Potential of Pm11 antimicrobial peptide against bovine mastitis pathogens.

OBJECTIVE: To investigate an alternative treatment for bovine mastitis by using Pm11 antimicrobial peptide. SAMPLE: 5 bovine mastitis pathogens that were previously isolated from cows affected by either clinical or subclinical mastitis. PROCEDURES: The current study introduces Pm11 antimicrobial peptide as an alternative treatment for bovine mastitis. The antibacterial activity of Pm11 was tested against Escherichia coli strain SCM1249, Klebsiella spp strain SCM1282, Staphylococcus aureus strain CM967, Streptococcus agalactiae strain SCM1084, and Streptococcus uberis strain SCM1310 using minimum bactericidal concentrations (MBCs) and time-kill kinetics. The pathogens' morphological changes were demonstrated using a scanning electron microscope (SEM). The cytotoxicity of Pm11 was assessed using the minimum hemolytic concentration assay. RESULTS: MBCs ranged from 2.5 to 10 µM and IC50 ranged from 0.32 to 2.07 µM. Time-kill kinetics at MBC demonstrated that Pm11 reduced viable cell counts of S agalactiae strain SCM1084 and S uberis strain SCM1310 from 105 to 0 CFU/mL within 1 h. E coli strain SCM1249 and S aureus strain CM967 were reduced from 105 to 0 CFU/mL within 4 h. The average Pm11-induced hemolytic activity was < 10% for all Pm11 concentrations tested except at the maximum concentration tested (160 µM: 10.19 ± 2.29%). Based on SEM, Pm11 induced morphological and cellular changes in S aureus and E coli. CLINICAL RELEVANCE: Pm11 antimicrobial peptide demonstrated in vitro antibacterial activity against the common bovine mastitis pathogens E coli, S aureus, S agalactiae, and S uberis, except Klebsiella spp, and should be further investigated in vivo.

Investigating the cellular antioxidant and anti-inflammatory effects of the novel peptides in lingzhi mushrooms.

The lingzhi mushroom (Ganoderma lucidum) is well known for its medicinal properties and has long played a role in traditional oriental medicine due to its health-giving benefits and potential to extend life expectancy. The mushroom contains a number of highly bioactive compounds and can also act as an excellent source of protein. This research investigated the peptides obtained from the protein hydrolysates of lingzhi mushrooms to assess their free radical scavenging abilities. These peptides were acquired via different proteases (Alcalase, Neutrase, papain, and pepsin-pancreatin) and were tested at a range of different concentrations (1.0%, 2.5%, and 5.0% w/v). The highest levels of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) radical scavenging activities were presented by lingzhi mushroom hydrolysate using 2.5% (w/v) pepsin-pancreatin after 6 h of digestion. The hydrolysate was then fractionated using 10, 5, 3, and 0.65 kDa molecular weight cut-off membranes. The results showed that the MW 0.65 kDa fraction had the highest level of free radical scavenging activity. Further analysis of this MW 0.65 kDa fraction began with another RP-HPLC fractionation technique to obtain three further sub-fractions. De novo peptide sequencing using electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS/MS) was chosen as the optimum method for studying the F3 sub-fraction. DRVSIYGWG and ALLSISSF were discovered as new peptides with different antioxidant properties. Adenocarcinoma colon (Caco-2) cells showed the antioxidant action of these synthesized peptides. This activity was linked to peptide concentration. The peptides and their pure synthetic counterparts were found to reduce NO generation by RAW 264.7 macrophages without causing cytotoxicity. The results of gene expression reveal that the DRVSIYGWG and ALLSISSF peptides were able to cut the expression of the proinflammatory cytokine genes iNOS, IL-6, TNF-α, and COX-2 in the context of RAW 264.7 macrophages.

Aqueous Extracts of Lemon Basil Straw as Chemical Stimulator for Gray Oyster Mushroom Cultivation.

