Lentils protein isolate as a fermenting substrate for the production of bioactive peptides by lactic acid bacteria and neglected yeast species.

In the current trend where plant-based foods are preferred over animal-based foods, pulses represent an alternative source of protein but also of bioactive peptides (BPs). We investigated the pattern of protein hydrolysis during fermentation of red lentils protein isolate (RLPI) with various lactic acid bacteria and yeast strains. Hanseniaspora uvarum SY1 and Fructilactobacillus sanfranciscensis E10 were the most proteolytic microorganisms. H. uvarum SY1 led to the highest antiradical, angiotensin-converting enzyme-inhibitory and antifungal activities, as found in low molecular weight water soluble extracts (LMW-WSE). The 2039 peptide sequences identified by LMW-WSE were screened using BIOPEP UWM database, and 36 sequences matched with known BPs. Fermentation of RLPI by lactic acid bacteria and yeasts generated 12 peptides undetected in raw RLPI. Besides, H. uvarum SY1 led to the highest abundance (peak areas) of BPs, in particular with antioxidant and ACE-inhibitory activities. The amino acid sequences LVR and LVL, identified in the fermented RLPI, represent novel findings, as they were detected for the first time in substrates subjected to microbial fermentation. KVI, another BP highly characteristic of RLPI-SY1, was previously observed only in dried bonito. 44 novel potential BPs, worthy of further characterization, were correlated with antifungal activity.

Inhibitory effect of four novel synthetic peptides on food spoilage yeasts.

The spoilage of foods caused by the growth of undesirable yeast species is a problem in the food industry. Yeast species such as Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Debaryomyces hansenii, Kluyveromyces lactis and Saccharomyces cerevisiae have been encountered in foods such as high sugar products, fruit juices, wine, mayonnaise, chocolate and soft drinks. The demand for new methods of preservations has increased because of the negative association attached to chemical preservatives. The sequence of a novel short peptide (KKFFRAWWAPRFLK-NH2) was modified to generate three versions of this original peptide. These peptides were tested for the inhibition of the yeasts mentioned above, allowing for the better understanding of their residue modifications. The range of the minimum inhibitory concentration was between 25 and 200 µg/mL. Zygosaccharomyces bailii was the most sensitive strain to the peptides, while Zygosaccharomyces rouxii was the most resistant. Membrane permeabilisation was found to be responsible for yeast inhibition at a level which was a two-fold increase of the MIC (400 µg/mL). The possibility of the production of reactive oxygen species was also assessed but was not recognised as a factor involved for the peptides' mode of action. Their stability in different environments was also tested, focusing on high salt, pH and thermal stability. The newly designed peptides showed good antifungal activity against some common food spoilage yeasts and has been proven effective in the application in Fanta Orange. These efficient novel peptides represent a new source of food preservation that can be used as an alternative for current controversial preservatives used in the food industry.

Identification and characterization of a new antifungal peptide in fermented milk product containing bioprotective Lactobacillus cultures.

Mold and yeast contamination constitutes a major problem in food commodities, including dairy products, hence new natural preventive measures are in high demand. The aim of the current study is to identify and characterize novel antifungal peptides produced by lactic acid bacteria (LAB) in sour cream. By the use of a newly developed image-based 96-well plate fungal growth inhibition assay targeting Debaryomyces hansenii, combined with a range of analytical tools comprising HPLC-high-resolution mass spectrometry, ultrahigh-performance liquid chromatography-Triple Quadrupole MS and nuclear magnetic resonance spectroscopy, we successfully identified a new antifungal peptide (DMPIQAFLLY; 1211 Da) in sour cream enriched with two bioprotective LAB strains. This peptide represents a fragment of casein, the most abundant protein in milk. Presumably, the proteolytic activity of these bioprotective strains results in the observed 4-fold higher concentration of the peptide during storage. Both bioprotective strains are able to generate this peptide in concentrations up to 0.4 µM, independently of the sour cream starter culture employed. The peptide attenuates the growth rate of D. hansenii at concentrations ≥35 µM, and results in smaller cells and more compact colonies. Hence, the peptide is likely contributing to the overall preserving effect of the investigated bioprotective LAB strains.

