Trehalose, a naturally occurring osmolyte, is known to be an exceptional stabilizer of proteins and helps retain the activity of enzymes in solution as well as in the freeze-dried state. To understand the mechanism of action of trehalose in detail, we have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins differing in their various physico-chemical properties. Among them, RNase A has been used as a model enzyme to investigate the effect of trehalose on the retention of enzymatic activity upon incubation at high temperatures. 2 M trehalose was observed to raise the transition temperature, T m of RNase A by as much as 18°C and Gibbs free energy by 4.8 kcal mol ؊1 at pH 2.5. There is a decrease in the heat capacity of protein denaturation (⌬C p ) in trehalose solutions for all the studied proteins. An increase in the ⌬G and a decrease in the ⌬C p values for all the proteins points toward a general mechanism of stabilization due to the elevation and broadening of the stability curve (⌬G versus T). A direct correlation of the surface tension of trehalose solutions and the thermal stability of various proteins has been observed. Wyman linkage analysis indicates that at 1.5 M concentration 4 -7 molecules of trehalose are excluded from the vicinity of protein molecules upon denaturation. We further show that an increase in the stability of proteins in the presence of trehalose depends upon the length of the polypeptide chain. The pH dependence data suggest that even though the charge status of a protein contributes significantly, trehalose can be expected to work as a universal stabilizer of protein conformation due to its exceptional effect on the structure and properties of solvent water compared with other sugars and polyols.Sugars have been known to protect proteins against loss of activity (1, 2), chemical (3, 4), and thermal denaturation (5-9). Among several sugars, ␣,␣-trehalose (␣-D-glucopyranosyl(131)-␣-D-glucopyranoside) has been known to be a superior stabilizer in providing protection to biological materials against dehydration and desiccation (10, 11). It is a compatible osmolyte that gets accumulated in organisms under stress conditions (12, 13). Because of this unique property, tremendous interest has been generated in understanding the molecular basis of stress management through induction of trehalose biosynthesis (13, 14).Trehalose has also been found to be very effective in the stabilization of labile proteins during lyophilization (15, 16) and exposure to high temperatures in solution (2,8,9). Sugars in general protect proteins against dehydration by hydrogen bonding to the dried protein by serving as water substitute (15, 17). Several studies carried out by Timasheff and coworkers (9,18) show that sugars and polyols stabilize the folded structure of proteins in solution as a result of greater preferential hydration of the unfolded state compared with the native state. The mechanism is fundamentally different from stabil...

BackgroundProbiotic microorganisms favorably alter the intestinal microflora balance, promote intestinal integrity and mobility, inhibit the growth of harmful bacteria and increase resistance to infection. Probiotics are increasingly used in nutraceuticals, functional foods or in microbial interference treatment. However, the effectiveness of probiotic organism is considered to be population-specific due to variation in gut microflora, food habits and specific host-microbial interactions. Most of the probiotic strains available in the market are of western or European origin, and a strong need for exploring new indigenous probiotic organisms is felt.Methods and FindingsAn indigenous isolate Lp9 identified as Lactobacillus plantarum by molecular-typing methods was studied extensively for its functional and probiotic attributes, viz., acid and bile salt tolerance, cell surface hydrophobicity, autoaggregation and Caco-2 cell-binding as well as antibacterial and antioxidative activities. Lp9 isolate could survive 2 h incubation at pH 1.5–2.0 and toxicity of 1.5–2.0% oxgall bile. Lp9 could deconjugate major bile salts like glycocholate and deoxytaurocholate, indicating its potential to cause hypocholesterolemia. The isolate exhibited cell-surface hydrophobicity of ∼37% and autoaggregation of ∼31%. Presence of putative probiotic marker genes like mucus-binding protein (mub), fibronectin-binding protein (fbp) and bile salt hydrolase (bsh) were confirmed by PCR. Presence of these genes suggested the possibility of specific interaction and colonization potential of Lp9 isolate in the gut, which was also suggested by a good adhesion ratio of 7.4±1.3% with Caco-2 cell line. The isolate demonstrated higher free radical scavenging activity than standard probiotics L. johnsonii LA1 and L. acidophilus LA7. Lp9 also exhibited antibacterial activity against E. coli, L. monocytogenes, S. typhi, S. aureus and B. cereus.ConclusionThe indigenous Lactobacillus plantarum Lp9 exhibited high resistance against low pH and bile and possessed antibacterial, antioxidative and cholesterol lowering properties with a potential for exploitation in the development of indigenous functional food or nutraceuticals.

