Naturally occurring and synthetic peptides: Efficient tyrosinase inhibitors.

Tyrosinase is a copper-containing enzyme involved in the biosynthesis of melanin pigment, which is the most important photo protective agent against skin photo carcinogenesis. Excess production of melanin causes hyperpigmentation leading to undesired browning in human skin, fruits, and vegetable as well as plant-derived foods. Moreover, the role of tyrosinase in the onset and progression of various diseases such as cancers, Alzheimer's, and Parkinson diseases has been well documented in the literature. In this respect, tyrosinase inhibitors have been in the center of attention particularly as the efficient skin whitening agents. Among a wide range of compounds possessing anti-tyrosinase activity, peptides both natural and synthetic derivatives have attracted attention due to high potency and safety.

Anticancer potential of an exopolysaccharide from Lactobacillus helveticus MB2-1 on human colon cancer HT-29 cells via apoptosis induction.

This study aimed at investigating the anticancer activity of an exopolysaccharide (EPS) isolated from Lactobacillus helveticus MB2-1. The crude EPS from L. helveticus MB2-1 (LHEPS) was fractionated into three fractions, namely LHEPS-1, LHEPS-2 and LHEPS-3. LHEPS-1 exhibited the most effective anti-proliferative activity, which was associated with a stronger inhibition rate and increased lactate dehydrogenase leakage of human colon cancer HT-29 cells. Flow cytometry analysis and colorimetric assay revealed that LHEPS-1 induced cell cycle arrest by preventing G1 to S transition and increased the apoptosis rate. Furthermore, LHEPS-1 enhanced the production of intracellular reactive oxygen species (ROS) and the activity of caspases-8/9/3, increased the levels of pro-apoptotic Bax and mitochondrial cytochrome c, while decreased the anti-apoptotic Bcl-2 level, indicating that LHEPS-1 might induce the apoptosis of HT-29 cells through a ROS-dependent pathway and a mitochondria-dependent pathway. These findings suggest that LHEPS-1 may be developed as an effective food and/or drug for the prevention and therapeutics of cancer, especially human colon cancer.

Characterization of casein-derived peptide bioactivity: Differential effects on angiotensin-converting enzyme inhibition and cytokine and nitric oxide production.

Lactic acid bacteria (LAB) are used as starter cultures in the production of fermented dairy products and have the potential to confer bioactivity relevant to cardiovascular health, as they possess extensive proteolytic systems that liberate small bioactive peptides from larger milk proteins. Certain casein-derived peptides released by various LAB strains during fermentation have been shown to reduce hypertension and to modulate the immune system. We investigated the growth and peptide production of 2 LAB strains, Lactobacillus helveticus R0389 and Lactocaseibacillus rhamnosus R0011, their immunomodulatory activities, as well as their abilities to inhibit the angiotensin-converting enzyme (ACE). Peptide fractions collected from the cell-free supernatant of both medium-grown and milk fermentation cultures were assessed for ACE-inhibitory activity and their effects on the production of proinflammatory and regulatory cytokines by human THP-1 monocytes. Cultures were grown in medium, with or without supplementation with 0.1% casein, or in 3.25% milk fermented with each LAB strain. Casein supplementation increased the growth rate of both LAB strains, and significantly increased ACE-inhibitory activity of peptide fractions collected from both L. helveticus R0389 and L. rhamnosus R0011 cultures grown for 12 h. Fermentation peptide fractions of L. rhamnosus R0011 showed comparable ACE-inhibitory activity to known ACE inhibiting peptides Val-Pro-Pro and Ile-Pro-Pro (up to 79% inhibition) with a significant difference between culture peptide fractions and acidified and nonacidified control fractions collected after 6 d of fermentation. Many milk and casein-derived peptides reported in previous studies have been identified as part of a larger bioactive fraction. We synthesized a group of these peptides to individually assess both ACE-inhibitory and immunomodulatory activity. The known ACE inhibitors Val-Pro-Pro and Ile-Pro-Pro showed similar ACE inhibition to previously published results, while also inducing the production of the regulatory cytokine IL-10 by monocytes in the presence and absence of a proinflammatory stimulant. These synthesized peptides could also induce the production of nitric oxide (NO), a potent vasodilator, in human endothelial cell cultures. Investigating the relationships among these bioactive properties could improve the use of probiotic organisms and their secreted products in the food industry.

