Proteomics of E.coli Nissle 1917 in Responce to Cocos nucifera sap and Wine

In the present study, we described the protein profile experimentally by 2D-PAGE and MALDI analysis to understand the stress mechanisms of cocoti sap and wine on E.coli Nissle 1917. We isolated one newly expressed protein from cocoti wine treated gel which is not present in both control and cocoti sap treated sample i.e. P21 prophage-derived head-stabilizing proteinVG03_ECOL6 (3n1) also called as Head protein gp3. This protein mainly activities related to the viral life cycle. It helps to attach the viral gene into host. The growth rate was delayed in cocoti wine treated E.coli Nissle 1917 when compared to control and cocoti sap treated samples. Stress mechanism induce many proteins they are involved in metabolic process, hydrolase activity, lyase activity, quinone binding, phosphotransferase system, carbohydrate metabolism, DNA binding, DNA repair, transferase activity, oxidoreductase, purine metabolism, transcription antitermination, transcription regulation and other related activities. We proved that the predicted protein structure quality, resolution, density and error plot values by QMEAN analysis. Based on these results, only two differentially expressed proteins under sap stress showed that the significant results, which were N-acetylgalactosamine-specific phosphotransferase enzyme IIB component 1, PTPB1_ECOLI and DinI-like protein Z3305/ECs2939 in prophage CP-933VDINI1_ECO57. In case of wine stress, the differentially expressed proteins were Transcription anti-termination protein RFAHECO57 NusA and PUR7eco24phosphoribosylamidazole-succinocarboxamide synthase showed significant results. ProtParam analysis indicating that the multiple physico-chemical characters of differentially expressed proteins were differed and compared. The phylogenetic tree represents the relationship in-between the differentially expressed proteins, were showed siblings (related) as well as monophytic clade. 2 Volume 41

gaining more and more interest as alternatives for antibiotics or anti-inflammatory drugs.

Probiotic bacteria:
Probiotics involving a number of different bacterial species and strains, mainly lactic acid bacteria (LAB). Lactic acid Bacteria considered as "Generally Regarded as safe" (GRAS) and there were no reports of any harmful effects from the consumption of these bacteria (Gilliland, 1990).
Probiotics are the beneficial microorganisms administered in a sufficient number to survive in the intestinal ecosystem. They must have a positive effect on the host (Gismondo et al., 1999). The term 'probiotic' was first used by Lilly and Stillwell in 1965 to describe the 'substances secreted by one microorganism that stimulate the growth of another. Now-a-days probiotics available commercially as supplements like tablets, capsules and powders (Weese et al., 2002). Most commercially available probiotic products sold for use in companion animals contain Lactobacillus spp. or Bifidobacterium spp (Weese et al., 2011). Certain species of enterococci are also commonly used. Probiotic dosing varies depending on the product and specific indication. No consensus exists about the minimum number of microorganisms that must be ingested to obtain a beneficial effect (Farnworth et al., 2008). In the digestive system, probiotics improve food digestion directly by International Letters of Natural Sciences Vol. 41 helping the body to assimilate nutrients, through contributing to the metabolism of bile acids and accelerating their elimination, as well as by producing digestive enzymes.

Prebiotics:
Non-digestible substances that provide a beneficial physiological effect for the host by selectively stimulating the favorable growth or activity of a limited number of indigenous bacteria are known as prebiotics. Most prebiotics are used as food ingredients in biscuits, cereals, chocolate, spreads, and dairy products. Lactulose is a synthetic disaccharide used as a drug for the treatment of constipation and hepatic encephalopathy. The prebiotic oligo-fructose is found naturally in many foods, such as wheat, onions, bananas, honey, garlic, and leeks. Oligofructose can also be isolated from chicory root or synthesized enzymatically from sucrose.

Normal intestinal micro flora:
The human intestinal tract is a very complex internal environment. It filled with various species of bacteria and yeasts that should assist digestion kill harmful or pathogenic infections and even help to produce many vitamins and other chemical substances needed for our health and long life. The name given to these organisms that live in our intestines is called intestinal flora. The GI tract is a biologically diverse and complicated system which contains around 10 14  hormones are conserved structures throughout evolution . The gastrointestinal tract is also a prominent part of the immune system (Richard Coico et al., 2003). The immune system must work hard to prevent pathogens from entering into blood and lymph. Intestinal bacteria serve to prevent the overgrowth of potentially harmful bacteria in the gut. A ratio of 80-

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85% beneficial to 15-20% potentially harmful bacteria generally is considered normal within the intestines.

Surface micro flora of human body:
Human body surface tissues, i.e. skin and mucous membranes are constantly in contact with environmental organisms and become readily colonized by various microbial species. The mixture of organisms regularly found at any anatomical site is referred to as the normal flora, except by researchers in the field who prefer the term "indigenous micro biota". The normal flora of humans consists of a few eukaryotic fungi and protists, but bacteria are the most numerous and obvious microbial components of the normal flora. Mycoplasmas + + + +/-+ Note: ++ = nearly 100 % ; + = common (about 25 %); +/-= rare (less than 5%) ; * = potential pathogen.

Role of probiotics on human health:
Probiotics have formed a vital part of Mediterranean and Middle Eastern diets for thousands of years, in the form of fermented milk and vegetable products such as yogurt and pickles (Mateos  Cocoti wine: Palm wine generally refers to a group of alcoholic beverages obtained by fermentation from the saps of palm trees (Agu et al., 1999). Palm wine is an alcoholic beverage obtained from the fermentation of the sugary sap of various palm samples by the presence of various microorganisms especially the bacteria and yeast. During fermentation, the sugars in the palm sap are metabolized to alcohol and organic acids with the results that the sap loses its sweetness (Okafor, 1977). After fermentation, oyster colour change in white and sweet taste change into sour. The natural fermented palm wine contains 5 -6 % v/v ethanol (Nwokeke, 2001). The major chemical constituents of palm wine are sugar, protein, water-soluble vitamin of the B-group, titratable organic acids, alcohol and water (Eschie, 1978;Ojimelukwe, 2000). Palm wine and its distillate are important solvent in herbal medicinal administration; pregnant women consume it fresh for the sweetness and nutrition while nursing mothers drink it warm to enhance breast milk production. The microorganisms on palm wine fermentation produce lactic acid and CO2 that make the palm wine anaerobic and leaven the International Letters of Natural Sciences Vol. 41 product. It has been established that consumption of alcohol during pregnancy results in profound developmental and behavioral effects on the fetus and offspring, called Fetal Alcohol Syndrome (FAS) (Desroches et al., 1987;Breese et al., 1993). Alcoholism among women has increased during the last few decades. The excess intake of alcohol for a long time period causes fatty liver (Ramakrishnan et al., 1976) and accumulation of fat in the heart and kidneys (Ramakrishnan et al., 1973) and in brain (Ramakrishnan et al., 1983). Previous studies have shown that maternal consumption of alcohol/Toddy reduced the body weight of both dams and fetuses and altered carbohydrate metabolism (Lal et al., 1997). Fermented palm wine exposure could cause prenatal osteo-inhibitory effects on bones (Eluwa et al., 2010).Consumption of ethanol during pregnancy causes reduction in the weight of the fetus and hepatic glycogen content (Desroches et al., 1987;Breese et al., 1993). Palm wine caused significant decrease in testosterone levels. Similar report was given by Das et al., (2009) in rats treated with Aeglemermelos extract.

Health effects:
According to recent studies by the World Health Organisation (WHO), alcohol consumption is a leading contributor to chronic disease and recognized as a strong risk factor affecting health in developed countries such as the United States and Canada (Rehm et al., 2006). The WHO global burden of disease project estimated that in developed countries alcohol was responsible for 9.2% burden of disease, behind tobacco (12.2%) and high blood pressure (10.9%).There are many kinds of palm wines especially in Africa, with various names. Palm wine contains many components such as a heavy suspension of fungi and bacteria, fermentation agents which give the palm wine a milky white flocculent appearance (Morah,1995), different kind of volatile constituents (Uzochukwu et al., 1994), chemical basis for aroma (Lasekan et al., 2007) mineral elements which may change from one production to another and must of all alcohol. Toddy caused reduction in weight gain and weight of fetuses and also altered carbohydrate metabolism (Lal et al., 1997).

