Simultaneous Extraction of Oil and Protein from Silkworm (Bombyx mori L.) Pupae (Lueng Parroj var.) and Their In Vitro Skin Moisturization.

Oil and protein from silkworm (Bombyx mori var. Leung Pairoj) pupae, by-product from sericulture, were extracted and evaluated for their potential uses as skin biomoisturizer. The silkworm pupae (SWP) oil and protein were simultaneously extracted by using three-phase partitioning (TPP) method and determined for their physicochemical properties including fatty acid and amino acid content, respectively. The highest yields of oil and protein at 8.24 ± 0.21% and 8.41 ± 0.26% w/w, respectively were obtained from 18 h extraction. Fatty acid analysis of SWP oil was rich in linolenic acid (37.81 ± 0.34%), oleic acid (28.97 ± 0.13%), palmitic acid (21.27 ± 0.05%), stearic acid (6.60 ± 0.09%) and linoleic acid (4.73 ± 0.21%). The clear yellow SWP oil possessed saponification value of 191.51 mg/g, iodine value of 119.37 g I2/g and peroxide value of 2.00 mg equivalent O2/kg. The SWP protein composed of 17 amino acids which aspartic acid, glutamic acid, glycine and serine were the major residues. SDS-PAGE analysis revealed that the SWP protein consisted of distinct protein at around 51, 70, 175 and over 175 kDa. Cytotoxicity of the SWP oil and protein was evaluated by using MTT assay and they showed low cytotoxicity toward keratinocyte cell (HaCat cell line). The SWP oil provided moisturizing effect on pig skin comparable to olive oil, while 1% and 2% of SWP protein showed higher moisturizing efficacy than 3% hydrolyzed collagen. The study indicated that the SWP oil and protein could be potential biomoisturizers for cosmetic products.

Amino acids imprinted ZIF-8s for the highly efficient and selective adsorption of antioxidant peptides from silkworm pupa protein.

Antioxidant peptides from edible protein sources have attracted great attention due to their high activity, easy adsorption and low toxicity. The purpose of this study was to develop a new MOF material with specific recognition and capture ability to achieve the aim of highly efficient and selective antioxidant peptides from silkworm pupa protein hydrolysate-N (SPPH-N). Firstly, Histidine (His) widely presented in antioxidant peptides was used to in-suit construct the modified ZIF-8 material (ZIF-His) with imprinted defective mesopores, based on "molecular imprinting strategy". Then, it was applied to extract antioxidant peptides from SPPH-N. ZIF-His exhibited high adsorption uptake for antioxidant peptides and especially presented specific adsorption property for peptides contained His, tryptophan, phenylalanine and tyrosine. Three peptides with the highest antioxidant activity (FKVPNMY, AVNMVPFPR and VNMVPFPR) from ZIF-His eluent were identified by Nano-LC-MS/MS. These peptides were synthesized and presented the excellent free radicals scavenging activity and the Ferric reducing power.

Chemical and Molecular Composition of the Chrysalis Reveals Common Chitin-Rich Structural Framework for Monarchs and Swallowtails.

The metamorphosis of a caterpillar into a butterfly is an awe-inspiring example of how extraordinary functions are made possible through specific chemistry in nature's complex systems. The chrysalis exoskeleton is revealed and shed as a caterpillar transitions to butterfly form. We employed solid-state NMR to evaluate the chemical composition and types of biomolecules in the chrysalides from which Monarch and Swallowtail butterflies emerged. The chrysalis composition was remarkably similar between Monarch and Swallowtail. Chitin is the major polysaccharide component, present together with proteins and catechols or catechol-type linkages in each chrysalis. The high chitin content is comparable to the highest chitin-containing insect exoskeletons. Proteomics analyses indicated the presence of chitinases that could be involved in synthesis and remodeling of the chrysalis as well as cuticular proteins which play a role in the structural integrity of the chrysalis. The nearly identical 13C CPMAS NMR spectra of each chrysalis and similar structural proteins supports the presence of underlying design principles integrating chitin and protein partners to elaborate the chrysalis.