Influences of drying methods on the structural, physicochemical and antioxidant properties of exopolysaccharide from Lactobacillus helveticus MB2-1.

In this study, in order to evaluate influences of different drying methods on the structural characteristics, physicochemical properties and antioxidant activities of exopolysaccharides (EPS) from Lactobacillus helveticus MB2-1, three drying methods, including spray-drying (SD), freeze-drying (FD) and spray freeze-drying (SFD), were applied to dry EPS. Results showed that different drying procedures had no significant influence on the primary structure and constituent monosaccharides of EPSs. However, the surface morphology of the three dried EPSs varied greatly in size and shape due to different drying processes. Among three dried EPSs, the particle size distribution of spray freeze-dried EPS (SF-EPS) was relatively narrower and uniform. Additionally, SF-EPS behaved better apparent viscosity and emulsifying property than spray-dried EPS (S-EPS) and freeze-dried EPS (F-EPS). SF-EPS exhibited stronger antioxidant activities when compared with S-EPS and F-EPS, according to the results of scavenging activities on different radicals and chelating activity on ferrous ion. Overall, SFD was the appropriate method for industrial production of EPS from Lactobacillus helveticus MB2-1 with better physicochemical properties and antioxidant activities.

Structural characterization and immunomodulatory activity of an exopolysaccharide produced by Lactobacillus helveticus LZ-R-5.

Exopolysaccharide (R-5-EPS) was isolated from the fermented milk of Lactobacillus helveticus LZ-R-5 and purified by DEAE-52 cellulose anion-exchange column, and characterization of the structure was conducted. Results showed that R-5-EPS was a heteropolysaccharide containing linear repeating units of →6)-ß-D-Galp-(1→3)-ß-D-Glcp-(1→3)-ß-D-Glcp-(1→3)-ß-D-Glcp-(1→3)-ß-D-Glcp-(1→ with an average Mw of 5.41 × 105 Da. Furthermore, at a cellular level, R-5-EPS showed immunostimulatory activity due to its strong effect on increasing proliferation of RAW264.7 macrophages and enhancing phagocytosis, acid phosphatase activity, nitric oxide production and cytokines production in macrophages. These results suggest that R-5-EPS have a potent immunostimulatory activity and may be explored as a potential immunomodulatory agent.

Surface Layer of Lactobacillus helveticus MIMLh5 Promotes Endocytosis by Dendritic Cells.

Surface layers (S-layers) are proteinaceous arrays covering the cell walls of numerous bacteria. Their suggested properties, such as interactions with the host immune system, have been only poorly described. Here, we aimed to elucidate the role of the S-layer from the probiotic bacterial strain Lactobacillus helveticus MIMLh5 in the stimulation of murine bone-marrow-derived dendritic cells (DCs). MIMLh5 induced greater production of interferon beta (IFN-ß), interleukin 10 (IL-10), and IL-12p70, compared to S-layer-depleted MIMLh5 (naked MIMLh5 [n-MIMLh5]), whereas the isolated S-layer was a poor immunostimulator. No differences in the production of tumor necrosis factor alpha (TNF-α) or IL-1ß were found. Inhibition of the mitogen-activated protein kinases JNK1/2, p38, and ERK1/2 modified IL-12p70 production similarly in MIMLh5 and n-MIMLh5, suggesting the induction of the same signaling pathways by the two bacterial preparations. Treatment of DCs with cytochalasin D to inhibit endocytosis before the addition of fluorescently labeled MIMLh5 cells led to a dramatic reduction in the proportion of fluorescence-positive DCs and decreased IL-12 production. Endocytosis and IL-12 production were only marginally affected by cytochalasin D pretreatment when fluorescently labeled n-MIMLh5 was used. Treatment of DCs with fluorescently labeled S-layer-coated polystyrene beads (Sl-beads) resulted in much greater uptake of beads, compared to noncoated beads. Prestimulation of DCs with cytochalasin D reduced the uptake of Sl-beads more than plain beads. These findings indicate that the S-layer plays a role in the endocytosis of MIMLh5 by DCs. In conclusion, this study provides evidence that the S-layer of L. helveticus MIMLh5 is involved in endocytosis of the bacterium, which is important for strong Th1-inducing cytokine production.IMPORTANCE Beneficial microbes may positively affect host physiology at various levels, e.g., by participating in immune system maturation and modulation, boosting defenses and dampening reactions, thus affecting the whole homeostasis. As a consequence, the use of probiotics is increasingly regarded as suitable for more extended applications for health maintenance, not only microbiota balancing. This implies a deep knowledge of the mechanisms and molecules involved in host-microbe interactions, for the final purpose of fine tuning the choice of a probiotic strain for a specific outcome. With this aim, studies targeted to the description of strain-related immunomodulatory effects and the identification of bacterial molecules responsible for specific responses are indispensable. This study provides new insights in the characterization of the food-origin probiotic bacterium L. helveticus MIMLh5 and its S-layer protein as a driver for the cross-talk with DCs.