Bacillus amyloliquefaciens Q-426 as a potential biocontrol agent against Fusarium oxysporum f. sp. spinaciae.

In recent years, Bacillus species have received considerable attention for the biological control of many fungal diseases. In this study, Bacillus amyloliquefaciens Q-426 was tested for its potential use against a variety of plant pathogens. Our screen for genes involved in the biosynthesis of antifungal agents revealed that the fen and bmy gene clusters are present in the Q-426 genome. Lipopeptides such as bacillomycin D, fengycin A, and fengycin B were purified from the bacterial culture broth and subsequently identified by ESI-mass spectrometry. The minimal inhibitory concentration of fengycin A against Fusarium oxysporum f. sp. spinaciae W.C. Snyder & H.N. Hansen O-27 was determined to be 31.25 µg ml(-1) . However, exposure of fungal cells to 50 µg ml(-1) of fengycin A did not allow permeation of fluorescein diacetate into the cytoplasm through the cell membrane. Moreover, leakage of intracellular inorganic cations, nucleic acid and protein were also not detected, indicating that the fungal cell membrane is not the primary target of action for fengycin A. Profound morphological changes were observed in the F. oxysporum strain and spore germination was completely inhibited, suggesting that 50 µg ml(-1) of fengycin A acts, at least, as a fungistatic agent.

[Antistress cross-protection of UV irradiated yeast cells with participation of extracellular peptide factors].

Antistress effect of extracellular peptides on UV irradiated yeast of different phylogenetic groups was studied. Yeast from different ecotopes and taxonomic groups exposed to UV radiation of a lethal intensity showed a protective effect and reactivating effect with participation of extracellular peptides. The highest protective activity was found in peptide reactivation factors (RFs) of bakery yeast-Saccharomyces cerevisiae, Kluyveromyces fragilis, and Candida utilis; the highest reactivating activity was exhibited by factors of the above-mentioned cultures and Debariomyces hansenii. Cross-protective and reactivating effects of RFs of yeast belonging to different taxonomic groups were demonstrated. Cross-protection increased two to three times after preexposure of reactivation factors to UV light (activation) in contrast to their reactivating effect.

Reversal of T cell anergy in leprosy patients: in vitro presentation with Mycobacterium leprae antigens using murabutide and Trat peptide in liposomal delivery.

Mycobacterium leprae, the causative agent of leprosy resides and multiplies within the host monocytes and macrophages, thereby evading host immune system. Cell-mediated immune response (CMI) plays a vital role as evidenced from the high CMI in BT/TT (borderline and tuberculoid) patients and conversely low in BL/LL (borderline and lepromatous) patients. In the present study, an attempt was made to immunomodulate the anergized T cells of lepromatous leprosy patients by presenting the mycobacterial antigen in combination with T cell adjuvant, murabutide (active analog of muramyl' dipeptide, MDP-BE) and a Trat peptide (T cell epitope of Integral membrane protein (Trat) from Escherichia coli) in particulate form (liposomes) or soluble form (media). PBMNC of normal, BT/TT and BL/LL were stimulated in vitro with five mycobacterial antigens (Ag) in the following formulations, Ag, Ag+murabutide, Ag+murabutide+Trat peptide either in liposomes or in medium. All the five antigen(s) when delivered in liposomes containing murabutide and Trat peptide showed a very high lymphoproliferative response (p<0.001) in all the three groups. IFN-gamma and IL-2 were significantly (p<0.001) high in these culture supernatants compared to IL-10 and IL-4 confirming a shift from CD4+Th2 to Th1 response in leprosy patients with particulate mode of antigen presentation. Interestingly, PBMNC derived from lepromatous patients also showed consistent T cell proliferation with all the formulations. Further, the mechanism of liposomal processing of antigens was studied using different inhibitors that interfere at different stages of antigen presentation. Results indicate that this study may pave way for an immunotherapeutic approach for reverting the anergic T cells of lepromatous patients to proliferating T cells with the release of Th1 cytokines thereby restoring the CMI response in these patients.

Heat shock protein Hsp60-reactive gamma delta cells: a large, diversified T-lymphocyte subset with highly focused specificity.