Nature׳s gifts to medicine: The metabolic effects of extracts from cocoons of Larinus hedenborgi (Coleoptera: Curculionidae) and their host plant Echinops cephalotes (Asteraceae) in diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Trehala manna (TM), the edible cocoons of several weevil species, e.g. Larinus hedenborgi Boheman, 1845 (Coleoptera: Curculionidae) and their host plant, i.e. Echinops cephalotes DC. (EC) (Asteraceae), are traditionally used to treat pain, inflammation, infectious diseases, as well as respiratory, renal, reproductive and metabolic disorders. AIM OF THE STUDY: This study investigated the metabolic effects of aqueous extracts from TM and EC on diabetic male Wistar albino rats. MATERIALS AND METHODS: Animals were orally gavaged with the extracts (75, 150, and 300 mg/kg), normal saline, and glibenclamide (Glbn), for 28 days. The serum levels of glucose, insulin, lipid profile, and hepatic enzymes, plus the body weight of rats were measured at the beginning and the end of study. The proximate composition of the extracts was determined, additionally. The antioxidant and cytotoxic potency of the extracts were evaluated by radical scavenging/ferric reducing and viability assays, respectively. RESULTS: Treatment of diabetic rats with the extracts significantly altered metabolic biomarkers compared with diabetic, control and Glbn-treated groups, but not in a dose-dependent manner. However, the antihyperglycemic effects of TM75/EC300, the antiobesity effects of EC150, and the hepatoprotective effects of TM150/EC150 were even stronger than those of Glbn. TM/EC-treated groups represented normal cell architecture in the pancreatic and renal tissues. Nutrient analysis displayed that TM is rich in sugar and magnesium, whereas EC is abundant in protein, sodium, potassium, and calcium. The extracts showed no antioxidant and cytotoxic effects, as compared to the control groups. CONCLUSIONS: The findings suggest that active ingredients in the extracts evaluated are responsible for the metabolic effects by lowering blood sugar and restoring the damaged islets of Langerhans. The close trophic relationship of the TM-producing beetle with the host thistle justifies the overlaps of the bioactivity of the TM and EC extracts.

A Study of Cuticular Hydrocarbons of All Life Stages in Sarcophaga peregrina (Diptera: Sarcophagidae).

Sarcophaga peregrina (Robineau-Desvoidy, 1830), a synanthropic flesh fly species found in different parts of the world, is of medical and forensic importance. Traditional methods of inferring developmental age rely on the life stage of insects and morphological changes. However, once the larvae reach the pupal and adult stage, morphological changes would become barely visible, so that the classic method would be invalid. Here, we studied the cuticular hydrocarbon profile of S. peregrina of the whole life cycle from larval stage to adult stage by GC-MS. Sixty-three compounds with carbon chain length ranging from 8 to 36 were detected, which could be categorized into four classes: n-alkanes, branched alkanes, alkenes, and unknowns. As developmental increased, branched alkanes dominant, and the content of high-molecular-weight hydrocarbons is variable, especially for 2-methyl C19, DiMethyl C21, docosane (C22), and tricosane (C23). This study shows that the composition of CHC could be used to determine the developmental age of S. peregrina and aid in postmortem interval estimations in forensic science.

A Fast and Reliable Process to Fabricate Regenerated Silk Fibroin Solution from Degummed Silk in 4 Hours.

Silk fibroin has a high potential for use in several approaches for technological and biomedical applications. However, industrial production has been difficult to date due to the lengthy manufacturing process. Thus, this work investigates a novel procedure for the isolation of non-degraded regenerated silk fibroin that significantly reduces the processing time from 52 h for the standard methods to only 4 h. The replacement of the standard degumming protocol by repeated short-term microwave treatments enabled the generation of non-degraded degummed silk fibroin. Subsequently, a ZnCl2 solution was used to completely solubilize the degummed fibroin at only 45 °C with an incubation time of only 1 h. Desalting was performed by gel filtration. Based on these modifications, it was possible to generate a cytocompatible aqueous silk fibroin solution from degummed silk within only 4 h, thus shortening the total process time by 48 h without degrading the quality of the isolated silk fibroin solution.

Effect of Drosophila suzukii on Blueberry VOCs: Chemical Cues for a Pupal Parasitoid, Trichopria anastrephae.

