Soybean peptide (SP) exhibited low intestinal absorption at oral administration due to its fragile structure under gastric digestion. Therefore, we have attempted to encapsulate peptide by cross-linkage interaction between positive charged chitosan (CS) or chitosan oligosaccharide (CSO) and negative charged peptide. The CS (or CSO) with SP nanoparticles were prepared by using ultrasonification technique. The objective of this study was to find the optimal processing method by changing concentration, pH, and homogenizing conditions. We measured physicochemical properties such as particle size, zeta-potential, encapsulation efficiency (EE%), release rate (RR) and antioxidant ability of samples. The results showed that the optimal processing method was using 0.5% (w/v) CSO (diluted by pH 3 Acetic acid buffer) mixed with 0.5% (w/v) SP (diluted by pH 6 buffer) by 9:1 ratio. Afterwards, using high-speed mixer at 12,000 rpm for 3 min, and then passed 2 times through an ultrasonicator (50% power, 3 min). In this way for processing, the particle sizes of CSO/SP nanoparticles were approximately 300 nm, zeta-potential were approximately 45 mV. In addition, the EE% and RR of CS/SP nanoparticles was higher than the CSO/SP nanoparticles. The increase in antioxidant ability of SP was attributed to the affected by CS/CSO microcapsules. In conclusion, this research can befoundation for the manufacturing process of CS/SP nanoparticles, and it was expected that the future application of this nanoparticle in food matrix.

Some bioactive peptides have been identified by activity-guided fractionation. However, it is difficult to prepare or synthesize enough amounts of active peptides for food ingredients and for animal or human trials. The objective of the present study was to prepare peptide fractions of sufficient amounts with high antioxidant activity which could be applied to the food and animal model. Oyster hydrolysate was fractionated the basis of the amphoteric nature of sample peptides by preparative isoelectric focusing (autofocusing). To monitor the fractionation of peptides by autofocusing, amino acid analysis of the autofocusing fractions was performed. Each autofocusing fraction was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl (OH) radical scavenging activities, oxygen radical absorbance capacity (ORAC), and Fe 2+ chelatingassays. More than 40% of the peptides were distributed between Fr. 4 and 7 (pH<5.0). Approximately 16% of the peptides were recovered in basic fractions (pH>10.0; Fr. 9-10). 11 % of peptides were recovered in the neutral fraction (6.0<pH<7.0; Fr. 8). The peptides in the acidic and basic fractions are characterized by a higher content of acidic and basic amino acids, respectively. Acidic fraction showed higher ORAC values than crude oyster hydrolysate. Also, the acidic fraction showed high DPPH and OH radical scavenging activity. On the other hand, basic fraction showed higher chelating ability than crude oyster hydrolysate. In the present study, oyster peptide fractions of large-scale with high antioxidant activity were successfully fractionated by autofocusing for food additives and animal trials.

Bee venom contains a variety of peptide constituents that have various biological, toxicological, and pharmacological actions. However, the biological actions of secapin, a venom peptide in bee venom, remain largely unknown. Here, we provide the first evidence that the Asiatic honeybee (Apis cerana) secapin (AcSecapin-1) exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities. AcSecapin-1 functions as a serine protease inhibitor-like peptide that has inhibitory effects against plasmin, elastases, microbial serine proteases, trypsin, and chymotrypsin. Consistent with these functions, AcSecapin-1 inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products, thus indicating the role of AcSecapin-1 as a clotting factor. AcSecapin-1 also inhibited both human neutrophil and porcine pancreatic elastases. Furthermore, AcSecapin-1 exhibited anti-microbial activity against fungi and Gram-positive and Gram-negative bacteria. Taken together, our data demonstrated that AcSecapin-1 has a multifunctional role as an anti-fibrinolytic agent, an anti-elastolytic agent, and an anti-microbial peptide, and our data suggested novel functions for the biological actions of the bee venom peptide, secapin.

The honeybee inhibitor cysteine knot (ICK) peptide acts as an antifungal peptide and insecticidal venom toxin. However, the ICK peptide from bumblebees has not been characterized. Here, we report the molecular cloning and antifungal activity of a bumblebee (Bombus ignitus) ICK peptide (BiICK). We identified a BiICK that contains an ICK fold. The BiICK was expressed in the epidermis, fat body, and venom gland of B. ignitus worker bees. A 6.7-kDa recombinant BiICK peptide was expressed in baculovirus-infected insect cells. Recombinant BiICK peptides directly bound to Beauveria bassiana, Ascosphaera apis, and Fusarium graminearum, but they did not bind to Escherichia coli, Paenibacillus larvae, or Bacillus thuringiensis. Consistent with this finding, BiICK exhibited antifungal activity against fungi. These results demonstrate that BiICK acts as an antifungal peptide.

A positional scanning synthetic peptide combinatorial library (PS-SCL) was screened in order to identify antimicrobial peptides against the cariogenic oral bacteria, Streptococcus mutans. Activity against Streptococcus gordonii and Aggregatibacter actinomycetemcomitans was also examined. The library was comprised of six sub-libraries with the format O(1-6)XXXXX-NH2, where O represents one of 19 amino acids (excluding cysteine) and X represents equimolar mixture of these. Each sub-library was tested for antimicrobial activity against S. mutans and evaluated for antimicrobial activity against S. gordonii and A. actinomycetemcomitans. The effect of peptides was observed using transmission electron microscopy (TEM). Two semi-mixture peptides, RXXXXN-NH2 (pep-1) and WXXXXN-NH2 (pep-2), and one positioned peptide, RRRWRN-NH2 (pep-3), were identified. Pep-1 and pep-2 showed significant antimicrobial activity against Gram positive bacteria (S. mutans and S. gordonii), but not against Gram negative bacteria (A. actinomycetemcomitans). However, pep-3 showed very low antimicrobial activity against all three bacteria. Pep-3 did not form an amphiphilic α-helix, which is a required structure for most antimicrobial peptides. Pep-1 and pep-2 were able to disrupt the membrane of S. mutans. Small libraries of biochemically-constrained peptides can be used to generate antimicrobial peptides against S. mutans and other oral microbes. Peptides derived from such libraries may be candidate antimicrobial agents for the treatment of oral microorganisms.