Previously, we detected a subset of gamma delta T cells in the newborn mouse thymus that responded to the mycobacterial heat shock protein Hsp60, as well as with what seemed to be a self-antigen. All of these cells expressed V gamma 1, most often in association with V delta 6+. It was not clear, however, whether similar, mature gamma delta cells with Hsp60 reactivity are common outside of the thymus, or rather, whether they are largely eliminated during development. From the data presented here, we estimate that gamma delta cells responding to Hsp60 comprise 10-20% of normal splenic and lymph node gamma delta T cells. Such cells, derived from adult spleen, always express a V gamma 1-J gamma 4-C gamma 4 gamma chain, although not all cells with this gamma chain show Hsp60 reactivity. Many of these V gamma 1+ cells also express V delta 6-J delta 1-C delta, though fewer than in V gamma 1+ cells from the newborn thymus. Extensive diversity is evident in both the gamma and delta chain junctional amino acids of the receptors of these cells, indicating that they may largely develop in the thymus of older animals or undergo peripheral expansion. Finally, we found that all such cells responding to both a putative self-antigen and to mycobacterial Hsp60 respond to a 17-amino acid synthetic peptide representing amino acids 180-196 of the Mycobacterium leprae Hsp60 sequence. This report demonstrates that a large subset of Hsp60-reactive peripheral lymphoid gamma delta T cells preexists in normal adult mice, all members of which respond to a single segment of this common heat shock protein.

Characterization of the functional properties of the 70-kDa protein of Mycobacterium bovis.

A number of mycobacterial proteins have been shown to induce strong humoral and cellular immune responses, including the 70-kDa antigen (p70) of Mycobacterium leprae and Mycobacterium bovis. On the basis of sequence homology and an ATP binding ability, p70 has previously been tentatively allocated to the 70-kDa family of heat shock proteins (hsp70). We have purified the M. bovis p70 antigen and described ATPase and Ca(2+)-dependent autophosphorylating activities. These co-purified with p70 on gel chromatography and were up-regulated by native proteins and down-regulated by peptides. Inhibitory peptides were shown to bind p70. These data imply close functional similarities of mycobacterial p70 to other members of the hsp70 family, the Escherichia coli homologue dnaK in particular.

A preformed, ordered structure of a 25-residue peptide derived from a major histocompatibility complex class I antigen is required to affect insulin receptor function.

It was recently shown that a 25-residue peptide, Dk-(61-85), derived from the alpha 1 domain of a murine major histocompatibility class I molecule (H-2Dk), affects insulin receptor functions (Hansen, T., Stagsted, J., Pedersen, L., Roth, R. A., Goldstein, A., and Olsson, L. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3123-3126; Stagsted, J., Reaven, G. M., Hansen, T., Goldstein, A., and Olsson, L. (1990) Cell 62, 297-307). We now report that this peptide can reversibly assume a biologically active or inactive state as measured in the rat adipocyte glucose uptake assay, implying that the peptide has at least two interconvertible conformations. The peptide has an ordered conformation in 0.1 M HCl or 0.1 M NaCl stock solution as shown by circular dichroism, but has a disordered molecular structure and is inactive when dissolved in H2O. The biologically active peptide forms liquid crystals at the stock solution concentration (1 mM), so the CD spectra do not provide information on the secondary structure. Under all conditions tested, biological activity (measured after transfer to assay buffer) is associated with an ordered conformation in stock solution. Biological activity and an ordered conformation of the peptide in H2O stock solution can be induced by increasing ionic strength (greater than 100 mM NaCl for maximal effect) or increasing pH (greater than 5 for maximal effect). The induction rate of the ordered conformation is slow with a half-maximal value obtained after approximately 20 min. Both biological activity and the ordered structure are lost upon heating of stock solution to 90 degrees C or upon transfer to assay buffer. A similar correlation of ordered structure with biological activity was observed with two truncated peptides derived from Dk-(61-85). It is inferred from these results that the Dk-(61-85) peptide and related peptides only affect insulin-stimulated glucose uptake in rat adipocytes if they have assumed an ordered conformation in stock solution prior to transfer to assay buffer and exposure to cells.