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Palm wine caused non-significant changes in body weight of rats after treatment for 30 days, this suggests that palm wine was not toxic to the animals as well as non-androgenic in nature, since androgens are known to possess anabolic activities. Similar report was given by (Gonzales et al., 2006) in rats. Palm wine caused significant decrease in sperm motility (Verma et al., 2002).Treatment of rats with palm wine caused mild interstitial congestion and oedema. Similar results were reported by (Manna et al., 2005) as well as (Mohammed et al.,2007) in rats treated with deltamethrin and sumithion. These could be due to (i) increased hydrostatic pressure (ii) reduced oncotic pressure (iii) lymphatic obstruction or (iv) Sodium retention (Kumar et al., 1999).

Genomics and proteomics:
The genome is defined as the complete set of genes inside a cell. Genomics is "the study of function and interactions of all the genes in the genome, including their interactions with environmental factors (Collins et al., 2006).Completion of human genome sequencing in 2006 started a new era of science. Since that time genomes of many organisms have been deciphered.
The word "PROTEOMICS" might come from the word "GENOMICS". By the study of genome full DNA sequence of several biological species including human has been determined. Under these circumstances, the focus of life science is moving from genome to proteins, which are biologically synthesized from genome. Aim of proteomics is to study the structure and function of all proteins of a biological species to reconstruct the total biological function of the life is called "PROTEOMICS". Metabolomics is the "systematic study of the unique chemical fingerprints that specific cellular processes leave behind" -specifically, the study of their small-molecule metabolite profiles. Metabolomics may provide information on an additional level of regulation called, metabolic regulation (Rossell et al., 2006). Metabolomics is the valuable technique for the all- What is the need of proteomics: The study of genomics we are not getting accurate results in all cases. It is impossible to make clear and (more) comprehensible mechanisms of disease, aging and effects of the environment only by studying the genome. Only through the study of proteins protein modifications can be characterized and the targets of drugs are to be identified.

Proteomics:
The terms "proteomics" and "proteome" were coined by Marc Wilkins and colleagues in the early 1990s and mirror the terms "genomics" and "genome," which describe the entire collection of genes in an organism. Proteomics is a tool for studying the proteome, i.e., the set of proteins expressed under a defined physiological condition in an organism or cell or tissue. Proteomics is the large scale of study of proteins, particularly their function and structure. Proteins are the vital parts of the living organisms (Naven, 2002;Twyman, 2004). Organisms respond at the molecular level  International Letters of Natural Sciences Vol. 41 21 Functional proteomics: Functional proteomics is the large-scale study of proteins at the functional activity level, such as expression and modification. Currently proteome investigations are focused on mainly two major areas i.e. expression and functional proteomics. Expression proteomics to indicate the down regulation and up regulation of protein levels, in functional proteomics to characterise protein activates, multi protein complexes, and signaling pathways (Pandey et al., 2000;Hinsby et al., 2003).

Structural proteomics:
The major challenges in structural proteomics include the determination and prediction of atomic resolution 3-D structures of proteins on a genome-wide scale for better understanding their structure-function relationships (Smith, 2000). Recent advances in the fields of X-ray crystallography and NMR spectroscopy (Montelione et al., 2000;Abola et al., 2002) have allowed structural biologists to gloss the structures and biological functions of proteins by determining their atomic coordinates.

Differential proteomics:
The aim of differential proteomics is to obtain information about all proteins in a sample. It provides difference between healthy and treated samples. (Bantscheff et al., 2007;Nikolov et al., 2012). Several approaches can be used and these typically involve electrophoresis and chromatography combined with mass spectrometry.

Protein expression studies:
In recent years, the analysis of mRNA expression by various methods has become increasingly popular. These methods include serial analysis of gene expression (SAGE) (Velculescu, 1995) and DNA microarray technology (Shalon, 1996). However, the analysis of mRNA is not a direct reflection of the protein content in the cell. Consequently, many studies have now shown a poor correlation between mRNA and protein expression levels (Abbott, 1999;Ideker, 2001).

Tools Used in Proteomics field:
In Proteomics field, combinations of Analytical techniques are used to analyse the protein samples. The first step in all proteomic studies is the separation of a mixture of proteins. This can be carried out using 2-D gel electrophoresis technique in which proteins are separated based on their individual Molecular weight and charges. The spots obtained in 2-D electrophoresis are separated for subjected to mass spectrometric analysis of each protein present in the mixture. Then the proteins were denatured, reduced, alkylated and digested with trypsin. Tryptic peptides were analysed by using MALDI-TOF.

Matrix:
Matrix is the supporting material in sample analysis, the choice of the matrix is crucial for success in MALDI experiments. Matrix consists of crystallized molecules, matrix act as a first absorber of the UV laser radiation and breaks down, expanding into the gas phase. A good matrix obey the following characters i.e. they are a fairly low molecular weight, but are large enough not to evaporate during sample preparation or while standing in the spectrometer. They are often acidic, therefore act as a proton source to encourage ionization of the analyte. Basic matrices have also been reported. They have a strong optical absorption in either the UV or IR range so they rapidly and efficiently absorb the laser irradiation. This efficiency is commonly associated with chemical structures incorporating several conjugated double bonds, as seen in the structure of cinnamic acid. International Letters of Natural Sciences Vol. 41

MS/MS analysis:
Mass spectrometry is an analytical technique that produces spectra of masses of the atoms or molecules comprising a sample of material. Mass spectrometry is an important newly emerging method for the characterization of proteins. In MS analysis a sample which may be solid, liquid or gas is ionized. The ions are separated according to their mass-to-charge ratio (Sparkman et al., 2000). Two types of methods are used to ionization of whole protein are electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Total protein mass analysis is preliminary conducted using either time-of-flight (TOF) MS or Fourier transform ion cyclotron resonance (FT-ICR). Protein sample enzymatically digested into smaller peptides using trypsin.
These peptides introduced into mass spectrometer and identified by peptide mass fingerprinting (PMF), tandem mass spectrometry (TMS). MALDI-TOF is often the preferred instrument because it allows a high sample throughput and several proteins can be analyzed in a single experiment, if complemented by MS/MS analysis.
For each MS/MS spectrum, software is used to determine which peptide sequence in a database of protein or nucleic acid sequences gives the best match.

Vaporization of proteins:
In MALDI-TOF analysis, the analyte is first co-crystallized with matrix compound, usually an ultraviolet (UV) absorbing weak organic acid, after which laser radiation of this analyte-matrix mixture results in the vaporization of the matrix which carries the analyte with it. The matrix therefore plays a key role by strongly absorbing the laser light energy and causing indirectly, the analyte to vaporize. The matrix also serves as a proton donor and receptor, acting to ionize the analyte in both positive and negative ionization modes, respectively.

Protein identification:
Protein identification is a major process in proteomics field. Two major techniques are used to identification of proteins i.e. MALDI-TOF and LC-MS/MS based on protein fingerprinting, peptide sequencing. In MALDI-TOF, sample is digested with trypsin mixed with matrix then allowed to examine under MS spectrum, it generates the massed of all peptides. Protein identification and analysis software performs a central role in the investigation of proteins from two-dimensional (2-D) gels and mass spectrometry. For protein analysis, information in protein database can be used to predict certain properties about a protein which can be useful for its empirical investigation.
Two main MALDI-MS based identification methods are used to describe the protein sample i.e. i) Peptide mass fingerprinting ii) Post-source decay (PSD) analysis.

Peptide mass fingerprinting:
Peptide mass fingerprinting is also known as peptide mass mapping/ protein fingerprinting it is an analytical technique for protein identification that was developed by several groups independently (Pappin et al., 1993;Henzel et al., 1993). In this method, sample first sample cleaved into smaller peptides, masses can be accurately measured with a mass spectrometer such as MALDI-TOF. These masses are then compared to either a database containing known protein sequences or even the genome. This is analysed by using computer programs/ software tools that translate the known genome of the organism into proteins, then theoretically cut the proteins into peptides, and measure the accurate masses of the peptides from each protein. Then compare to the masses of the peptides of the unknown protein to the theoretical peptide masses of each protein encoded in the genome. High-mass accuracy is required for this method to be of use. Sometimes no matches are found or the level of certainty is too low. The results are statistically analyzed to find the best match.