Like vertebrate insulins, insulin-like peptides (ILPs) play crucial roles in controlling immature growth, adult lifespan, and plasma sugar level in some insects. An ILP gene (SeILP1) was predicted from a transcription database of Spodoptera exigua. SeILP1 encodes 95 amino acid sequence, which shares sequence homologies (33~83%) with other insects ILPs. The predicted B and A chains possess six cysteine residences. SeILP1 was expressed in all developmental stages of S. exigua. However, its expression was detected in fat body, gut and epidermis, but not in hemocytes. Its expression increased with feeding activity. Plasma trehalose levels of fifth instar larvae maintained at relatively stable concentration of 2.31±0.62 mM. However, starvation induced a significant increase of plasma trehalose level by more than two fold in 48 h, at which SeILP1 expression kept at a low level. RNA interference of SeILP1 induced a significant increase of plasma trehalose level. Interestingly, a bovine insulin decreased plasma trehalose level in a dose-dependent manner. These results indicate mat SeILP1 plays a role in suppressing plasma trehalose level in S. exigua.

Inhibitor cysteine knot (ICK) peptides exhibit ion channel blocking, insecticidal, and antimicrobial activities, but currently, no functional roles for bee-derived ICK peptides have been identified. In this study, a bee (Apis cerana) ICK peptide (AcICK) that acts as an antifungal peptide and as an insecticidal venom toxin was identified. AcICK contains an ICK fold that is expressed in the epidermis, fat body, or venom gland and is present as a 6.6-kDa peptide in bee venom. Recombinant AcICK peptide (expressed in baculovirus-infected insect cells) bound directly to Beauveria bassiana and Fusarium graminearum, but not to Escherichia coli or Bacillus thuringiensis. Consistent with these findings, AcICK showed antifungal activity, indicating that AcICK acts as an antifungal peptide. Furthermore, AcICK expression is induced in the fat body and epidermis after injection with B. bassiana. These results provide insight into the role of AcICK during the innate immune response following fungal infection. Additionally, we show that AcICK has insecticidal activity. Our results demonstrate a functional role for AcICK in bees: AcICK acts as an antifungal peptide in innate immune reactions in the body and as an insecticidal toxin in venom. The finding that the AcICK peptide functions with different mechanisms of action in the body and in venom highlights the two-pronged strategy that is possible with the bee ICK peptide.

Biological properties of antimicrobial peptides (AMPs) of hemimetabolous insect are poorly characterized in innate immunity field. To investigate the biochemical properties of hemimetabolous insect’s AMPs, we purified the pyrrhocoricin-like AMP from the hemolymph of Riptortus pedestris and then named as riptocin. We successfully determined the primary protein structure and its cDNA sequence. Interestingly, the determined cDNA revealed that riptocin precursor is composed of 12 repeating units of active riptocins, which implied that riptocin precursor might require to be processed to generate active riptocins by several unidentified processing enzymes. In order to characterize the bio-processing mechanisms of riptocin precursor, we generated the antibody against active riptocin. Using quantitative PCR and Western blot analyses, we showed that gene of riptocin was started to express from the fatbody after three hours post bacterial infection. To address our hypothesis that active riptocin is generated from riptocin precursor by several processing enzymes, we need to obtain the riptocin precursor. Currently, we are expressing the recombinant riptocin precursor using in vitro translation system. Meanwhile, we investigated whether naive hemolymph (naive HL), which may contain precursor riptocin, can generate active riptocin when riptocin precursor was co-incubation with bacteria-challenged hemolymph (active HL), which may contain all processing enzymes. Actually, when naive HL was incubated with active HL, antimicrobial activity was dramatically increased, suggesting that processing enzymes in active HL may induce processing of riptocin precursor to generate active riptocins.

This peptide has antibacterial activity against several Gram-positive and Gram-negative bacteria. BmCecB1 is antimicrobial peptides from Bombyx mori and belongs to cecropin family. Antimicrobial peptides are important components of the innate immune systems in all living organism. To produce the BmCecB1 antimicrobial peptide, we constructed transgenic silkworm that expressed BmCecB1 gene under the control BmA3 promoter using piggyBac vector. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor vector and helper vector were micro-injected into 600 eggs of bivoltin silkworms, Baegokjam. In total, 49 larvae (G0) were hatched and allowed to develop into moths. The resulting G1 generation consisted of 22 broods, and we selected 2 broods containing at least 1 EGFP-positive embryo. The rate of successful transgenesis for the G1 broods was 11%. We identified 9 EGFP-positive G1 moths and these were backcrossed with wild-type moths. With the aim of identifying a BmCecB1 as antimicrobial peptide, we investigated the Radical diffusion Assay (RDA) and then demonstrated that BmCecB1 possesses high antibacterial activities against Gram-negative bacteria.