Biocontrol agents such as parasitic wasps use long-range volatiles and host-associated cues from lower trophic levels to find their hosts. However, this chemical landscape may be altered by the invasion of exotic insect species. The spotted-wing drosophila (SWD), Drosophila suzukii (Diptera: Drosophilidae), is a highly polyphagous fruit pest native to eastern Asia and recently arrived in South America. Our study aimed to characterize the effect of SWD attack on the volatile organic compounds (VOCs) of blueberries, a common host fruit, and to correlate these odor changes with the olfactory-mediated behavioral response of resident populations of Trichopria anastrephae parasitoids, here reported for the first time in Uruguay. Using fruit VOC chemical characterization followed by multivariate analyses of the odor blends of blueberries attacked by SWD, we showed that the development of SWD immature stages inside the fruit generates a different odor profile to that from control fruits (physically damaged and free of damage). These differences can be explained by the diversity, frequency, and amounts of fruit VOCs. The behavioral response of T. anastrephae in Y-tube bioassays showed that female wasps were significantly attracted to volatiles from SWD-attacked blueberries when tested against both clean air and undamaged blueberries. Therefore, T. anastrephae females can use chemical cues from SWD-infested fruits, which may lead to a successful location of their insect host. Since resident parasitoids are able to locate this novel potential host, biological control programs using local populations may be plausible as a strategy for control of SWD.

Identification of potential allergens in larva, pupa, moth, silk, slough and feces of domestic silkworm (Bombyx mori).

The silkworm (Bombyx mori) is an important economic insect that can be used as food in many countries in Asia. However, silkworms and their metabolites are an important source of allergens, which can induce severe allergic reactions. So far, there are no systematic studies on the potential allergens in silkworm and its metabolites. These studies have important guiding significance for the prevention, diagnosis, and treatment of silkworm allergy. The aim of this study was to identify the potential allergens from larva, pupa, moth, silk, slough and feces of silkworm and analyze the sequence homology of silkworm allergens with other allergens identified in the Allergenonline database. We have found 45 potential allergens in silkworm. The results of the homology comparison suggested that silkworm allergens likely cross-react with those of Dermatophagoides farinae, Aedes aegypti, Tyrophagus putrescentiae, Triticum aestivum and Malassezia furfur.

Nutritional targeting modification of silkworm pupae oil catalyzed by a smart hydrogel immobilized lipase.

To prepare a nutritional supplement using silkworm pupae oil (SPO) as a feedstock, a microfluidic reactor with a smart hydrogel immobilized lipase was first constructed to reduce the relative content of palmitic acid at sn-1,3 and improve the nutritional function. The effects of flow rate, reaction temperature, and substrate molar ratio were investigated. In vitro digestion and pH-stat models were employed to analyze the digestion feature after the modification of SPO, while HPLC-ELSD, zeta potential, DSC, and TGA were used to evaluate the nutritional function. The relative content of "OOO" and "OPO" type triglycerides was increased by 49.48% and 107.67%, and that of palmitic acid at sn-1,3 was decreased by 49.61% in 10 s. After the verification of the in vitro digestion model, the fatty acid release rate of the modified SPO was significantly improved by 22.07%, indicating the nutritional function improvement of SPO. Therefore, the nutritional function of SPO has been improved successfully by the application of a microchannel reactor with photo-immobilized lipase, which could set a reference for the utilization of insect oil resources.

Purification and Identification of Novel Antioxidant Peptides from Enzymatically Hydrolysed Samia ricini Pupae.

The emergence of excessive free radicals leads to the destruction of various systems within the body. These free radicals also affect nutritional values, color, taste, and emit an odor akin to rancid food. Most food industries use synthetic antioxidants, such as BHT (butylated hydroxytoluene) or BHA (butylated hydroxy anisole). However, high doses of these can be harmful to our health. Therefore, an antioxidant compounds, such as bioactive peptides from edible animals or plants, have emerged to be a very promising alternative as they reduce potential side effects. This study focused on the purification and identification of antioxidant peptides from protein hydrolysates of wild silkworm pupae (Samia ricini). Antioxidant peptides were purified from the hydrolysate by ultrafiltration and RP-HPLC. The results showed that protein hydrolysate from S. ricini pupae by trypsin with a molecular weight lower than 3 kDa and highly hydrophobic property, exhibited strong DPPH radical scavenging activity and chelating activity. Further identification of peptides from the fraction with the highest antioxidant activity was carried out using LC-MS/MS. Three novel peptides, i.e., Met-Ley-Ile-Ile-Ile-Met-Arg, Leu-Asn-Lys-Asp-Leu-Met-Arg, and Glu-Asn-Ile-Ile-Leu-Phe-Arg, were identified. The results of this study indicated that the protein hydrolysate from S. ricini pupae possessed potent biological activity, and the novel antioxidant peptides could be utilized to develop health-related antioxidants in food industry.