Polyols and sugars are known to increase the thermal stability of proteins in aqueous solutions and lead to their preferential hydration. While the mechanism of action of sugars has been proposed to be essentially due to the increase in the surface tension of water in their presence, polyols including glycerol have been reported to lower the surface tension of water and act via the solvophobic effect. Surface tension measurements of polyols in water carried out by us, on the contrary, indicate a substantial increase in the surface free energy of water. These increments are comparable to those found in the case of sugars. To investigate the role of the surface tension of the solvent medium on the thermal stability of proteins, we have carried out a comprehensive study on the effects of a series of polyols with varying numbers of hydroxyl groups and stereochemistry, such as mannitol, inositol, sorbitol, xylitol, and adonitol, on the thermal stability of five proteins varying in their physicochemical characteristics, viz., ribonuclease A, R-chymotrypsinogen, lysozyme, cytochrome c, and trypsin inhibitor. The studies have been carried out at pH 2.5, 4.0, and 7.0 to elucidate the contribution of the surface charges toward the polyol-mediated thermal stability of proteins. A very good correlation between an increase in the surface tension of water in the presence of a polyol and an increase in the themal stability of proteins has been observed. Correlation of the thermal stability of proteins with physicochemical properties such as net charge and net hydrophobic surface exposed on unfolding indicates that none of these properties is a dominant factor in governing protein thermal stability. The results indicate that polyol-induced water-mediated effects such as an increase in the surface tension of water play a major role in the stabilization of proteins and that the preferential hydration of proteins observed in their presence is a consequence of the increase in the surface free energy of water.

Lactic acid bacteria (LAB) have taken centre stage in perspectives of modern fermented food industry and probiotic based therapeutics. These bacteria encounter various stress conditions during industrial processing or in the gastrointestinal environment. Such conditions are overcome by complex molecular assemblies capable of synthesizing and/or metabolizing molecules that play a specific role in stress adaptation. Thiols are important class of molecules which contribute towards stress management in cell. Glutathione, a low molecular weight thiol antioxidant distributed widely in eukaryotes and Gram negative organisms, is present sporadically in Gram positive bacteria. However, new insights on its occurrence and role in the latter group are coming to light. Some LAB and closely related Gram positive organisms are proposed to possess glutathione synthesis and/or utilization machinery. Also, supplementation of glutathione in food grade LAB is gaining attention for its role in stress protection and as a nutrient and sulfur source. Owing to the immense benefits of glutathione, its release by probiotic bacteria could also find important applications in health improvement. This review presents our current understanding about the status of glutathione and its role as an exogenously added molecule in food grade LAB and closely related organisms.

Aims: To characterize a bacteriocin‐like factor from Bacillus licheniformis 26 L‐10/3RA isolated from buffalo rumen. Methods and Results: The culture supernatant exhibited the antibacterial activity against a number of indicator organisms in a cut‐well agar assay under anaerobic conditions. The inhibitory component was purified by following ammonium sulphate precipitation, gel filtration and ion exchange chromatography and confirmed to be a single peptide. A single band on tricine‐sodium dodecyl sulphate‐polyacrylamide gel electrophoresis confirmed that the peptide was purified to homogeneity and having an estimated molecular mass of approximately 1400 dalton. Complete amino acid sequence of the peptide yielded 12 amino acids from the N‐terminal end (ISLEICXIFHDN). No homology with previously reported bacteriocins was observed and has been designated as Lichenin. Lichenin was found to be hydrophobic, sensitive to atmospheric oxygen, retained biological activity even after boiling for 10 min and was active over a pH range of 4·0–9·0. Conclusions: The Lichenin represents the first anaerobiosis specific expression of bacteriocin‐like compound isolated from Bacillus licheniformis 26 L‐10/3RA of buffalo rumen origin. Significance and Impact of the Study: Lichenin could be a potential condidate for manipulating the rumen function at molecular level intended for improving the productivity of the ruminant.