Evaluation of casein & whey protein hydrolysates as well as milk fermentates from Lactobacillus helveticus for expression of gut hormones.

BACKGROUND & OBJECTIVES: Milk proteins play a beneficial role in the regulation of food intake, postprandial glycaemia and enteroendocrine hormone secretions and thus are receiving considerable attention for the management of metabolic inflammatory disorders such as type 2 diabetes mellitus (T2DM). The objective of this study was to evaluate the efficacy of peptide/s obtained from milk proteins (casein and whey) as well as from the milk fermented with Lactobacillus helveticus as secretagogues for gut hormones and to purify and characterize the active peptides. METHODS: Effect of hydrolysates of casein protein (CP) and whey protein (WP) and L. helveticus fermented milk on the expression of proglucagon, pro-gastric inhibitory peptide (GIP) and cholecystokinin (CCK) genes was monitored by real-time quantitative polymerase chain reaction. The active glucagon-like peptide-1 (GLP-1) secretion was also quantitatively measured using ELISA. RESULTS: Hydrolysates of CP and WP as well as fermentates of L. helveticus induced the proglucagon, pro-GIP and CCK expression and secretion of GLP-1 in STC-1 (pGIP/Neo) cells. However, intact casein exhibited maximum GLP-1 secretion and proglucagon expression. Two active peptides (F5 and F7) derived from CP1 and WP3 hydrolysates having the ability to upregulate the GLP-1 secretion by 1.6 and 1.8 folds were obtained, and the mass was found to be 786 and 824 Da, respectively, as determined by electrospray ionization-mass spectrometry. However, no single active peptide from L. helveticus fermented milk could be obtained. INTERPRETATION & CONCLUSIONS: Casein as well as fermentates obtained from L. helveticus fermented milk showed higher potential for GLP-1 induction. These can be explored as novel therapeutics to T2DM effectively after demonstrating their in vivo efficacy in appropriate animal models.

Characterization of a novel polysaccharide with anti-colon cancer activity from Lactobacillus helveticus MB2-1.

The present study aimed at investigating the potential anti-colon cancer activity of three purified exopolysaccharides fractions (LHEPS-1, LHEPS-2 and LHEPS-3) from the Lactobacillus helveticus MB2-1. The experimental evidence showed that LHEPS-1 significantly inhibited cell proliferation of human colon cancer Caco-2 cells in both time- and concentration-dependent manners. In contrast, no significant improvements of the inhibitory effects of LHEPS-2 and LHEPS-3 on Caco-2 cells were observed with increasing sample concentrations or prolonged incubation time. Furthermore, the structure of LHEPS-1 was elucidated using methylated analysis, gas chromatography-mass spectroscopy (GC-MS) and nuclear magnetic resonance spectroscopy (NMR), including one- and two-dimensional nuclear magnetic resonance (1D and 2D NMR). Results indicated that the LHEPS-1 consisted of a decasaccharide repeating unit with the following structure (n ≈ 122)： Our results suggested that the LHEPS-1 produced by L. helveticus MB2-1 might be suitable for using as natural anti-colon cancer drugs and functional foods ingredients.

Structural characterization and anticancer activity of cell-bound exopolysaccharide from Lactobacillus helveticus MB2-1.