Effect of the silkworm pupa protein-glucose conjugate on the thermal stability and antioxidant activity of anthocyanins.

Anthocyanin (cyanidin-3-O-glucose) is a natural water-soluble pigment with a robust antioxidant capacity. However, its poor stability and bioavailability limits its application as a functional food ingredient. This study explored the ability of the silkworm pupa protein-glucose (Spp-Glu) conjugate, developed under wet-heating conditions, to improve the thermal stability and antioxidant activity of cyanidin-3-O-glucose (C3G) at pH 3.0 and 6.8. The characterization experiments suggested that C3G complexed with the Spp-Glu conjugate could modify the protein's microenvironment and cause unfolding of the protein's secondary structures under varied pH conditions. Spectroscopic techniques further revealed the formation of complexes via hydrophobic interactions and static quenching processes when C3G was bound to Spp or Spp-Glu. The formation of these complexes effectively attenuated C3G degradation, thereby enhancing its stability under heat treatment over a range of pH values, and the experiments measuring antioxidant activity suggested that the Spp-Glu conjugate formed does not affect the efficacy of C3G after complexation. Therefore, our study suggests that Spp-Glu has the potential to effectively protect and deliver anthocyanins during industrial application for functional food formulation.

Production of natural chitin film from pupal shell of moth: Fabrication of plasmonic surfaces for SERS-based sensing applications.

Commercially available types of chitin or chitin isolate are usually in powder form and are nanofibrous in microstructure. However, the surface characteristics of natural chitin in the body of insects are currently understudied. Herein, natural chitin film was successfully produced from bio-waste of insect pupae of the Japanese giant silkworm. Two different surface morphologies of the chitin film were observed. We report for the first time a micropapillary surface structure of chitin which was observed on the dorsal side of the film. To further potential of the micropapillary structured natural chitin in sensing applications, we develop a protocol for generating a nanoscopic film of Ag using thermal evaporation. The Ag-deposited natural chitin films exhibited surface-enhanced Raman scattering (SERS) activity to an extent depending on the structure of the film. In conclusion, materials science has been expanded by addition of a natural, three-dimensional chitin film with utilizable properties.

An alternative solution for α-linolenic acid supplements: in vitro digestive properties of silkworm pupae oil in a pH-stat system.

α-Linolenic acid (ALA) is recognised to have a regulatory effect on cardiovascular diseases. Due to the low bioavailability of linseed oil (LINO), which is the most common ALA supplement, it is necessary to find a replacement for ALA supplements that is more easily accepted by the human body. The content of ALA in silkworm pupae oil (SPO) is 32.60 ± 0.67%, and SPO can be substituted as a dietary lipid to meet the demand of the human body. In the present study, a pH-stat system was used to investigate the release degree of free fatty acids (FFAs) from SPO and construct a first-order kinetic model. Digestion experiments in vitro with different lipids showed that the maximum release FFA levels were SPO > SO (soybean oil) > LO (lard oil) > MSO (mulberry seed oil) > LINO, and the first-order kinetic apparent rate constants were LINO > SPO > LO > SO > MSO. Triacylglycerol (TAG) and fatty acid composition are the decisive factors in determining the level of lipid digestion. Therefore, the maximum level of FFAs released from SPO (84.34 ± 1.37%) was much higher than that of LINO (49.78 ± 0.52%) when the hydrolysis rates were 0.2114 s-1 and 0.2249 s-1, respectively. In addition, the smaller emulsion droplet size (609.24 ± 43.46 nm) and weaker surface charge (-17.93 ± 0.42 mV) also resulted in higher levels of SPO under in vitro digestion conditions. Meanwhile, due to low melting and crystallisation temperature, SPO is quickly absorbed by the human body. Overall, SPO can be used as a new alternative for ALA supplements based on its superior digestive properties.