To reduce the burning of lemon basil straw (LBS)-the byproduct of basil seed production-we propose utilizing LBS as a replacement substrate for mushroom cultivation. LBS can stimulate both mycelial growth and percentage biological efficiency; however, the rigidity of this material limits particle size reduction. In this work, aqueous extractions were facilely performed without using either hazardous chemicals or complex procedures to valorize LBS as a stimulator for gray oyster mushroom cultivation. An aqueous extraction at solid-to-liquid of 50 g/L was employed. The macerated-LBS and decocted-LBS extracts were tested for mycelial growth in potato dextrose agar and sorghum grains. Following this, both aqueous extracts were applied as a wetting agent in cylindrical baglog cultivation to estimate mycelial growth, biological efficiency, and productivity. It was found that LBS extracts insignificantly enhanced the mycelia growth rate on all media, while the diluted LBS (1:1 v/v) extracts improved 1.5-fold of percentage biological efficiency. Gas chromatograph-mass spectrometer results indicated 9-octadecaenamide is a major component in LBS aqueous extract. Results demonstrated that the LBS extract is a good stimulator for the production of Pleurotus mushroom.

A novel angiotensin I-converting enzyme inhibitory peptide derived from the trypsin hydrolysates of salmon bone proteins.

When fish are processed, fish bone becomes a key component of the waste, but to date very few researchers have sought to use fish bone to prepare protein hydrolysates as a means of adding value to the final product. This study, therefore, examines the potential of salmon bone, through an analysis of the benefits of its constituent components, namely fat, moisture, protein, and ash. In particular, the study seeks to optimize the process of enzymatic hydrolysis of salmon bone with trypsin in order to produce angiotensin-I converting enzyme (ACE) inhibitory peptides making use of response surface methodology in combination with central composite design (CCD). Optimum hydrolysis conditions concerning DH (degree of hydrolysis) and ACE-inhibitory activity were initially determined using the response surface model. Having thus determined which of the salmon bone protein hydrolysates (SBPH) offered the greatest level of ACE-inhibitory activity, these SBPH were duly selected to undergo ultrafiltration for further fractionation. It was found that the greatest ACE-inhibitory activity was achieved by the SBPH fraction which had a molecular weight lower than 0.65 kDa. This fraction underwent further purification using RP-HPLC, revealing that the F7 fraction offered the best ACE-inhibitory activity. For ACE inhibition, the ideal peptide in the context of the F7 fraction comprised eight amino acids: Phe-Cys-Leu-Tyr-Glu-Leu-Ala-Arg (FCLYELAR), while analysis of the Lineweaver-Burk plot revealed that the FCLYELAR peptide can serve as an uncompetitive ACE inhibitor. An examination of the molecular docking process showed that the FCLYELAR peptide was primarily able to provide ACE-inhibitory qualities as a consequence of the hydrogen bond interactions taking place between ACE and the peptide. Furthermore, upon isolation form the SBPH, the ACE-inhibitory peptide demonstrated ACE-inhibitory capabilities in vitro, underlining its potential for applications in the food and pharmaceutical sectors.

Investigation of major amino acid residues of anti-norfloxacin monoclonal antibodies responsible for binding with fluoroquinolones.

It is important to understand the amino acid residues that govern the properties of the binding between antibodies and ligands. We studied the binding of two anti-norfloxacins, anti-nor 132 and anti-nor 155, and the fluoroquinolones norfloxacin, enrofloxacin, ciprofloxacin, and ofloxacin. Binding cross-reactivities tested by an indirect competitive enzyme-linked immunosorbent assay indicated that anti-nor 132 (22-100%) had a broader range of cross-reactivity than anti-nor 155 (62-100%). These cross-reactivities correlated with variations in the numbers of interacting amino acid residues and their positions. Molecular docking was employed to investigate the molecular interactions between the fluoroquinolones and the monoclonal antibodies. Homology models of the heavy chain and light chain variable regions of each mAb 3D structure were docked with the fluoroquinolones targeting the crucial part of the complementarity-determining regions. The fluoroquinolone binding site of anti-nor 155 was a region of the HCDR3 and LCDR3 loops in which hydrogen bonds were formed with TYR (H:35), ASN (H:101), LYS (H:106), ASN (L:92), and ASN (L:93). These regions were further away in anti-nor 132 and could not contact the fluoroquinolones. Another binding region consisting of HIS (L:38) and ASP (H:100) was found for norfloxacin, enrofloxacin, and ciprofloxacin, whereas only ASP (H:100) was found for ofloxacin.