Post-source decay:
Post-source decay (PSD) is a process specific to the ion source utilizing matrix-assisted laser desorption/ ionisation and operating in vacuum. In the post-source decay, parent ions fragment in a process of laser-induced fragmentation. Time interval suitable for observation of the post-source decay in the reflection starts after the precursors leave the ion source and ends prior to the moment when the precursors enter the ion mirror (Kaufmann et al., 1994). The kinetic energy of fragment of mass m in the post-source decay significantly differs from that of parent ions of mass M is proportional to m/M. So, the distribution of kinetic energies for the PSD ions is extremely large.
Not surprisingly, it cannot be compensated in "classic" single or doublestage reflections. To achieve acceptable mass resolution for PSD ions which masses typically distributed over broad mass range, these ions are accelerated to energies substantially (Kurnosenko et al., 2010) exceeding the initial energy of precursor ions.

Post-translational modifications (PTM):
Proteins are created by ribosomes translating m-RNA into polypeptide chains. These polypeptide chains undergo PTM, (such as folding, cutting and other processes), before becoming the mature protein product. A protein is a chain of amino acids. During protein synthesis, 20 different amino acids can be incorporated to become a protein. Escherichia coli is a host 28 Volume 41 commonly used for expression of proteins in research, diagnostic, therapeutic, and industrial applications (Baneyx, 1999). Non-enzymatic post translational modifications could be the result of unavoidable interference between bacterial metabolic pathways and the abundant heterologously expressed protein. Such modifications result in heterogeneity of the expressed protein (Mark et al., 2014). In the case of therapeutic proteins, efforts have to be made to correct such metabolic interference to help ensure the highest level of protein quality. Non-enzymatic glycation is one type of post translational modification with important implications. This type of reaction has been extensively evaluated in higher eukaryotes (Lindsay et al., 1997) and in prokaryotes (Casey et al., 1995;Geoghegan et al., 1999;Yan et al., 1999;Kim et al., 2001;Mironova et al., 2003).

Homology modelling:
Homology modeling is also known as comparative modeling of protein, the technique used for the identification of one or more known protein structures likely to resemble the structure of the query sequence to residues in the template sequence (Kaczanowski et al., 2010 No pair wise interactions considered in scoring function. iii) No optimal solution guarantee. iv) Exponential run-time.

Peptide mass finger printing:
Peptide mass finger printing is also known as Peptide mass mapping (or) Protein finger printing . It is an analytical technique for protein identification of proteins following separation by 2-D gel electrophoresis, SDS-PAGE (Sodium Dodecyl Sulphate) or liquid chromatography (Wang et al., 2003). The advantage of this method is that masses of the peptides have to be known. 2-D gel electrophoresis is the most preferred method for protein separation prior to peptide mass finger printing. After the cleavage of proteins, digest with enzymes commonly used enzyme is trypsin. Trypsin is the favoured enzyme for peptide finger printing; it is relatively cheap, highly effective and generates peptides with average size amino acids, ideally suited for analysis by MS. The masses of the sample compared with known protein sequence or the genome (Shevchenko et al., 1996). The mass of these peptide fragments is then calculated and compared to the peak list of measured peptide masses.

Role of Bio-informatics tools in proteomics:
Bioinformatics is the branch of science which uses the applications of information FASTA is a short common pattern in query and database sequences and joins these into alignment.
BLAST is similar to FASTA, but gains a further increase in speed by searching only for rarer, more   Secondary structure refers to highly regular local sub-structures. The most common secondary structures are alpha helices and beta sheets. A rough estimation of a polymer is 40% αhelix and 20% βsheets can often be estimated spectroscopically.

Tertiary structure:
Tertiary structure refers to 3-D structure of a single, double, or triple bonded protein molecule. Tertiary structure will have a single polypeptide chain "backbone" with one or more protein secondary structures, protein domains. α-helix and βsheets are folded into a component globular structure.

Quaternary structure:
Quaternary structure is the three-dimensional structure of a multi-subunit protein. Structures of the quaternary protein were determined by a variety of experimental techniques that require a sample of protein in a variety of experimental conditions. A Variety of bonding interaction including hydrogen bonding interactions, salt bridges, and disulphide bonds hold the various chains into a particular geometry. There are two major categories of proteins with quaternary structurefibrous and globular.

Root Mean Square Deviation:
The Root mean square deviation (RMSD) is a frequently used measure of the differences between values predicted by a model (or) an estimator and the values actually observed. In the study of globular protein conformations one customarily measures the similarity in 3-D structure by the RMSD of the Cα atomic coordinate after optimal rigid body superposition. The applications of RMSD are in Bio-informatics the RMSD is the measure of the average distance between the atoms of superimposed proteins. In protein nuclear magnetic resonance spectroscopy, the RMSD is used as a measure to estimate the quality of the obtained bundle of structures. In structure based drug design, the RMSD is a measure of the difference between a crystal conformation of the ligand conformation and a docking prediction.

Phylogenetic analysis:
Phylogenetic tree is a branching tree showing the relationships among various biological species. The similarity of biological functions and molecular mechanisms in living organisms strongly suggests that species from a common ancestor. Molecular phylogenetics uses the structure and function of molecules and how they change over time infer these evolutionary relationships (Liddell et al., 1968).
Based on evolutionary theory, all organisms on earth have descended from a common ancestor, which means that any set of species, extant or extinct is related. This relationship is called a phylogeny, and is represented by phylogenetic trees (Linder et al., 2005).
The confidence statements made about such trees will be main focus. Biologists have also begun to adapt Bayeesian methods based on Markow chain Moute Carlo computations using parametric evolutionary models (Li et al., 2000).

Protparam analysis:
The tool ProtParam is allows the computation of various physical and chemical properties that can be deducted from a protein sequence (http://web.expasy.org/protparam/). The computed parameters include the molecular weight, theoretical pI, amino acid composition, atomic

Probiotics and Gut Microflora:
Gut microflora controls several aspects of bodily function including certain type of cancer (LAB) and Bacillus sp. In the gastrointestinal microflora by consumption of probiotics or by oral administration of specific non-digestible substrates, such as oligofructose, termed as prebiotics (Parracho et al., 2007).  (Grozdanov et al., 2003) and even low-coverage genomic shotgun sequencing (Sun et al., 2005). Until now, however, the whole genome sequence has been inaccessible.

E. coli
Serotyping of E. coli Nissle 1917 has identified the presence of a K5 antigen, which is known to be Nissle 1917 produces significantly more CPS than E. coli K5, making the organism attractive as a production strain for bioengineered heparin.

Important aspects when select as a probiotic strain:
The significance of human origin has been debated recently, but currently successful strains are indicated to be of human origin. It can also argue that a probiotic strain can function better in a similar environment like human gut to where it was originally isolated from; safety aspects include the following specifications.
1) Strains for human use are preferable of human origin.
2) They are isolated from healthy human GI Tract G. Peretz in 1933 (Fitzpatrick et al., 2008). This strain used extensively in humans as a therapy for GI diseases such as colitis, inflammatory bowel disease and infections. Anti-colitis action of EC-M17 is mediated by modulation of immune processes attributed to an inhibitory effect on NF-kB signaling.

Cocoti sap and wine:
Isolation of microorganisms from palm wine Saccharomyces cerevisiae dominated in yeast, Vitamins, Vitamin C as well as FOS. The protein quantification assays revealed that the proteins content was about 0.2g/ml which was comparable with that of the coconut palm exudates 0.1g/ml but was lower than the proteins concentrations of cucumber (60g/l) and of pumpkin (35g/l) phloem sap ( Walz et al., 2002;Nakamura et al., 2004).

International Letters of Natural Sciences Vol. 41
Physico -chemical stress on E.coli: E.coli in response to physical (heat) and chemical (benzyl alcohol) stress elucidate the common and differing elements of the stress response originating in cellular membranes caused by external stress signals of a different nature (high temperature and membrane fluidising agent), by observing overlapping changes at the membrane level. It is expected that signals generated within the membranes might cause HSR and acquisition of cellular thermotolerence in a similar manner independently from the nature of the membrane perturber. The present study addressed the validity of the membrane sensor hypothesis in E.coli, which was chosen as our model organism due to its different cellular simplicity and because it is biochemically and genetically well characterised. A reporter system was also developed to study the transcription of heat shock genes, including heterologous promoter sequences of cyanobacterial heat shock genes recognised in an E.coli host (Georgopoulos et al., 1993). In proteome analysis total 93 proteins are identified that are phosphorylated in E.coli upon heat shock. These are include chaperones, signaling molecules, ionchannels, proteins involved in transcription and translation process, in amino acid biosynthesis, oxidoreduction, energy metabolism, cell motility and cell membrane structure. Changes in stress signaling pathways are achieved mostly through the activation of protein-tyrosine kinases (Kim et al., 2002).