A novel cell-bound exopolysaccharide (c-EPS) was isolated from Lactobacillus helveticus MB2-1 by ultrasonic extraction, anion exchange, and gel filtration chromatography before being structurally characterized. The c-EPS is a heteropolysaccharide with an average molecular weight of 1.83 × 10(5) Da and is composed of glucose, mannose, galactose, rhamnose, and arabinose at a molar ratio of 3.12:1.01:1.00:0.18:0.16. Methylation analysis and nuclear magnetic resonance analysis revealed that the c-EPS is a linear glucomannogalactan containing repeating units of → 6)-ß-D-Manp-(1 → 3)-ß-D-Glcp-(1 → 3)-ß-D-Glcp-(1 → 3)-ß-D-Glcp-(1 → 4)-α-D-Galp-(1 → and trace amounts of Rhap-(1 → and (1 → 4)-Arap residues. Complex formation with Congo red demonstrated a triple-strand helical conformation for the c-EPS. Scanning electron microscopy of the c-EPS revealed many regular feather-like structural units. Topographical examination of c-EPS by atomic force microscopy revealed that the c-EPS formed rounded-to-spherical lumps with different sizes and chain formations. Furthermore, preliminary in vitro tests revealed that c-EPS significantly inhibited the proliferation of HepG-2, BGC-823, and especially HT-29 cancer cells.

Proteome analysis of Lactobacillus helveticus H9 during growth in skim milk.

Lactobacillus helveticus H9 was isolated from traditionally fermented yak milk in Tibet (China) with the ability to produce the antihypertensive peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) during milk fermentation. To understand the changes in the protein expression of L. helveticus H9, proteome analysis was performed at 3 different growth stages, lag phase (pH 6.1), log phase (pH 5.1), and stationary phase (pH 4.5) using 2-dimensional electrophoresis (2-DE). Further analysis showed that 257 differential protein spots were found and 214 protein spots were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The cellular functions of the differentially expressed proteins were complex. Interestingly, the proteolytic system-related proteins aminopeptidase N (PepN), aminopeptidase E (PepE), endopeptidase O2 (PepO2), and oligopeptide transport system permease protein (OppC) were observed only on the maps of pH 5.1 and pH 4.5, which was consistent with the presence of angiotensin I-converting enzyme (ACE)-inhibitory peptides VPP and IPP during these 2 growth stages (log phase and stationary phase). These results, combined with a previous study of gene expression of the proteolytic system, led us to conclude that the Opp transport system, pepE, and pepO2 are likely related to the production of ACE-inhibitory peptides.

Characterization of an antiproliferative exopolysaccharide (LHEPS-2) from Lactobacillus helveticus MB2-1.

In this study, we reported that three purified exopolysaccharides fractions (LHEPS-1, LHEPS-2 and LHEPS-3) and crude LHEPS from the Lactobacillus helveticus MB2-1 inhibited the proliferation potential of human gastric cancer BGC-823 cells. We found that all the LHEPS fractions and crude LHEPS exerted concentration and time dependent inhibitory activities in vitro on BGC-823 cells. LHEPS-2 exhibited higher antiproliferative activity against BGC-823 cells in vitro than LHEPS-1, LHEPS-3 and crude LHEPS. With the consideration of the outstanding antiproliferative effect of LHEPS-2 on BGC-823 cells, more fine structural characterization of LHEPS-2 was further identified. The structure of LHEPS-2 was elucidated by methylated analysis, GC-MS, (1)H NMR and (13)C NMR spectroscopy, together with two-dimensional (2D) COSY, TOCSY, HSQC, HMBC and NOESY spectroscopy. Results showed that the LHEPS-2 was consisted of the following repeating units:

The structure and immunomodulatory activity on intestinal epithelial cells of the EPSs isolated from Lactobacillus helveticus sp. Rosyjski and Lactobacillus acidophilus sp. 5e2.

