Promising antifungal effect of some Euro-Asiatic plants against dangerous pathogenic and toxinogenic fungi

BACKGROUND: Increasing evidence of fungicide‐resistant toxinogenic and pathogenic fungal species is obvious. Looking for new possibilities of antifungal treatment or sources of antifungal substances is a major problem. Some medicinal plants exert strong antifungal properties and could be conveniently used as a promising alternative source for presently problematic antifungal treatment in many areas with respect to their natural origin. Methanol extracts of 46 medicinal plants from the Eurasian area were used in a screening assay for antifungal activity in this study. The growth inhibitory effect was tested against six significant pathogenic and toxinogenic fungal species: Fusarium oxysporum, F. verticillioides, Penicillium expansum, P. brevicompactum, Aspergillus flavus and A. fumigatus. RESULTS: For 14 plant species, the possibility of using them as natural fungicides was indicated. The extract from Grindelia camporum showed significant activity against all target fungal species. The most sensitive target fungus was the toxinogenic and human pathogenic species A. fumigatus. CONCLUSION: This study has identified 14 extracts of medicinal plants with a potential use as an antifungal treatment in various areas. One of them showed promising efficiency against all selected significant pathogenic and toxinogenic fungal species. Copyright © 2010 Society of Chemical Industry     

Moisture‐Absorption and Water Dynamics in the Powder of Egg Albumen Peptide,Met‐Pro‐Asp‐Ala‐His‐Leu

Moisture absorbed into the powder of Met‐Pro‐Asp‐Ala‐His‐Leu (MPDAHL)—a novel egg albumen antioxidant peptide—profoundly affects its properties. In this study, we elucidated water dynamics in MPDAHL using DVS, DSC, and low‐field ¹H NMR. Based on the DVS data, we found that MPDAHL sorption kinetics obey a parallel exponential model. DSC results indicated that both water and heating could change the microstructure of MPDAHL. The T₂ parameters of NMR reflected the different phases of moisture absorption revealed that there were 4 categories of water with different states or mobility in the MPDAHL during the moisture absorption process. The fastest fraction T_2b mainly dominated the hygroscopicity of MPDAHL and the absorbed water significantly changed the proton distribution and structure of MPDAHL. Thus, this study shows that DVS, DSC, and low‐field ¹H NMR are effective methods for monitoring water mobility and distribution in synthetic peptides. It can be used to improve the quality assurance of functional peptides.     

Malting Barley Grain Non‐specific Lipid‐Transfer Protein (ns‐LTP): Importance for Grain Protection

A basic protein (pI < 9) was isolated to homogeneity from a domestic cultivar of malting barley grain (Hordeum vulgare). In its unreduced form it exists as a dimer of a 9 kDa protomer with four disulphide bridges. These characteristics together with protein sequence data revealed that the isolated protein belongs to the class of ns‐LTP. The antifungal potency of malting barley grain ns‐LTP was examined on Saccharomyces cerevisiae, Candida albicans and the plant pathogen Fusarium solani growth in vitro. It was found that ns‐LTP inhibits Saccharomyces and Fusarium growth; the concentration required for 50% inhibition after 24 h of incubation (IC₅₀) was 100 and 80 μg/mL, respectively. On the basis of these results, the importance of ns‐LTP for barley grain protection from fungal diseases has been discussed.     

Novel Antifungal Peptides Produced by Leuconostoc mesenteroides DU15 Effectively Inhibit Growth of Aspergillus niger

The ability of Leuconostoc mesenteroides DU15 to produce antifungal peptides that inhibit growth of Aspergillus niger was evaluated under optimum growth conditions of 30 °C for 48 h. The cell‐free supernatant showed inhibitory activity against A. niger. Five novel peptides were isolated with the sequences GPFPL, YVPLF, LLHGVPLP, GPFPLEMTLGPT, and TVYPFPGPL as identified by de novo sequencing using PEAKS 6 software. Peptide LLHGVPLP was the only positively charged (cationic peptides) and peptide GPFPLEMTLGPT negatively charged (anionic), whereas the rest are neutral. The identified peptides had high hydrophobicity ratio and low molecular weights with amino acids sequences ranging from 5 to 12 residues. The mode of action of these peptides is observed under the scanning electron microscope and is due to cell lysis of fungi. This work reveals the potential of peptides from L. mesenteroides DU15 as natural antifungal preservatives in inhibiting the growth of A. niger that is implicated to the spoilage during storage.     