Cellular cross-protection by stress:
Cellular cross-protection occurs when the stress response induced by one specific type of stress, gives cells increased resistance to other types of stress (Mary, 2003;Vattanaviboon, 2003).
An example of this kind of protection is demonstrated with stress-induced thermo-tolerance, where Escherichia coli cells given a non-lethal heatshock (42 °C) down-regulated normal protein production and begin production of HSPs, and so are later able to survive what would otherwise be a lethal heat shock (46 °C). This is due to the up-regulation of stress proteins at many levels (e.g. mRNA synthesis and stability, translational efficiency) that can protect cells from other stress.
Cross-protection is not universal, and it can also occur in specific ways. For example, heat shock may protect against hydrogen peroxide.
Booth (2002) proposed that "Stress is any change in the genome, proteome or environment that imposes either reduced growth or survival potential". The cellular response depends on the severity of the stress. Under slight stress, growth continues at the same rate and cells fully adapt to 42 Volume 41 the new conditions. Under severe stress the growth rate is reduced but cells adapt and tolerate the conditions while under extreme stress, growth cease and cells switch to a survival mode (Storz et al., 2000). The key aspects to surviving environmental stress are the cell's ability to maintain the integrity of the cell membrane, the integrity of DNA and the ability to properly fold proteins (Booth, 2002). An understanding of the physiological, biochemical and molecular mechanisms involved in response of E.coli to environmental stresses is essential for assessing, predicting, and minimizing the health risks and can offer insight into designing effective methods to control their growth.
When the bacteria expose to high temperature into less time period it produce heat shock response proteins these proteins are unfolded and damaged proteins, such as exposure to harmful chemicals (antibiotics, solvents) or overproduction of endogenous and recombinant proteins. In E.coli, heat shock response consists of the induction of more than 20 different heat shock proteins (HSPs), the majority of proteases that degrade misfolded and abnormal proteins. Bacterial cells exposed to one type of stress it can also condition them against other, seemingly unrelated, stresses, when bacteria are challenged with high osmolality (Fletcher et al., 2001).

Proteomics of stress responses of potentially probiotic bacteria:
Proteomics is an excellent approach for studying changes in bacterial metabolism and, e.g., stress responses during the progression of growth. The proteome of the potential probiotic L.plantarum WCFS1 was mapped at mid-and late-exponential and early-and late-stationary phases, and growth phase-dependent differences were detected in the abundances of 154 protein spots (Cohen et al., 2006). In a study of L. plantarum REB1, isolated from fermented feed, and the potential probiotic L. plantarum MLBPL1, isolated from white cabbage, both the growth phase (lag, early exponential, late exponential, and early stationary phases) dependent and strain-dependent differences in the proteomes were compared . Proteome maps of L. casei Zhang cells grown until the exponential and stationary phases were also compared. Forty-seven protein spots showed growth phase-dependent production, and the major up-regulated proteins in the stationary phase were stress proteins and proteins involved in carbohydrate and energy metabolism, and they were suggested to be involved in the stress response mechanisms of L. casei

International Letters of Natural Sciences Vol. 41
When E.coli under hyperosmolarity stress results in rapid loss of water (plasmolysis), loss of turgor, and shrinkage of the cell (Weber et al., 2005). Within the minutes, respiration ceases, both the intracellular ATP concentration and the cytoplasmic pH increases. Many studies reported the cellular membrane of E.coli is a vital factor that allows for cells acclimate to external stresses and is also one of the components highly affected by organic solvents like alcohol (Isaac et al., 2005).
Most of the researchers have proposed that the plasma membrane is the most affected target of organic solvents and plays a significant role in adapting to stress, alcohols are sensitive toxins to E.coli as tolerances of n-butanol and ethanol are only 0.5-1% and 4-5% respectively.
Proteomics is an excellent approach for studying changes in bacterial metabolism and response. The proteome of the potential probiotic L. plantarum WCFS1 was mapped at mid-and late-exponential and early-and late-stationary phases, and growth phase-dependent differences were detected in the abundances of 154 protein spots (Cohen et al., 2006). In a study of L.
plantarum REB1, isolated from fermented feed, and the potential probiotic L. plantarum MLBPL1, isolated from white cabbage, both the growth phases dependent and strain-dependent differences in the proteomes were compared . Proteome maps of L. casei Zhang cells grown until the exponential and stationary phases were also compared. 47 protein spots showed growth phase-dependent production and the major up-regulated proteins in the stationary phase were stress proteins and proteins involved in carbohydrate and energy metabolism and they were suggested to be involved in the stress response mechanisms of L. casei (Wu et al., 2009).

Culture collection:
The

Collection of palm sap and wine:
Fresh palm sap samples were collected (Wilson, 1996)  Physical factors: Colour, Odour, Taste, pH and Turbidity.
Chemical factors: Acidity and Alcohol.

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Colour: Four test tubes were taken. One tube was filled with cocoti wine and another filled with cocoti sap. Remaining tubes filled with standard solutions like potassium chloroplatinate and cobaltous chloride and observed the colour parallel to the eye with white background (Yohannes et al., 2004).

Odour:
Take one liter wide mouth bottle and cleaned it with hydrochloric acid pour it out and find the smell it. Then rinsed with distilled water two times washed out and find the smell it. The sample rinsed and observed the odour (Nordin et al., 2004).

Taste:
After

3-D)
Turbidity: Turbidity indicates the growth of microorganisms in the samples. Distilled water is taken into a cuvette and inserted it into the holder and calibrate by using zero knob to set zero. By using standard solutions to set the calorimeter finally and introduced the samples and analyse the turbidity of sap and wine (Baton Rouge, 2007).

Acidity:
5 ml of wine/sap sample was taken into clean conical flask and homogenise by gentle shaking. To this, added 2 drops of phenolphthalein indicator and it was titrated against 0.1N NaOH.
A clean burette was taken and filled with NaOH and initial volume was recorded by drop wise release of NaOH burette to wine/ sap sample at complete neutralisation. Turning of a pink colour development was recorded. Likewise, three concurrent values were recorded by taking same wine/ sap sample. By taking standard neutralisation of 0.1N NaOH (100 ml) can neutralise 9 gms of lactic acid. The amounts of lactic acid present in the given wine/ sap was evaluated. To calculate acidity,

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we can use the following formula. The concentration of acetic acid in the media was determined by titration following the protocol described by Accolas et al., (1977).
% total acidity = ml of alkali x Normality of alkali x 9 Weight of sampling

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The minimum inhibitory concentration i.e. the lowest concentration of metal required to cause inhibition of bacterial growth. Which concentration shows approximately 50% growth inhibition was selected as stress concentration for the subsequent proteomic analysis.

Protein Extraction:
The proteomes of three different samples were compared. The first sample served as the centrifugation. Protein pellet was collected, discarded the supernatant and dried the protein pellet at room temperature. Reconstitutes the dried pellet with rehydration buffer and stored overnight at -20 0 C before carry out protein quantification.

Protein quantification:
Protein quantification was done by three methods i.e. Lowry's method and Bradford method and BCA kit method when compare to three methods BCA kit method gives better comprehensive results than the other methods.

Lowry's method:
In Lowry's method (Jakob et al., 2002) first prepared Bovine serum albumin (BSA) working standards in test tubes upto 1 ml by using distilled water. To this solution, 4.5 ml of reagent -1(Na2CO3, NaOH, Na-K Tartrate, and CuSO4.5 H2O) was added and allowed to incubation for 10 minutes. After incubation 0.5ml of reagent-II (Folin-Phenol) was added and allowed to incubate for 30 minutes. Blank was also maintained and prepared unknown solutions as mentioned above. The absorbance was measured at 660nm and standard graph was plotted. The amounts of proteins presents in the unknown sample were estimated using standard graph. O.D values on Y-axis and concentrations of known samples on X-axis were taken and calculate the unknown concentrations.