Protein films from black soldier fly (Hermetia illucens, Diptera: Stratiomyidae) prepupae: effect of protein solubility and mild crosslinking.

BACKGROUND: This work evaluated the performances of protein-based bioplastics obtained from black soldier fly (Hermetia illucens) prepupae. Protein films were synthesized by film casting, using both the whole proteins and their soluble fraction at pH = 10. The effects of glycerol as a plasticizer and of citric acid as a mild crosslinker on film properties were also evaluated. RESULTS: Films obtained using the soluble protein fraction were the strongest, as well as the most homogeneous and transparent ones. Protein mild crosslinking improved film tensile properties, especially in films obtained with the whole protein fraction. Non-crosslinked samples had a high affinity with water while crosslinking almost eliminated the ability of films to absorb water. All protein-based films proved to be effective barriers to red light (transmittance less than 2%). CONCLUSIONS: Bioplastics derived from black soldier fly prepupae may find applications in the agricultural sector (biodegradable pots, mulching films, utensils) and deserve to be tested for food and non-food packaging. © 2021 Society of Chemical Industry.

Volatile compounds reveal age: a study of volatile organic compounds released by Chrysomya rufifacies immatures.

Age determination of insects collected from vertebrate remains is an essential step in estimating time since colonization as related to the post-mortem interval. Long-established methods for making such estimates rely on determining age related to stage of development at the time of collection in relation to conditions experienced. However, such estimates are based on the completion of a stage of development. Methods allowing for more precise estimates of age (i.e., within a stage of development) are sorely needed. This study examined the potential of volatile organic compounds emitted by blow fly, Ch. rufifacies (Macquart), immatures to determine stage of development, which could potentially be used to estimate the age. Volatile organic compounds (VOCs) from the larval and pupal stages of Ch. rufifacies were collected by headspace solid-phase micro-extraction followed by gas chromatography-mass spectrometry (GC-MS). Analyses indicated 37 compounds shift quantitatively, as well as qualitatively, as the larvae and pupae age. Furthermore, compounds, such as 2-ethyl-1-hexanol, phenol, butanoic acid, hexadecanoic acid, octadecanoic acid, 2-methyl propanamide, and 2-methyl butanoic acid, serve as indicator compounds of specific stages within Ch. rufifacies development. This information could be important to determine if these compounds can be used in the field to predict the presence of certain developmental stages, in order to determine the potential of using volatile markers to estimate time of colonization.

Two-step enzymatic synthesis of α-linolenic acid-enriched diacylglycerols with high purities from silkworm pupae oil.

In this study, α-linolenic acid-enriched diacylglycerols (ALA-DAGs) were prepared via a two-step enzymatic way by combi-lipase using silkworm pupae oils as substrates. Firstly, several factors including temperature, mass ratio of water to oil, pH and enzyme loading were optimized for the hydrolysis of silkworm pupae oil. The maximum fatty acid content (96.51%) was obtained under the conditions: temperature 40 °C, water/oil 3:2 (w/w), pH 7, lipase TL100L loading 400 U/g, lipase PCL loading 30 U/g. Then, ALA was enriched by urea inclusion, with an increased ALA content of 82.50% being obtained. Secondly, the ALA-enriched silkworm pupae DAG oil (SPDO) was prepared by lipase PCL-catalyzed esterification reaction. After molecular distillation, the final SPDO product contained contents of DAGs (97.01%) and ALA (82.50%). This two-step enzymatic way for production of ALA-DAGs was successfully applied in a 100-fold scale-up reaction. Overall, our study provides a promising way for the preparation of ALA-DAGs.

Simultaneous sex and species classification of silkworm pupae by NIR spectroscopy combined with chemometric analysis.

BACKGROUND: Most studies only focus on the sex discrimination of silkworm pupae. However, species differentiation of silkworm pupae is also needed in sericulture. To classify the sex and species at the same time, the present study adopts near infrared (NIR) spectroscopy combined with multivariate analysis. RESULTS: First, spectra samples were acquired using an NIR sensor, comprising female and male silkworm pupae from three species. Second, three different variables selection approaches were used, including a successive projections algorithm, competitive adaptive reweighted sampling (CARS) and interval partial least squares (iPLS). Third, identification models were built based on random forest and partial least squares discriminant analysis (PLSDA). The experimental results show that iPLS-PLSDA model (95.24%) gives a high performance when using the one of the three variable selection methods alone. To further increase the performance, the variable selection methods are optimized. The accuracy of the iPLS-CARS-PLSDA model is as high as 98.41%. CONCLUSION: The present study demonstrates that the optimized variable selection method in combination with NIR spectroscopy represents a suitable strategy for sex and species identification of silkworm pupae. © 2020 Society of Chemical Industry.