Development of active plastic crate and evaluation of phytopathogenic and pathogenic food spoilage micro‐organism inhibition

BACKGROUND: The plastic crates used in fruit and vegetable shipping can be vehicles of disease dissemination among production fields, since there is a chance of phytopathogenic micro‐organism adhesion on the crate surfaces when in contact with soil, contaminated produce or handling. The aim of this study was to develop an active plastic crate incorporated with a triclosan‐based antimicrobial agent and to evaluate its efficiency of micro‐organism inhibition. RESULTS: Staphylococcus aureus (a human pathogen), Clavibacter michiganensis ssp. michiganensis and Erwinia carotovora ssp. carotovora (phytopathogens) were the most sensitive micro‐organisms when in contact with samples of plastic crate incorporated with 30 g kg^?1 of antimicrobial agent. They presented diameters of approximately 5.0, 3.5 and 3.5 cm respectively in the halo test. Mean specific growth rates decreased in samples with 30 g kg^?1 of antimicrobial agent, compared with control samples, from 1.13 to 0 h^?1 for S. aureus, from 1.26 to 0.47 h^?1 for Escherichia coli and from 1.75 to 0.18 h^?1 for Listeria monocytogenes. The antimicrobial agent did not influence the mechanical properties of the crates. CONCLUSION: The active plastic crate has great potential to contribute to the safety of horticultural produce by restraining the proliferation of micro‐organisms among production fields. Copyright © 2008 Society of Chemical Industry     

NY‐ESO‐1 protein glycosylated by yeast induces enhanced immune responses

Vaccine strategies that target dendritic cells to elicit potent cellular immunity are the subject of intense research. Here we report that the genetically engineered yeast Saccharomyces cerevisiae, expressing the full‐length tumour‐associated antigen NY‐ESO‐1, is a versatile host for protein production. Exposing dendritic cells (DCs) to soluble NY‐ESO‐1 protein linked to the yeast a‐agglutinin 2 protein (Aga2p) protein resulted in protein uptake, processing and MHC class I cross‐presentation of NY‐ESO‐1‐derived peptides. The process of antigen uptake and cross‐presentation was dependent on the glycosylation pattern of NY‐ESO‐1‐Aga2p protein and the presence of accessible mannose receptors. In addition, NY‐ESO‐1‐Aga2p protein uptake by dendritic cells resulted in recognition by HLA‐DP4 NY‐ESO‐1‐specific CD4⁺ T cells, indicating MHC class II presentation. Finally, vaccination of mice with yeast‐derived NY‐ESO‐1‐Aga2p protein led to an enhanced humoral and cellular immune response, when compared to the bacterially expressed NY‐ESO‐1 protein. Together, these data demonstrate that yeast‐derived full‐length NY‐ESO‐1‐Aga2p protein is processed and presented efficiently by MHC class I and II complexes and warrants clinical trials to determine the potential value of S. cerevisiae as a host for cancer vaccine development. Copyright © 2010 John Wiley & Sons, Ltd.     

Antioxidant and anti‐acetylcholinesterase activities of anchovy (Coilia mystus) protein hydrolysates and their memory‐improving effects on scopolamine‐induced amnesia mice

Anchovy protein hydrolysates (APHs) were prepared through hydrolysis for 2, 4 or 8 h (APH‐2, APH‐4 and APH‐8, respectively). The chemical analyses, in vitro assessments [antioxidant activity and acetylcholinesterase (AchE) inhibitory activity] and in vivo mice tests were evaluated. Results revealed that APH‐8 exhibited the strongest reducing power and AchE inhibitory capacity (IC₅₀ = 159.76 ± 0.03 mg mL^?1), which may be due to its specific amino acid composition and newly formed peptides. In addition, AchE inhibitory kinetics of amino acids suggested that lysine was featured of both competitive and noncompetitive inhibitors. Furthermore, the results of in vivo study showed that all APHs exhibited memory‐improving action on scopolamine‐induced amnesia mice especially, APH‐8, indicating that anchovy protein is a potential source for health‐promoting peptides.     