Bradford method:
Bradford method is a colorimetric analysis method used to measure the concentration of protein (Bradford, 1976 proportional to the amount of protein in the sample. The BCA Protein assay is suitable for measuring protein concentration in the range of 0.5-30 μg protein (0.01-0.6 mg/ml).

2-D clean-up method:
Protein samples were collected which contains some salts and detergents. So, it requires Washed the pellet by using de-ionised water to this pellet and 25 μl of de-ionised water was added vortex the pellet for 5-10 sec and discarded the water. 1ml of chilled wash buffer and 5 μl wash additive were added to this pellet vortex the tube 20-30sec every 10 minutes, repeated this step at least 3-5 times. Chill the wash buffer at -20 0 C for at least one hour before starting the experiment.
Again centrifuged the tube and discard the supernatant, pellet allowed to air dry. Resuspend the pellet in rehydration or sample solution of choice. Now the sample is ready to load on gel.

2-D gel electrophoresis:
Two-dimensional gel electrophoresis (2-D electrophoresis) is a powerful and widely used method (http://www.bio-rad.com) for the analysis of complex protein mixtures extracted from cells, tissues or other biological samples. This technique separates proteins in two steps, based on charge/ isoelectric point (pI) and molecular weight (MW).

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Isoelectric Focusing: Isoelectric focusing was carried out on 18 cm immobilize strips which provided a linear gradient from pH 4 to 7 (Bio-Rad) by a PROTEAN IEF Cell (Bio-Rad). For isoelectric focusing (IEF), the first dimension of 2-DPAGE, we used immobilized pH gradient (IPG) strips at both pH 4 to 7, linear gradient and carried out the experiment by using a protean IEF Cell (Bio-Rad). In the first step of IEF, we applied our protein samples and the rehydration procedure was carried out in IEF focusing tray. After rehydration, we carried out focusing. We used length focusing tray suitable for our IPG strips which was cleaned before use. First of all, we placed paper wicks above the electrodes and soaked them with 8μl ultra-pure water.
Then we diluted our protein samples to 500μg with rehydration buffer and 2 % Ampholytes were mixed with the sample. We loaded both control and stress E.coli Nissle 1917 sap and wine treated samples. Then we placed 320μl of our protein sample into a certain point in IEF focusing tray and avoided bubble formation. Then IPG strips were taken out of -20 ºC and allowed to thaw for 5 minutes at room temperature. Afterwards the protective layer above the IPG strips was removed and the strips were positioned in the IEF focusing tray gel side down and positioned with the positive end of the strip to the positive electrode and the negative end to the negative electrode.
To minimize evaporation and urea crystallization, the strip was then covered with 2 ml of mineral oil. Finally, the focusing tray was covered and allowed to stand for one hour at room temperature. After one hour, the IEF tray was placed into Protean IEF Cell then IPG strips were actively rehydrated with 50V application per strip for 12 hours. After rehydration, we carried out 6stepped focusing which was as follows: phase 1, linear gradient up to 250

Equilibration of Strips:
After IEF, the strips containing the focused proteins were equilibrated. This procedure is applied for formation of SDS-protein complexes, reducing the disulphide bonds and to alkylate the sulfhydryl groups. After we applied this procedure, proteins had completely unfolded structure and carried only negative charges. We used two equilibration buffers both of them containing SDS, Tris-Hcl pH 8.8, glycerol, and urea. Equilibration buffer-I contained DTT and equilibrium buffer-II contains Iodoacetamide instead of DTT. DTT is a reducing agent required for cleavage of disulphide bonds between cysteine residues. Iodoacetamide is an alkylation agent used for preventing disulphide bond formation by alkylating free sulfhydryl groups in cysteine residues.   Step-n-hold -50 V 10:00 hrs S2

Preparation of equilibrium buffer
Step-n-hold -500V for 1:00 h S3 Gradient -1000V during 1:00 h S4 Gradient -8000V during 3:00 hrs S5 Step-n-hold -8000V for 56000 V hrs International Letters of Natural Sciences Vol. 41 55 The entire IEF takes 24h to finish. At the end of an IEF run strips can be stores at -80 0 C for later use. It is convenient to start it in the next day so that it is ready for running the second dimension during the next day.

SDS-PAGE:
SDS-PAGE experiments were performed in Bio-Rad PROTEAN II XL electrophoresis cell.
Dimensions of the glass plates were 16 x 20 cm for inner plate and18.3 x 20 cm for outer plate.
Prior to analysis, the glass plates were assembled according to the instructions in the manufacturer's manual (http://www.lifetechnologies.com).

Pouring SDS-Polyacrylamide Gels:
As it is stated above, SDS-polyacrylamide gel contains only the resolving gel but not the stacking gel. The resolving gel for SDS-PAGE should be prepared the day before SDS-PAGE analysis, and kept at 4°C overnight. The composition of acrylamide for the resolving gel was chosen as 12% so that components of the gel could be prepared according to this value. Preparation of the resolving gel components were described below.

Preparation of Acrylamide mixture:
Acrylamide 30.0 g and Bis-acrylamide 0.8 g were dissolved in distilled water and made up to 100 ml. pH of the mixture was adjusted to 7.0 and stored at 4 0 C.

Preparation of 1.5M Tris-HCl Buffer pH 8.8:
18.5 g of Tris base dissolved in 80ml of distilled water, pH adjusted to 8.8 with 1 n HCL made up to 100ml with water , stored at 4 0 C.
Preparation of 0.5M Tris-HCl Buffer pH 6.8: 6.0g of Tris base was dissolved in 60 ml of distilled water pH adjusted to 6.8 with in HCL made up to 100 ml with water , stored at 4 0 C.
Preparation of 10% ammonium persulfate: 1 g of ammonium persulfate (Sigma) was dissolved in 10 ml of distilled water. 2-D polyacrylamide gels were prepared for samples of 15°C and 30°C in a comparative manner. Since the volume of each gel amount was approximately 35 ml, necessary volume of each solution component was adjusted to prepare 110 ml of 12%SDS-polyacrylamide gel. After the preparation of all solvents they were mixed in a beaker in the above order, gel has polymerised, decant the overlay, prepare the staking monomer, add TEMED and then ammonium persulfate (APS) because to avoid degasing and pour into the gel apparatus. Insert the comb and allow polymerizing completely before running.
The final mixture was then swirled rapidly and poured into the gap between the glass plates without delay. After the completion of polymerization (~30 minutes), upper side of the gels were covered with distilled water and they were kept at 4°C overnight.

Running the Gel:
After period of equilibration of IPG strip, a 100 ml-graduated cylinder was filled with 1X Tris/glycine/SDS buffer and any bubbles on the surface of the buffer was removed using Pasteur pipette. Then, equilibrated IPG strips were dipped briefly into the graduated cylinder respectively to be rinsed in the buffer. After that, each strip was laid gel side up on to the longer (back) glass plate, International Letters of Natural Sciences Vol. 41 57 and connected with the gel without any air bubble at the interface. The glass plates were then held vertically by placing them in the gel box, and one ml of preheated overlay agarose solution was pipetted into the IPG well of each gel. After allowing the overlay agarose solution to solidify for 5 minutes, the reservoir of the gel box and the gap between the gels were filled with 1X Tris/glycine/SDS running buffer. Water circulation for controlled cooling of electrophoresis was enabled and electrophoresis was started according to the conditions. The migration of the Bromophenol Blue which is present in the overlay agarose solution was used to monitor the progress of the electrophoresis. When it reached the bottom of the gel, electrophoresis was stopped.
The given Table (13) illustrates the running conditions of SDS-PAGE. 15.0 g of Tris-base and 72 g of glycine were dissolved in 900 ml of distilled water. To this mixture, 50 ml of 10% (w/v) SDS was added on and the final volume was adjusted to 1000 ml with distilled water. 200 ml of this solution was taken and diluted to 1000 ml with distilled water before use.