Effect of heat, enzymatic hydrolysis and acid-alkali treatment on the allergenicity of silkworm pupa protein extract.

Silkworm pupae are edible insects with high-quality nutrition in many Asian countries, but consumption of silkworm pupae can cause severe IgE-mediated allergic disease. The aim of this study was to investigate the effect of heat, enzymatic hydrolysis and acid-alkali treatment on the allergenicity of silkworm pupa protein extract (SPPE). Heating reduced the allergenicity of SPPE when the temperature was higher than 60 °C. Spectroscopy studies suggested an unfolded conformation of SPPE with heating, dependent on temperature and time. Enzymatic hydrolysis revealed that SPPE at 25 to 33 kDa contained pepsin- and trypsin-resistant allergens. The results of acid-alkali treatment suggested that low pH can promote hydrolysis of SPPE and decrease its allergenicity. Thus, heat, enzymatic hydrolysis and acid-alkali treatment can significantly decrease the allergenicity of SPPE, with heat-, enzyme- and acid-alkali-resistant allergens at 25 to 33 kDa SPPE. This study can help in the development of methods to prepare silkworm pupa protein.

Characteristics of chitin extracted from black soldier fly in different life stages.

Chitin was collected and extracted along different lifecycle stages of the Black Soldier Fly (BSF) (larvae, prepupae, pupae, flies, shedding & cocoons). The chitin content in the collected biomass ranged between 8% and 24%, with sheddings and cocoons being most rich in chitin. Purified chitin was subjected to a physicochemical evaluation based on FTIR, XRD, and TGA as well as a deacetylation step. The data indicated that BSF chitin was α-chitin with FTIR profiles matching closely to shrimp chitin and showing some differences compared to squid pen chitin (ß-chitin). Small physicochemical differences were observed among the different BSF samples. Prepupae and cocoon chitin was more crystalline while chitin from larvae and sheddings had a lower thermal degradation temperature. In addition, sheddings were more difficult to purify. Further processing to chitosan showed that a deacetylation degree of 89% could be obtained for all samples after 3 h, although sheddings were found to be less reactive in the deacetylation process. Overall, the small differences in physicochemical properties that were detected between the BSF chitin samples did not prevent further processing of chitin to chitosan with the same degree of deacetylation via the same treatment.

Changes in composition and levels of hemolymph proteins during metamorphosis of Manduca sexta.

The tobacco hornworm, Manduca sexta, is a lepidopteran model species widely used to study insect biochemical processes. Some of its larval hemolymph proteins are well studied, and a detailed proteomic analysis of larval plasma proteins became available in 2016, revealing features such as correlation with transcriptome data, formation of immune complexes, and constitution of an immune signaling system in hemolymph. It is unclear how the composition of these proteins may change in other developmental stages. In this paper, we report the proteomes of cell-free hemolymph from prepupae, pupae on day 4 and day 13, and young adults. Of the 1824 proteins identified, 907 have a signal peptide and 410 are related to immunity. Drastic changes in abundance of the storage proteins, lipophorins and vitellogenin, for instance, reflect physiological differences among prepupae, pupae, and adults. Considerably more proteins lacking signal peptide are present in the late pupae, suggesting that plasma contains relatively low concentrations of intracellular components released from remodeling tissues during metamorphosis. The defense proteins detected include 43 serine proteases and 11 serine protease homologs. Some of these proteins are members of the extracellular immune signaling network found in feeding larvae, and others may play additional roles and hence confer new features in the later life stages. In summary, the proteins and their levels revealed in this study, together with their transcriptome data, are expected to stimulate focused explorations of humoral immunity and other physiological systems in wandering larvae, pupae, and adults of M. sexta and shed light upon functional and comparative genomic research in other holometabolous insects.