Ursolic acid,a naturally occurring triterpenoid,suppresses migration and invasion of human breast cancer cells by modulating c‐Jun N‐terminal kinase,Akt and mammalian target of rapamycin signaling

Metastasis is one of the most important factors related to breast cancer therapeutic efficacy. Ursolic acid, a naturally occurring triterpenoid, has various anticancer activities. In this study, we first observed that ursolic acid exerted a dose‐ and time‐dependent inhibitory effect on the migration and invasion of highly metastatic breast MDAMB231 cells at non‐cytotoxic concentrations. This effect was associated with reduced activities of metalloproteinase‐2 (MMP‐2) and u‐PA, which correlated with enhanced expression of tissue inhibitor of MMP‐2 and plasminogen activator inhibitor‐1, respectively. Ursolic acid suppressed the phosphorylation of Jun N‐terminal kinase, Akt and mammalian target of rapamycin, but had no effect on the phosphorylation of ERK and p38. Ursolic acid also strongly reduced the levels of NFκB p65, c‐Jun and c‐Fos proteins in the nucleus of MDAMB231 cells. A time‐dependent inhibition of the protein levels of Rho‐like GTPases, growth factor receptor‐bound protein 2, Ras and vascular endothelial growth factor in cytosol by ursolic acid treatment was also observed. In conclusion, we demonstrated that the anti‐invasive effects of ursolic acid on MDAMB231 cells might be through the inhibition of Jun N‐terminal kinase, Akt and mammalian target of rapamycin phosphorylation and a reduction of the level of NFκB protein in the nucleus, ultimately leading to downregulation of MMP‐2 and u‐PA expression. These results suggest that ursolic acid has potential as a chemopreventive agent for metastatic breast cancer.     

γ‐Glu‐Met synthesised using a bacterial glutaminase as a potential inhibitor of dipeptidyl peptidase IV

This study describes γ‐glutamyl dipeptides as competitive inhibitors of dipeptidyl peptidase‐IV (DPP‐IV), and the feasible synthesis of γ‐Glu‐Met through transpeptidation catalysed by a commercial glutaminase of Bacillus amyloliquefaciens. γ‐Glu‐Met, γ‐Glu‐Leu, γ‐Glu‐Phe, γ‐Glu‐Trp or γ‐Glu‐Tyr exhibited a competitive inhibitory effect on DPP‐IV. The yield of γ‐Glu‐Met was 26.16% under optimised conditions: 200 mm Gln, 20 mm Met, 0.1 U mL^?1 enzyme, pH 9.0, 37 °C and reaction time 3 h. For the first time, the side products containing characteristic sequences, that is poly‐γ‐glutamyl short chains with a terminal Met residue (γ‐Glu‐γ‐Glu‐Met and γ‐Glu‐γ‐Glu‐γ‐Glu‐Met) were identified. The superiority of the commercial glutaminase in the synthesis of DPP‐IV‐inhibitory peptides can enable the application of this novel process for manufacturing γ‐glutamyl‐peptides as potential functional ingredients in the type 2 diabetic diet.     

“Potential Health Benefits of Lunasin: A Multifaceted Soy‐Derived Bioactive Peptide”

Bioactive peptides are small protein fragments derived from enzymatic hydrolysis of food proteins, fermentation with proteolytic starter cultures, and gastrointestinal digestion. These peptides have positive impacts on a number of physiological functions in living beings. Lunasin, a soy‐derived bioactive peptide, is one of the most promising among them. Lunasin encoded within 2S albumin (GM2S‐1) gene, identified as a novel peptide extracted from soybean seed. It is composed of 43 amino acid residues with a molecular weight of 5.5 kDa. Extensive scientific studies have shown that lunasin possesses inherent antioxidative, anti‐inflammatory, anticancerous properties and could also play a vital role in regulating of cholesterol biosynthesis in the body. Its high bioavailability and heat stable nature allow its potential use as dietary supplement. The present review summarizes some of the potential health and therapeutic benefits of lunasin reported hitherto.     