Protein gel staining techniques:
After 2-D PAGE, the separated proteins have to be visualized, either by "universal" or by "specific" staining methods. Since the concentrations of individual proteins in a single cell differ between six or seven orders of magnitude, ranging from several millions of copies/cell for some highly abundant proteins (e.g., glycolytic enzymes) to a few copies/cell for very low abundant proteins, these enormous variations in protein concentrations are a major challenge for almost all currently available protein detection methods. The most important properties of protein visualization methods are high sensitivity (low detection limit), high linear dynamic range (for quantitative accuracy), reproducibility, and compatibility with post electrophoretic protein identification procedures, such as mass spectrometry. Unfortunately, currently no staining method for 2-D gels meets all requirements for proteome analysis.
Here we use Colloidal coomassie staining technique, after electrophoresis was finished; gels were removed from glass plates and transferred to a large tray. Gels were rinsed with milli Q protein spot sets were created, pattern was chosen as a reference template, and spots in a standard gel were then matched across all gels. Spot quantity values were normalized in each gel dividing the raw quantity of each spot by the total quantity of all the spots included in the standard gel. In order to analyse gel similarities or experimental variations such as disparities in stain intensity or sample loading, one can produce Scatter plots for groups. Scatter plots give an idea of the relationship between the spot values from two gels by searching for the linear dependence between the spot values of one gel in comparison to another gel. Spot sizes, Mean, Standard deviation, Coefficient of Variation in each group were determined. Transferred the data square root to Sin -1 √p. After performing Sin -1 √p transformation independent samples ttest was conducted in order to compare the two groups and identify sets of proteins that showed a statistically significant difference with a confidence level of 0.05 and a minimum two fold of variation. The spots in these sets were excised from gels using Spot Cutter for further analysis of spots and explain the steps involved in using Image master 2D platinum 6.0.    Peptide mass fingerprinting by MALDI-TOF and sequencing by tandem mass spectrometry have evolved into the major methods for identification of proteins following separation by twodimensional gel electrophoresis. This technique, which is user friendly and quite fast. The standard approach to identify proteins includes separation of proteins by gel electrophoresis. In gel electrophoresis special caution must be taken to avoid contamination of the protein samples with keratin. To avoid contamination gels should be sealed as soon as possible after staining.
Subsequently, the proteins are cleaved with sequence specific end-proteases or Proteolytic enzymes such as trypsin, chymotrypsin, mainly trypsin is used for protein digestion to produce peptides with molecular masses in the optimal range for MS analysis. For protein identification, the experimentally obtained finger print masses are compared with the theoretical peptide masses of proteins stored in databases by means of mass search programs.

Peptide mass fingerprinting MALDI-TOF-MS:
Spots were picked from colloidal coomassie blue stained gels placed into tubes, add 200 μl of 25 mM NH4HCO3 / 50% acetonitrile and vortex these mixture for 10 min discard the supernatant. Repeat this step 3-4 times until the gel pieces are colorless and discard the supernatant.
Spots were treated with 100% acetonitrile for dehydration allowed to stand for few minutes until the gel pieces shrink and turn white. Remove the acetonitrile and the spots allowed for drying by using Speed Vac process. 25μg trypsin solution was added to the dried spots. Allow the gel pieces to rehydrate with trypsin at 4 0 C for 60 minutes and remove the supernatant was discarded to minimize auto-digested for 10 min. 25mM NH4HCO3 was added it will prevent the gel from drying, incubate at 37 0 C overnight (12-16 hrs).
Mass spectrometry was performed by using the (Model Voyager-DE STR, Applied Biosystems, Foster, CA, USA). The spectra measured for unknown peptides were compared against the mass peaks derived from calibration of internal standards. Spectra were collected over the mass

Generation of 3-D-structure models -Homology modeling:
In this study, the 3-D structures of the stress expressed proteins are modeled generated by using phyre-2 programme. The primary structure for the stress expressed proteins can be obtained from Many hits were displayed on the basis of the sequence identity and E value between the protein sequences.

Multiple sequence alignment:
Multiple alignments of protein sequences (MPS) are important tools in studying sequences. (Chenna et al., 2003). CLUSTAL X, W is a general purpose multiple sequence alignment programs for proteins. It produces biologically meaningful sequence alignment of divergent sequences. It calculate the best match for the selected sequences, it brings out both evolutionary and structural similarity among the proteins encoded by each sequence in the alignment. Evolutionary relationships can be seen via viewing Cladograms or Phylograms. Sequences can be aligned across their entire length or only in certain regions. This is true for pair wise alignment and multiple alignments. Global alignments need to use gaps while local alignments can avoid them, aligning regions between gaps.

International Letters of Natural Sciences Vol. 41
Target-template alignment: The alignments between the stress expressed proteins and templates are executed by SWISS MODELL. Target sequence, template structure matches are determined by aligning the target sequence profile against the template profiles, by using CLUSTALW and MUSCLE programmes (Schwede et al., 2003). In order to analyze the close relation between the target and template protein sequences we carry out the comparative modeling procedure. Comparative modeling requires the information on target template alignment. Now the matching parts of the template structure and unknown sequence were realigned by the use of Kalign-SBC, it is fast and accurate multiple sequence alignment tool.
This command executes a global dynamic programming method for comparison between the target-template sequences and also relies on the observation that evolution tends to place residue insertions and deletions in the regions that are solvent exposed, curved, outside secondary structure segments, and between two Cα-β positions close in space (Melo et al., 2002). Gaps are included between the target-template alignments, in order to get maximum correspondence between the protein sequences. Gaps in these regions of high correspondence are favoured by the variable gap penalty function that is executed from the template structure alone.
In principle, the error between the target-template alignments is greatly minimized almost by one-third relative to the present day sequence alignment methods. Models are built for each of the sequence-structure matches using SWISS MODEL. Nevertheless, there is clearly a need for even more accurate sequence-structure alignments and for using multiple template structures, so that more accurate models are obtained. The resulting models are then evaluated by a composite model quality criterion that depends on the compactness of a model, the sequence identity of the sequence-structure match and statistical energy Z-scores.

Homology Modeling Using the Swiss-Model Server:
In this section, we are discussing about the generation of the three dimensional structure for the unknown metal stress expressed protein sequence (target) with templates as its suitable structural homolog. SWISS MODELL is the software tool used for 3-D model building ( repeated until a satisfying modeling result is achieved. Each of the four steps requires specialized software and access to up-to-date protein sequence and structure databases. Protein sequence and structure database necessary for modeling are accessible from the workspace and are updated in regular intervals. Software tools for template selection, model building, and structure quality evaluation can be involved from within the workspace. The output of this module displays many restraints parameters between the target template alignments including the distances, main chain dihedral angles, side chain dihedral angles, disulphide dihedral angle, NMR distant restraints and non−bonded restraints between these two proteins (Mac Kerell et al., 1998). These relationships are expressed as conditional probability density functions (pdf's) and can be used directly as spatial restraints.
Homology modeling (Guex et al., 2009) is used to build three-dimensional models for a protein target based upon a single or multiple templates with known structure. The accuracy of homology modeling is dependent on the fact that evolutionarily related protein sequences often have similar threedimensional structures. The homology modeling procedures is a multi-step process that can be summarized in the following steps i.e. Sequence alignment of the target and template, target backbone generation, target loop modeling, target side-chain modeling and target model refinement.

Structure validation:
Validation refers to the procedure for assessing the quality of deposited atomic models Ramachandran's plot, the core or allowed regions indicates the preferred areas for psi/phi angle pairs for all residues in a protein. If the determination of protein structure is reliable, most pairs will be in the favoured regions of the plot, some pairs will be in the allowed region, and only a few will appear in 'disallowed' regions.

International Letters of Natural Sciences Vol. 41 67
Phylogenetic analysis: The phylogenetic tree (phylogeny) (http://www.phylogeny.fr/) or evolutionary tree is a branching diagram or tree showing the inferred evolutionary relationships between the studied data.
The data must be comprised of homologous types. Computational phylogenetics is the application of computational algorithms, methods and programs to phylogenetic analyses. The goal is to assemble a phylogenetic tree representing a hypothesis about the evolutionary ancestry of a set of genes, species, or other taxa. The studied data is allowed to phylogenetic analysis by using Bio-Edit tool version 7.0.9, it is used for several modes of alignment, Automated ClustalW alignment, Plasmid drawing and annotation.