An in vitro study of lipase inhibitory peptides obtained from de-oiled rice bran.

De-oiled rice bran (DORB) is a potentially useful by-product of the rice bran oil industry. DORB may prove to be an important protein source, and also contains many other micronutrients. This study has the principal aim of optimizing the process of DORB protein hydrolysate preparation, and then testing the hydrolysate to determine its lipase inhibitory activity. DORB underwent hydrolysis using Alcalase® and response surface methodology (RSM). The resulting degree of hydrolysis (DH) was then monitored along with the extent of any lipase inhibitory activity. The optimum levels of lipase inhibition were obtained at a temperature of 49.88 °C, a duration of 150.43 minutes, and 1.53% Alcalase® used for the sample 5% (w/v) solution. In these conditions, the DH value was 35.65%, and the IC50 value for lipase inhibitory activity was 2.84 µg mL-1. Five ranges of different molecular weights were obtained via fractionation, whereupon it was determined that the highest level of inhibitory activity was achieved by the <0.65 kDa fraction. This fraction was then further purified via RP-HPLC, and the resulting peak had a retention time of 21.75 minutes (F 2 sub-fraction) and exhibited high lipase inhibitory activity. Mass spectrometry was used to determine the amino acid sequence for this peak, identified as FYLGYCDY. This particular peptide is categorized as bitter, with a non-toxic profile, and having poor water solubility. The synthesized form of this peptide showed lipase inhibitory activity measured by an IC50 value of 0.47 ± 0.02 µM. The Lineweaver-Burk plot revealed that FYLGYCDY is a non-competitive inhibitor, while analysis of the docking results provided details of the FYLGYCDY peptide binding site with the porcine pancreatic lipase (PPL) complex, which is a competitive type. It can be inferred from these findings that DORB may prove a useful raw material source for the production of anti-obesity peptides which might enhance the therapeutic and commercial performance of functional foods and healthcare products.

Expression, purification and biological activity of monomeric insulin precursors from methylotrophic yeasts.

The methylotrophic yeasts Pichia pastoris and Hansenula polymorpha have been used for the production of recombinant monomeric insulin precursor (MIP). Recombinant plasmids with one, two and four cassettes of the MIP gene have been successfully constructed in the pPICZαA expression vector to study the effects of gene copy number on MIP production. The MIP protein can be detected by dot-blot analysis from the culture broth of P. pastoris KM71H 24 h after placement in MMH induction medium. The secretion levels of MIP protein in culture broth at 72 h after induction indicated that P. pastoris KM71H with one cassette of the MIP gene had highest MIP protein levels (4.19 ±â€¯0.96 mg L-1). The transcription levels of the MIP gene increased proportionately with copy number. However, the amount of secreted MIP protein showed no correlation. The MIP molecular mass was 5756.951 Da, as confirmed by typical MALDI-TOF mass spectrometry. The MIP protein in culture broth was purified by two steps purification including SP Sepharose Fast Flow chromatography followed by ultrafiltration (10 kDa MW cutoff). The percentage of MIP recovery after the two-step purification was 70%, with a single band in a native-PAGE. The biological activity of tryptic hydrolyzed MIP was determined via the expression of the glucose transporter 4 gene (GLUT4) in H9c2 (2-1) cell line by RT-qPCR, and the results demonstrated that the MIP protein can induce glucose uptake and upregulation of GLUT4 mRNA transcription at 3 h and that this activity was related to Humalog® insulin.