Survival,growth characteristics and bioactive potential of Lactobacillus acidophilus in a soy‐based cream cheese

BACKGROUND: Soy‐based products have received much attention lately as dairy replacers and carriers for probiotics, without the cholesterol and lactose intolerance factors. We have previously developed a soy cream cheese product and would like to evaluate its suitability as a carrier for probiotic microorganisms. Soy cream cheese is commercially uncommon, while a probiotic soy cream cheese is yet to be available in the market. RESULTS: Five strains of probiotics were screened for their α‐galactosidase activity. Lactobacillus acidophilus FTCC 0291 showed the highest α‐galactosidase‐specific activity and was incorporated into soy cream cheese for a storage study of 20 days at 25 and 4 °C. L. acidophilus FTCC 0291 in soy cream cheese at both storage temperatures maintained a viability exceeding 10⁷ CFU g^?1 over storage. Oligosaccharide and reducing sugar analyses indicated that L. acidophilus FTCC 0291 was capable of utilizing the existing reducing sugars in soymilk and concurrently hydrolyzing the oligosaccharides into simpler sugars for growth. L. acidophilus FTCC 0291 also produced organic acids, leading to decreased pH. Under low pH and high organic acid concentration, the growth of total aerobes and anaerobes was significantly (P < 0.05) suppressed compared to the control. The hydrolysis of protein in soymilk produced essential growth factors such as peptides and amino acids that may have promoted the growth of L. acidophilus FTCC 0291 and the release of bioactive peptides with in vitro angiotensin I‐converting enzyme inhibitory activity. CONCLUSION: This study showed that soy cream cheese could be used as a carrier for probiotic bacteria, with potential antihypertensive property. Copyright © 2009 Society of Chemical Industry     

Angiotensin‐converting enzyme‐inhibitory activity in protein hydrolysates from normal and anthracnose disease‐damaged Phaseolus vulgaris seeds

BACKGROUND: Bean seeds are an inexpensive source of protein. Anthracnose disease caused by the fungus Colletotrichum lindemuthianum results in serious losses in common bean (Phaseolus vulgaris L.) crops worldwide, affecting any above‐ground plant part, and protein dysfunction, inducing the synthesis of proteins that allow plants to improve their stress tolerance. The aim of this study was to evaluate the use of beans damaged by anthracnose disease as a source of peptides with angiotensin‐converting enzyme (ACE‐I)‐inhibitory activity. RESULTS: Protein concentrates from beans spoiled by anthracnose disease and from regular beans as controls were prepared by alkaline extraction and precipitation at isolelectric pH and hydrolysed using Alcalase 2.4 L. The hydrolysates from spoiled beans had ACE‐I‐inhibitory activity (IC₅₀ 0.0191 mg protein mL^?1) and were very similar to those from control beans in terms of ACE‐I inhibition, peptide electrophoretic profile and kinetics of hydrolysis. Thus preparation of hydrolysates using beans affected by anthracnose disease would allow for revalorisation of this otherwise wasted product. CONCLUSION: The present results suggest the use of spoiled bean seeds, e.g. anthracnose‐damaged beans, as an alternative for the isolation of ACE‐I‐inhibitory peptides to be further introduced as active ingredients in functional foods. © 2012 Society of Chemical Industry     

Substrate analysis of the Pneumocystis carinii protein kinases PcCbk1 and PcSte20 using yeast proteome microarrays provides a novel method for Pneumocystis signalling biology

Pneumocystis carinii (Pc) undergoes morphological transitions between cysts and trophic forms. We have previously described two Pc serine/threonine kinases, termed PcCbk1 and PcSte20, with PcSte20 belonging to a family of kinases involved in yeast mating, while PcCbk1 is a member of a group of protein kinases involved in regulation of cell cycle, shape, and proliferation. As Pc remains genetically intractable, knowledge on specific substrates phosphorylated by these kinases remains limited. Utilizing the phylogenetic relatedness of Pc to Saccharomyces cerevisiae, we interrogated a yeast proteome microarray containing >4000 purified protein based peptides, leading to the identification of 18 potential PcCbk1 and 15 PcSte20 substrates (Z‐score > 3.0). A number of these potential protein substrates are involved in bud site selection, polarized growth, and response to mating α factor and pseudohyphal and invasive growth. Full‐length open reading frames suggested by the PcCbk1 and PcSte20 protoarrays were amplified and expressed. These five proteins were used as substrates for PcCbk1 or PcSte20, with each being highly phosphorylated by the respective kinase. Finally, to demonstrate the utility of this method to identify novel PcCbk1 and PcSte20 substrates, we analysed DNA sequence data from the partially complete Pc genome database and detected partial sequence information of potential PcCbk1 kinase substrates PcPxl1 and PcInt1. We additionally identified the potential PcSte20 kinase substrate PcBdf2. Full‐length Pc substrates were cloned and expressed in yeast, and shown to be phosphorylated by the respective Pc kinases. In conclusion, the yeast protein microarray represents a novel crossover technique for identifying unique potential Pc kinase substrates. Copyright © 2011 John Wiley & Sons, Ltd.     