Analysis of physico-chemical parameters of a sequence:
Protparam is one among the protein analysis tool available on the ExPasy server.   The turbidity of cocoti sap was slightly transparent solution, but in the case of wine it is different. Turbidity indicating the growth rate of microorganisms (or) fermentation rate was enhanced due to higher fermentation processing. Old cell debris is settled down on bottom of the container. Cells within a culture scattering the light make it harder to see cloudy. It should be above 2 NTU (Nephelo Turbidity Units). For analysis of total acidity, titration method was employed in seven samples. The average total amount of acidity in cocoti sap and wine was 2.59g/100ml and 4.75g/100ml respectively. There is a sharp rise in titratable acidity of cocoti sap after 4 hours. This is probably due to the Acetobacter species. Conversion of alcohol to acetic acid may have occurred.
The causation of active fermentation creates aerobic conditions which favours this organism.      P-value for concentrations in greater than 0.05 whereas for time intervals is less than 0.05, we concluded that there is no significant difference occur between concentrations and there is high significance difference between time intervals.

Growth curves:
The present results indicating that the high concentration of cocoti sap and wine shows pathogenic effect on probiotic E.coli growth. Growth curves also evidenced to the said statement.

Proteins isolation and purification:
There are several methods to isolate proteins from the cell or tissue i.e. sonication, freezing, homogenization, thawing by high pressure, filtration or permeabilization by organic solvents. We used sonication process for isolation of proteins from E.coli cells. Re-suspended pellet was sonicated on ice is enable the bacterial cells to break. So that, the contents were released sonication involves the use of high energy sound waves that capable of breaking outer membrane of cell. All cells contains including protein of interest leak out of the disrupted membrane carry out the sonication procedure for 30 sec with the pulse of 1 sec at 40% amplitude, once it was completed, centrifuged the contents and collected the supernatant. Protein samples were purified because 2-D electrophoresis is very sensitive to salts and detergents.

Proteins quantification:
Protein quantification is a major step in 2-D electrophoresis because it is based on the quantification rate sample should be uploaded into the IPG strips.

Analysis of proteins by BCA kit method:
Total proteins of the control, sap, and wine treated E.coli through the BCA method were analysed and presented in the following table 28 and in figure-14.
Analysis of proteins by BCA kit method: Table-

Statistical analysis:
Statistical analysis was made using the statistical package for the social sciences (SPSS) 15.0 software. Data was analysed by one-way analysis of variance (ANOVA). In all statistical analyses, p<0.05 was taken as the level of significance.

Differentially expressed proteins in sap and wine treated E.coli identified by 2-D gel electrophoresis:
The effect of cocoti sap on E.coli Nissle 1917 is caused changes in protein synthesis. We analyzed the proteomic response to sap, to identify differentially expressed proteins, which could be important for resistance to this sample. E.coli Nissle 1917 cells were treated with 180µl sap.

Differentially expressed E.coli Nissle 1917 proteins regulation with response to cocoti sap and wine:
Analysis of differentially expressed proteins is one of the major challenges in proteomics.
Identification of expressed proteins, whose encoding genes are differentially expressed. Its

3-D view for protein spot 276 3-D view for protein spot 376
International Letters of Natural Sciences Vol. 41

1). UPF0401 protein ECP Y3010_ECOL5:
It is a phase protein, Uniprot id of the protein S1GQX9 and the taxonomic identifier number is -1182698 mostly the protein involves molecular functions like hydrolase activity acting on ester bonds and metabolic process. Esterase does exist different their substrate specifically, their protein structure and functions phosphatase is an enzyme that removes a phosphate group from its substrate by hydrolysing phosphoric acid monoesters into a phosphate ions and a molecule with a free hydroxyl group. This action is directly opposite to that of phosphorylases and kinases which attach phosphate groups to their substrates by using energetic molecule like ATP. Protein phosphorylation plays a crucial role in biological functions and controls nearly every cellular process, including metabolism, gene transcription and translation, cell-cycle propagation, cytoskeletal rearrangement,

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protein-protein interactions, protein stability, cell movement and apoptosis. This process dependent on the highly regulated and opposing actions of PKs and PPs, through changes in the phosphorylation of key proteins. Histone phosphorylation, along with methylation, ubiquitination, sumoylation and acetylation, also regulates access to DNA through chromatin reorganisation.
UPF0401 protein ECP Y3010_ECOL5 is a down regulated protein with response of cocoti sap treatment. We noticed the protein spot in pH range around 7 and the molecular mass close to 8744.9 Da. The percentage of sequence coverage, calculated pI and protein score were presented in table-31.

2). Transcriptional regulatory protein BAER_ECOL6:
Transcriptional regulatory protein involved in transcription regulation, DNA binding, phosphorelay response regulator activity. Taxonomic identification number of this protein is 1181761 and the Uniprot id L4RK56, total sequence length of this protein 240 AA, molecular weight of this protein 140.6kda. The percentage of sequence coverage, calculated pI and protein score were presented in table-31.
The protein responds to a phosphorelay sensor to initiate a change in cell state or activity.
This is a type of intracellular signal transduction, this process first elucidated by studies of the action of hormones such as epinephrine, which signals the breakdown of glycogen in anticipation of muscular activity. The activity of the response regulator is regulated by transfer of a phosphate from a histidine residue in the sensor, to an aspirate residue in the response regulator. Many but not all response regulators act as transcriptional regulators to elicit a response. These protein consist a membrane bound histidine kinase that senses a specific environmental stimulus and a corresponding response that mediates the cellular response, mostly through differential expression of a target genes  electrons, is also probable. Other proteins may also be involved in formation of the enzyme complex, which requires the presence of metal (nickel_ cobalt). This protein has homology with one of the subunits of NADH: ubiquinone Oxidoreductase of the respiratory chain and also involves Oxidoreductase activities (Sawers, 1994   carbohydrate active-transport system, catalyzes the phosphorylation of incoming sugar substrates concomitantly with their translocation across the cell membrane. This system is involved in Nacetylgalactosamine transport (Ray et al., 2004). AgaVWEF (N-acetyl-galactosamine PTS agaoperon (comprising agaVWEF), the cryptic galNAc PTS permease, belongs to the functional superfamily of the PEP-dependent, sugar transporting PTS. If all of its components were present, AgaVWEF would take up exogenous GalNAc, releasing the phosphate ester into the cell cytoplasm in preparation for metabolism (Brinkkotter et al., 2000). The percentage of sequence coverage, calculated pI and protein score were presented in table-31. Taxonomic identifier number of the protein is 1116033 and the Uniprot id of the protein is M8THA9.

7). t-RNA-Specific adenosine deaminase monomer:
Taxonomic identifier of the protein sample is 868141and Uniprot code H4L196, length of the sequence 167AA, molecular weight of this protein sample is 26kDa. TadA is a tRNA-specific adenosine deaminase that belongs to the family of adenosine at position 34 of tRNA Arg2 resulting in an inosine at this position, which is the wobble base of the anticodon. Substrate requirements have been evaluated, the anticodon stem and loop are found to be sufficient for inosine formation (Wolf et al., 2002). TadA can form a homodimer in vitro, but it is unknown whether it functions as a homodimer in vivo (Wolf et al., 2002). A crystal structure of TadA has been solved at 2.0 Å resolutions (Kim et al., 2006). Adenosine deaminase (ADA) is considered one of the key enzymes of purine metabolism (Glader et al., 1983). The high degree of amino acid sequence conservation suggests the crucial nature of ADA in the purine salvage pathway (Cristalli et al., 2001). The calculated pI, percentage of sequence coverage and protein score were presented in the table -31.

Sequence alignment of differentially expressed proteins under sap stress:
The target sequence was searched with BLAST search against Protein Data Bank, which one has a high level of sequence identity with target protein selected as a template protein

Global quality validation of proteins:
The analysis provides both global and site-specific measures of protein structure quality.
Global quality measures are reported as Z scores, based on calibration with a set of high-resolution X-ray crystal structures. PSVS is particularly useful in assessing protein structures determined by NMR methods, but is also valuable for assessing X-ray crystal structures or homology models.
RMS Deviation is a good measure of accuracy.

Tab -34: Validation scores of differentially expressed proteins under sap treatment.
Quality score of the model, Close contacts and deviations from ideal geometry were represented in the following Under  condensation of the host cell. A type l toxin antitoxin (TA) system where expression of the proteinaceous toxin is controlled by an antisense sRNA, in this case RdlA or RdlC (Kawano et al., 2002). Only a few of these TA systems have been mechanistically characterised, the mechanisms used to control expression of the toxin gene are not necessarily the same.