The Lactic acid bacteria (LAB) Lactobacillus acidophilus sp. 5e2 and Lactobacillus helveticus sp. Rosyjski both secrete exopolysaccharides (EPSs) into their surrounding environments during growth. A number of EPSs have previously been shown to exhibit immunomodulatory activity with professional immune cells, such as macrophages, but only limited studies have been reported of their interaction with intestinal epithelial cells. An investigation of the immunomodulatory potential of pure EPSs, isolated from cultures of Lactobacillus acidophilus sp. 5e2 and Lactobacillus helveticus sp. Rosyjski, with the HT29-19A intestinal epithelial cell line are reported here. For the first time the structure of the EPS from Lactobacillus helveticus sp. Rosyjski which is a hetropolysaccharide with a branched pentasaccharide repeat unit containing d-glucose, d-galactose and N-acetyl-d-mannosamine is described. In response to exposure to lactobacilli EPSs HT29-19A cells produce significantly increased levels of the proinflammatory cytokine IL-8. Additionally, the EPSs differentially modulate the mRNA expression of Toll-like receptors. Finally, the pre-treatment of HT29-19A cells with the EPSs sensitises the cells to subsequent challenge with bacterial antigens. The results reported here suggest that EPSs could potentially play a role in intestinal homeostasis via a specific interaction with intestinal epithelial cells.

Probiotics prevent enterohaemorrhagic Escherichia coli O157:H7-mediated inhibition of interferon-gamma-induced tyrosine phosphorylation of STAT-1.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 inhibits interferon (IFN)-gamma-stimulated tyrosine phosphorylation of signal transducer and activator of transcription (STAT)-1 in epithelial cells. We determined the effects of probiotics on EHEC-mediated disruption of IFN-gamma-stimulated STAT-1 activation in epithelial cell lines. Confluent Intestine 407, HEp-2 and Caco-2 epithelial cells were pre-treated (3 h) with either probiotics or surface-layer proteins derived from Lactobacillus helveticus R0052 prior to infection with EHEC O157:H7 strain CL56 (m.o.i. 100:1, 6 h, 37 degrees C in 5% CO2). Subsequently, cells were washed and stimulated with human recombinant IFN-gamma (50 ng ml(-1), 0.5 h, 37 degrees C) followed by whole-cell protein extraction and immunoblotting for tyrosine-phosphorylated STAT-1. Relative to uninfected cells, STAT-1-activation was reduced after EHEC O157:H7 infection. Pre-incubation with the probiotic L. helveticus R0052 followed by EHEC infection abrogated pathogen-mediated disruption of IFN-gamma-STAT-1 signalling. As determined using Transwell inserts, probiotic-mediated protection was independent of epithelial cell contact. In contrast, pre-incubation with boiled L. helveticus R0052, an equal concentration of viable Lactobacillus rhamnosus R0011, or surface-layer proteins (0.14 mg ml(-1)) did not restore STAT-1 signalling in EHEC-infected cells. The viable probiotic agent L. helveticus R0052 prevented EHEC O157:H7-mediated subversion of epithelial cell signal transduction responses.

Surface-layer protein extracts from Lactobacillus helveticus inhibit enterohaemorrhagic Escherichia coli O157:H7 adhesion to epithelial cells.

Adherence of intestinal pathogens, including Escherichia coli O157:H7, to human intestinal epithelial cells is a key step in pathogenesis. Probiotic bacteria, including Lactobacillus helveticus R0052 inhibit the adhesion of E. coli O157:H7 to epithelial cells, a process which may be related to specific components of the bacterial surface. Surface-layer proteins (Slps) are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. However, the ability of S-layer protein extract derived from probiotic bacteria to block adherence of enteric pathogens has not been investigated. Human epithelial (HEp-2 and T84) cells were treated with S-layer protein extract alone, infected with E. coli O157:H7, or pretreated with S-layer protein extract prior to infection to determine their importance in the inhibition of pathogen adherence. The effects of S-layer protein extracts were characterized by phase-contrast and immunofluorescence microscopy and measurement of the transepithelial electrical resistance of polarized monolayers. Pre-treatment of host epithelial cells with S-layer protein extracts prior to E. coli O157:H7 infection decreased pathogen adherence and attaching-effacing lesions in addition to preserving the barrier function of monolayers. These in vitro studies indicate that a non-viable constituent derived from a probiotic strain may prove effective in interrupting the infectious process of an intestinal pathogen.