Peptides from Pisum sativum L. enzymatic protein digest with anti‐adhesive activity against Helicobacter pylori: Structure–activity and inhibitory activity against BabA,SabA, HpaA and a fibronectin‐binding adhesin

Scope: Identification of anti‐adhesive peptides against Helicobacter pylori obtained by enzymatic hydrolysis of seed proteins from Pisum sativum L. (Fabaceae). Methods and results: Bioassay‐guided fractionation of protein tryptic digest by ultrafiltration, size exclusion chromatography (SEC) and reversed phase chromatography (RPC) were used. Identification of bioactive peptides was achieved by MALDI‐TOF‐MS. Adhesion of H. pylori was monitored by two different assays, using a quantitative in vitro assay on human AGS cells with evaluation of bacterial binding by flow cytometry, beside a semi‐quantitative in situ adhesion assay using FITC‐labelled H. pylori on human stomach tissue sections. From two highly active fractions (F3, F3.3) two anti‐adhesive peptides (S3, S5) were identified. Neither F3 nor S3 or S5 had any cytotoxic effect against H. pylori. By hemagglutination assay and semiquantitative dot blot overlay assay with immobilized ligands it was shown that F3 interacts specifically with H. pylori adhesins BabA, SabA, HpaA and a fibronectin‐binding adhesin, while S3 and S5 inhibit only BabA. It was demonstrated that BabA, usually interacting with carbohydrate motifs such as fucosylated blood group antigens, interacts with the peptide moieties. Conclusion: Bioactive peptides from pea protein could be applied as functional ingredients for protecting infants and children against infections such as H. pylori.     

Isolation and characterisation of a novel angiotensin I‐converting enzyme‐inhibitory peptide derived from douchi,a traditional Chinese fermented soybean food

BACKGROUND: Douchi, a traditional fermented soybean food, has recently attracted a great deal of attention owing to its superior physiological activity. In the present study the angiotensin I‐converting enzyme (ACE)‐inhibitory activity of typical douchi procured from various regions of China was analysed. An ACE‐inhibitory peptide derived from the most potent douchi was also isolated and characterised. The pattern of ACE inhibition and resistance to hydrolysis by gastrointestinal proteases of this peptide are described. RESULTS: ACE‐inhibitory activities were detected in all douchi samples, with IC₅₀ values ranging from 0.204 to 2.011 mg mL^?1. Among the douchi samples, a Mucor‐type douchi exhibited the most potent ACE‐inhibitory activity (IC₅₀ = 0.204 mg mL^?1). A novel ACE‐inhibitory peptide was then isolated from this Mucor‐type douchi using ultrafiltration followed by Sephadex G‐25 column chromatography and reverse phase high‐performance liquid chromatography. The amino acid sequence of the purified peptide was identified by Edman degradation as His‐Leu‐Pro (IC₅₀ = 2.37 µmol L^?1). The peptide is a competitive inhibitor and maintained its inhibitory activity even after incubation with some gastrointestinal proteases. CONCLUSION: The present study shows that peptides derived from soybean fermentation during douchi processing could be the main contributor to the ACE‐inhibitory activity observed. Copyright © 2009 Society of Chemical Industry     

Emodin,a natural inhibitor of protein kinase CK2, suppresses growth,hyphal development,and biofilm formation of Candida albicans

