fat Akawi cheese made from blends (100:0, 85:15, or 70:30) of bovine milk and camel milk and ripened for 28 d. Biological activities, such as antioxidant activities, amylase and glucosidase inhibition, angiotensin-converting enzyme inhibition, and antiproliferative of the WSE, and the 10 kDa permeable and nonpermeable fraction of the digesta were assessed. To identify the nature of the bioaccessible compounds, untargeted metabolomic analysis was carried out by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Higher o-phthaldialdehyde absorbances were observed in cheeses made of bovine-camel milk blends compared with cheese from bovine milk only. The WSE from these blends also exhibited higher angiotensin-converting enzyme inhibitory effects and higher antiproliferative effects than from bovine milk. The results from this study suggest that the use of blends of camel milk and bovine milk can modulate biological activities of low-fat Akawi cheese.

Invited review: Characterization of new probiotics from dairy and nondairy products-Insights into acid tolerance, bile metabolism and tolerance, and adhesion capability.

The selection of potential probiotic strains that possess the physiological capacity of performing successfully in the gastrointestinal tract (GIT) is a critical challenge. Probiotic microorganisms must tolerate the deleterious effects of various stresses to survive passage and function in the human GIT. Adhesion to the intestinal mucosa is also an important aspect. Recently, numerous studies have been performed concerning the selection and evaluation of novel probiotic microorganisms, mainly probiotic bacteria isolated from dairy and nondairy products. Therefore, it would be crucial to critically review the assessment methods employed to select the potential probiotics. This article aims to review and discuss the recent approaches, methods used for the selection, and outcomes of the evaluation of novel probiotic strains with the main purpose of supporting future probiotic microbial assessment studies. The findings and approaches used for assessing acid tolerance, bile metabolism and tolerance, and adhesion capability are the focus of this review. In addition, probiotic bile deconjugation and bile salt hydrolysis are explored. The selection of a new probiotic strain has mainly been based on the in vitro tolerance of physiologically related stresses including low pH and bile, to ensure that the potential probiotic microorganism can survive the harsh conditions of the GIT. However, the varied experimental conditions used in these studies (different types of media, bile, pH, and incubation time) hamper the comparison of the results of these investigations. Therefore, standardization of experimental conditions for characterizing and selecting probiotics is warranted.

Updates on understanding of probiotic lactic acid bacteria responses to environmental stresses and highlights on proteomic analyses.

Probiotics are defined as live microorganisms that improve the health of the host when administered in adequate quantities. Nonetheless, probiotics encounter extreme environmental conditions during food processing or along the gastrointestinal tract. This review discusses different environmental stresses that affect probiotics during food preparation, storage, and along the alimentary canal, including high temperature, low temperature, low and alkaline pH, oxidative stress, high hydrostatic pressure, osmotic pressure, and starvation. The understanding of how probiotics deal with environmental stress and thrive provides useful information to guide the selection of the strains with enhanced performance in specific situations, in food processing or during gastrointestinal transit. In most cases, multiple biological functions are affected upon exposure of the cell to environmental stress. Sensing of sublethal environmental stress can allow for adaptation processes to occur, which can include alterations in the expression of specific proteins.

Proteomic and ecophysiological responses of soybean (Glycine max L.) root nodules to Pb and hg stress.

BACKGROUND: Lead (Pb) and mercury (Hg) are persistent hazardous metals in industrially polluted soils which can be toxic in low quantities. Metal toxicity can cause changes at cellular and molecular level which should be studied for better understanding of tolerance mechanism in plants. Soybean (Glycine max L.) is an important oilseed crop of the world including India. Indian soils growing soybean are often contaminated by Pb and Hg. The aim of this study was to explore how soybean root nodule responds to Pb and Hg through proteomic and ecophysiological alterations in order to enhance tolerance to metal stress. RESULTS: Soybean plants were exposed to Pb (30 ppm PbCl2) and Hg (0.5 ppm HgCl2) to study histological, histochemical, biochemical and molecular response of N2-fixing symbiotic nodules. Both Pb and Hg treatment increased the level of oxidative stress in leaves and nodules. Chlorosis in leaves and morphological/anatomical changes in nodules were observed. Activities of ascorbate peroxidase, glutathione reductase and catalase were also modulated. Significant changes were observed in abundance of 76 proteins by Pb and Hg. Pb and Hg influenced abundance of 33 proteins (17 up and 16 down) and 43 proteins (33 up and 10 down), respectively. MS/MS ion search identified 55 proteins which were functionally associated with numerous cellular functions. Six crucial proteins namely catalase (CAT), allene oxide synthase (AOS), glutathione S-transferase (GST), calcineurin B like (CBL), calmodulin like (CML) and rapid alkalinisation factor (RAF) were selected for transcript abundance estimation. The qRT-PCR based real time expression exhibited a positive correlation with proteomics expression except for GST and RAF. CONCLUSION: Soybean root nodule responds to metal stress by increased abundance of defence, development and repair related proteins. An efficient proteomic modulation might lead to metal-induced stress tolerance in N2-fixing nodules. Although concentrations of Pb and Hg used in the study cannot be considered equimolar, yet Hg seems to induce more changes in nodule proteomic profile, and higher damage to both bacteroides and root anatomy.

A novel multicopper oxidase (laccase) from cyanobacteria: Purification, characterization with potential in the decolorization of anthraquinonic dye.

A novel extracellular laccase enzyme produced from Spirulina platensis CFTRI was purified by ultrafiltration, cold acetone precipitation, anion exchange and size exclusion chromatography with 51.5% recovery and 5.8 purification fold. The purified laccase was a monomeric protein with molecular mass of ~66 kDa that was confirmed by zymogram analysis and peptide mass fingerprinting. The optimum pH and temperature of the enzyme activity was found at 3.0 and 30°C using ABTS as substrate but the enzyme was quite stable at high temperature and alkaline pH. The laccase activity was enhanced by Cu+2, Zn+2 and Mn+2. In addition, the dye decolorization potential of purified laccase was much higher in terms of extent as well as time. The purified laccase decolorized (96%) of anthraquinonic dye Reactive blue- 4 within 4 h and its biodegradation studies was monitored by UV visible spectra, FTIR and HPLC which concluded that cyanobacterial laccase can be efficiently used to decolorize synthetic dye and help in waste water treatment.

Allium sativum Protease Inhibitor: A Novel Kunitz Trypsin Inhibitor from Garlic Is a New Comrade of the Serpin Family.

PURPOSE: This study was aimed to purify and characterize the Protease inhibitor (PI) from a plant Allium sativum (garlic) with strong medicinal properties and to explore its phytodrug potentials. METHODS: Allium sativum Protease Inhibitor (ASPI) was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD) spectroscopy. RESULTS: ASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max)" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2-12 showing a decline in the activity around pH 4-5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10-80°C) but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (µM/min) and Km value of 0.12 µM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM). The Far UV CD depicted 2.0% α -helices and 51% ß -sheets at native pH. CONCLUSIONS: To conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids present in the reactive sites. Therefore, ASPI with trypsin inhibitory property has the potential to be used as a non-cytotoxic clinical agents.