Xanthine dehydrogenase iron sulphur binding subunit -XDHC-Eco57:
Molecular weight of this protein is 16949.7 Daltons, Uniprot code of this protein is K3TSG9 and taxonomic identifier is 1005482. The protein involved in Xanthine dehydrogenase activity, electron carrier activity, iron-sulphur cluster binding, metal ion binding activity. Iron -sulphur are found metalloproteins, such as ferredoxins, coenzyme Q-cytochrome c reductase and nitrogenase.
Iron-sulphur clusters are best known for their role in the Oxidation-reductions of mitochondrial electron transport. Xanthine dehydrogenase can be converted to Xanthine oxidase by reversible sulfhydryl oxidation or by irreversible proteolytic modification. Xanthine dehydrogenase cause xanthinuria, may contribute to adult respiratory syndrome, and may potentiate influenza infection through an oxygen metabolite-dependent mechanism (Ichida et al., 1993). Calculated pI and sequence coverage identity and total protein score were presented in table -35.

Template alignment of differentially expressed proteins under wine stress:
Homology models were obtained from wine stress proteins based on MASCOT search data sequence, on wine treatment eight proteins were isolated from 2-D gel and overall all proteins gave good prediction structure. Differentially expressed proteins were presented in the above

Validation of protein samples:
Validation of the protein model was done with RAMPAGE server (Lovell et al., 2000).
After the refinement process, validation of the model was carried out using Ramachandran's plot.

Global quality validation of proteins:
The analysis provides both global and site-specific measures of protein structure quality.
Global quality measures are reported as Z scores, based on calibration with a set of high-resolution X-ray crystal structures. PSVS is particularly useful in assessing protein structures determined by NMR methods, but is also valuable for assessing X-ray crystal structures or homology models.
RMS Deviation is a good measure of accuracy. Phylogeny is the evolutionary history of a particular group of organisms or their genes.
Phylogeny can be represented in a phylogenetic tree which graphically represents the lines of descent among organisms or their genes. Phylogenetic analysis of differentially expressed E.coli Nissle 1917 proteins were represented in the following figure-36.

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The method of bootstrapping is the multinomial non-parametric bootstrap as applied in the binomial setting. The tree were represented in the following figure-36

ProtParam analysis:
Extinction coefficients indicate how much light absorbs a protein at a certain wavelength,   pdb structure by using pdb validation tool, Z-score and error value of structure were analysed by QMEAN server (http://swissmodel.expasy.org), finally the protein physico-chemical characters, like molecular weight, molecular formula, half-life of the protein, extinction coefficient, grand average of hydropathicity (GRAVY), total number of atoms and instability index were determined by using ProtParam server.
International Letters of Natural Sciences Vol. 41

CONCLUSION
In the present study, we described the protein profile experimentally by 2D-PAGE and MALDI analysis to understand the stress mechanisms of cocoti sap and wine on E.coli Nissle 1917.
We isolated one newly expressed protein from cocoti wine treated gel which is not present in both control and cocoti sap treated sample i.e. P21 prophage-derived head-stabilizing proteinVG03_ECOL6 (3n1) also called as Head protein gp3. This protein mainly activities related to the viral life cycle. It helps to attach the viral gene into host. The growth rate was delayed in cocoti wine treated E.coli Nissle 1917 when compared to control and cocoti sap treated samples.
Stress mechanism induce many proteins they are involved in metabolic process, hydrolase activity, lyase activity, quinone binding, phosphotransferase system, carbohydrate metabolism, DNA binding, DNA repair, transferase activity, oxidoreductase, purine metabolism, transcription antitermination, transcription regulation and other related activities.
We proved that the predicted protein structure quality, resolution, density and error plot values by QMEAN analysis. Based on these results, only two differentially expressed proteins under sap stress showed that the significant results, which were N-acetylgalactosamine-specific phosphotransferase enzyme IIB component 1, PTPB1_ECOLI and DinI-like protein Z3305/ECs2939 in prophage CP-933VDINI1_ECO57. In case of wine stress, the differentially expressed proteins were Transcription anti-termination protein RFAH-ECO57 NusA and PUR7-eco24-phosphoribosylamidazole-succinocarboxamide synthase showed significant results.
ProtParam analysis indicating that the multiple physico-chemical characters of differentially expressed proteins were differed and compared. The phylogenetic tree represents the relationship in-between the differentially expressed proteins, were showed siblings (related) as well as monophytic clade.
Finally we concluded that E.coli Nissle 1917 exhibited low resistance to cocoti sap. Three differentially expressed proteins showed under cocoti wine stress negative effect on human health.
P21 prophage-derived head-stabilizing proteinVG03_ECOL6 protein helps to attach the viral gene into host, Xanthine dehydrogenase iron sulphur binding subunit XDHC_Eco57 protein chance to cause xanthinuria (respiratory syndrome), may contribute to adult respiratory syndrome, and may potentiate influenza infection through an oxygen metabolite-dependent mechanism. Small toxic polypeptide LDRA_ECOLi protein under wine stress is influencing on cell-signaling.

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In my post-doctoral research, these three differentially expressed proteins 1) P21 prophagederived head-stabilizing proteinVG03_ECOL6 protein, 2) Xanthine dehydrogenase iron sulphur binding subunit XDHC_Eco57 and 3). Small toxic polypeptide LDRA_ECOLi are to be undergoing for drug designing which will be useful for bio-pharmaceutical industries to prepare the drugs against the toxicity of wine treatment.

International Letters of Natural Sciences Vol. 41
List of Tables   S.No  Title of the Table  1 Bacteria commonly found on the surfaces of the human body 2 Chemical composition of Palm Sap 3 Vitamin content of freshly-gathered cocoti Sap 4 Mineral content of cocoti Sap 5 Chemical constituents in palm wines (6% sugar) 6 Matrix compounds for MALDI-TOF analysis 7 Search engines for uninterrupted MS/MS data 8 Mascot parameters 9 Composition of Nutrient broth 10 2-D electrophoresis rehydration buffer components.

11
Types of Rehydration 12 12 % separating gel components for three gels Total acidity levels of cocoti sap and wine 20 Total alcohol content in cocoti sap and wine 21 By dilution methods, the O.D values represented the cocoti sap influence on E.coli 22 Two-way ANOVA for sap influence on E.coli 23 By dilution methods, the O.D values represented the cocoti wine influence on E.coli 24 Two way ANOVA for wine influence on E.coli 25 Growth curves of E.coli under cocoti sap and wine treatment.

26
Total protein concentration in control, cocoti sap and wine treated E.coli by Lowry's method 27 Total protein concentration in control, cocoti sap and wine treated E.coli by Bradford's method 28 Total proteins concentration in control, cocoti sap and wine treated E.coli by BCA kit analysis 29 Comparative statement of protein concentration in control, sap and wine treated samples.

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Analysis of Variance (ANOVA) of the protein concentrations 31 List Validation scores of differentially sap expressed proteins 1).UPF0401 protein ECP Y3010_ECOL5 protein global quality scores 1.1.Close contacts and deviations from ideal geometry 2).Transcriptional regulatory protein BAER_ECOL6 global quality scores 2.1. Close contacts and deviations from ideal geometry 3. Protein PmbA (PMBA-Eco57-protein pmba 0s E.coli) global quality scores 3.1. Close contacts and deviations from ideal geometry 4. Formatehydrogenlyase subunit HYCE_ECOLI global quality scores 4.1. Close contacts and deviations from ideal geometry 5). DinI-like protein Z3305/ECs2939 in prophage CP-933VDINI1_ECO57 global quality scores 5.1. Close contacts and deviations from ideal geometry 6).N-acetylgalactosamine-specific phosphotransferase enzyme IIB component 1. PTPB1_ECOLI Global quality scores 6.1). Close contacts and deviations from ideal geometry 7). t-RNA-Specific adenosine deaminase monomer global quality scores 7.1). Close contacts and deviations from ideal geometry 35 List of newly expressed proteins of Eschericia coli Nissle 1917 identified by Mass Spectrometry by using peptide mass fingerprinting (PMF) analysis under cocoti wine stress.