Emodin (1,3,8‐trihydroxy‐6‐methyl‐anthraquinone) is a natural secondary plant product, originally isolated from the rhizomes of Rheum palmatum. Many reports show its diuretic, vasorelaxant, antibacterial, antiviral, anti‐ulcerogenic, immunosuppressive, hepatoprotective, anti‐inflammatory and anticancer potential. Emodin is a pleiotropic molecule capable of interacting with several major molecular targets, e.g. NF‐κB, AKT/mTOR and STAT3. The compound can also act as an inhibitor of some protein kinases, with special affinity to protein kinase CK2. The aim of the presented report was to evaluate antifungal properties of emodin and its activity towards CK2 isolated from Candida cells. Our studies revealed that the compound suppressed growth of the cells of reference strains as well as clinical Candida strains, with minimal inhibitory concentration and minimal fungicidal concentration values between 12.5 and 200 μg/mL. Moreover, at a low concentration, the compound was able to effectively stop hyphal formation, thus showing a distinct antivirulent potential. Interestingly, we showed that emodin added to Candida culture inhibited the phosphorylation of many cellular proteins, presumably owing to the inhibition of protein kinase CK2. Notably, the enzyme isolated from the Candida cells was susceptible to emodin with IC₅₀ of 2.8 μg/mL. Indeed, our computational modelling revealed that emodin was able to occupy the ATP‐binding pocket of CK2. Copyright © 2017 John Wiley & Sons, Ltd.     

A study on the preparation and some functional properties of a cross‐linked casein–gelatin composite by a microbial transglutaminase

A microbial transglutaminase (TGase) was used in this work as biocatalyst to prepare a cross‐linked casein–gelatin composite, a modified protein product with 4‐hydroxyproline about 41 mg g^?1 peptides. Some cross‐linking conditions such as total protein content, the ratio of caseinate to gelatin and original pH were fixed at 5% (w/v), 4:1 (w/w) and 7.5, respectively. Other suitable conditions selected by single factor trials were TGase addition of 20 U g^?1 peptides, reaction temperature of 45 °C and reaction time of 4 h. Peptide profiles from SDS‐PAGE analysis showed the composite was peptide polymers. Compared to that of the original caseinate or the cross‐linked caseinate, the dispersion of the composite exhibited a markedly enhanced apparent viscosity, storage modulus and viscous modulus. Meanwhile, the composite also showed a better water holding capacity, unchanged oil binding capacity and lower enzymatic digestibility in vitro, conferring its applicability as a new protein ingredient.     

Purification of two bacteriocins produced by Enterococcus faecalis DBFIQ E24 strain isolated from raw bovine milk

Enterococcus faecalis DBFIQ E24 was formerly characterised as a producer of antibacterial and antifungal substances. This strain is vancomycin sensitive, nonhaemolytic and gelatinase negative. SDS‐PAGE revealed the presence of an active band located between 1060 and 3500 Da. After the application of a three‐step purification procedure, two antimicrobial peptides were isolated. One of them is mostly active against Bacillus cereus with a molecular mass of 1364 Da, and the other one with a molecular mass of 1686 Da is mainly active against Escherichia coli. These results confirm the technological potential of E. faecalis DBFIQ E24 strain as a GRAS food biopreservative.     

Preparation and characterisation of glyceollin‐enriched soya bean protein using solid‐state fermentation

Glyceollins, stress‐induced phytoalexins from parent soya bean isoflavones, were elicited with Aspergillus oryzae. This solid‐state fermentation facilitated the conversion of isoflavone glycosides into aglycones and glyceollins, which could mainly enrich in soya bean protein isolate (SPI) during protein preparation due to protein–polyphenol interactions. Glyceollin‐enriched SPI exhibited less flavour volatiles, higher solubility, lower whiteness and higher antioxidant activity than unfermented SPI. Fermented SPI was more easily to be digested during in vitro pepsin–trypsin digestion. This may be attributed to partial degradation of protein, especially α′ and α subunits of β‐conglycinin and acidic subunit of glycinin. The antioxidant activity of digestive products derived from fermented SPI was obviously enhanced with increasing digestion time due to simultaneous release of antioxidant peptides and glyceollins involved in the interior of protein molecule. These results suggest an effective technique for producing a nutrient‐enhanced SPI as novel functional ingredients applied in food industry.