Pepsin Digestion Research Articles

Allergenic characterization of Bomb m 4, a 30-kDa Bombyx mori lipoprotein 6 from silkworm pupa.

Silkworm pupa (SWP) food anaphylaxis has been described frequently in Asian countries. However, false-positive reactions by skin pricks and serum IgE (sIgE) tests to the extract complicate diagnosis, requiring identification of clinically relevant major allergens. In this study, we characterized a novel SWP allergen, Bomb m 4, a 30-kDa lipoprotein, and evaluated its diagnostic sensitivity. Bomb m 4 was identified by a proteomic analysis. This recombinant (r)Bomb m 4 was overexpressed in Escherichia coli, and the IgE reactivity by ELISA was compared with other reported allergenic proteins: Bomb m 1 (arginine kinase), 27-kDa glycoprotein, Bomb m 3 (tropomyosin) using the serum samples from 17 SWP allergic patients and 11 asymptomatic sensitized subjects. rBomb m 4-specific IgE was recognized by all 17 SWP allergic patients. The 27-kDa glycoprotein and Bomb m 1sIgE were found in 35.3% and 0%, respectively, in the SWP allergic patients. ELISA sIgE reactivity increased significantly, when 4M urea was added in serum samples. However, only 16% inhibition of sIgE reactivity to the whole SWP extract was exhibited by rBomb m 4, whereas more than 93% of self-inhibition of rBomb m 4sIgE was obtained, possibly due to the low abundance of Bomb m 4 in the extract. Three linear epitopes (81-95, 191-205 and 224-238 residues) of rBomb m 4 were identified. These epitopes are shown to be released by pepsin digestion. Receiver operator characteristic (ROC) analysis showed the highest diagnostic value of Bomb m 4 followed by Bomb m 1, 27-kDa glycoprotein and Bomb m 3. Bomb m 4 is the major allergen of SWP allergic patients. It has cryptic epitopes which are exposed to IgE antibodies with digestive enzymes. This recombinant Bomb m 4 allergen permits exact diagnosis of SWP allergy.

Evolution of in vitro digestibility techniques: a systematic review

The inability to reproduce certain digestive processes in vivo, high research costs and ethical aspects have led to the development of a large number of in vitro digestion models. These models allow us to take into account various factors of modeling complex multistage physiological processes occurring in the gastrointestinal tract, which makes them promising and widely used. A significant part of in vitro methods includes assessment by enzymatic digestion and are based on the calculation of nitrogen remaining after digestion in relation to the initial total nitrogen (according to the Dumas, Kjeldahl method, spectrophotometric or chromatographic method). There are also a number of titrometric methods (pH‑stat), which are mainly used to assess the digestibility of feed, most successfully for aquatic animals due to the simplicity of their digestive tract. Methods for assessing the digestibility of food products by enzymatic digestion have undergone various stages of evolution (since 1947) and have been widely modified by including various enzymes (pepsin, trypsin, pancreatin, erepsin, etc.) in model systems, indices for various products have been determined on their basis (pepsin-digest-residue (PDR) index, 1956; pepsin pancreatin digest (PPD) index, 1964; pepsin digest dialysate (PDD), 1989). As a result, a single protocol was formed to study the digestibility of food — INFOGEST (2014–2019), which includes three stages of digestion (oral, gastric and intestinal). It allows researchers to accurately reproduce the conditions of the human gastrointestinal tract and is widely used by scientists around the world.

Effect of high pressure shucking on the gel properties and in vitro digestibility of myofibrillar proteins from red swamp crayfish (Procambarus clarkii)

The effect of high pressure shucking (HPS) on heat-induced gelation properties of myofibrillar proteins from shucked crayfish meat mince was investigated, by comparing with conventionally industrial shucking methods (i.e., freeze-thawing and cooking). The texture, color, water holding capacity, water distribution, microstructure, protein patterns and secondary structure as well as the pepsin digestibility and antioxidant activity of gels were evaluated. Compared to conventional shucking, HPS at 300 MPa resulted in good-quality gels with improved mechanical properties and desirable reddish color. The treatment seemed to be conducive to the maintenance of astaxanthin, although it slightly reduced the water holding capacity and proportion of T22 water. SEM analysis showed that the gels treated by HPS at 300 MPa had a more orderly and uniform microstructure with reduced pore size. Under this scenario, more myosin heavy chains appeared to be involved in the crosslinking as revealed by SDS-PAGE. However, FTIR results suggested that no obvious change was found in the protein secondary structure of gels. Intriguingly, HPS of 300 MPa enhanced the pepsin digestibility and ABTS-radical scavenging activity of the gels during in vitro digestion. These findings underlined the advantages of HPS for creating new gel-type products based on crayfish meat.

Physicochemical characterization and identification of major linear epitopes of sarcoplasmic calcium-binding protein (SCP) allergen from Pacific oyster (Crassostrea gigas).

Food allergy is a serious public nutritional health problem that has attracted extensive worldwide attention. Shellfish allergy is a long-lasting disorder that has a lifelong impact on health. Sarcoplasmic calcium-binding protein (SCP) plays a vital role in cell and muscle functions and has been identified as an allergen in oyster. In this study, recombinant SCP (rSCP) with a molecular mass of 21 kDa was produced and identified based on SCP amino acid sequencing of Pacific oyster (Crassostrea gigas), and was used as a follow-up experimental material. Its physicochemical characterization showed that purified rSCP is highly stable to heat and acid-alkali and trypsin digestion but less resistant to pepsin digestion. We established an animal sensitization model and rSCP displayed stronger Immunoglobulin E (IgE)-binding activity with rat serum in the rSCP + cholera toxin (CT) group compared with the CT group and a control group. Five epitope peptides were identified as linear immunodominant epitopes by indirect competitive enzyme-linked immunosorbent assay (icELISA) for the first time. We also found that conformational epitopes may play a major role in the immunoreactivity of SCP. These results are significant for understanding hypersensitization of humans to oyster and offer available preventive measures and treatment programs in further research. © 2021 Society of Chemical Industry.

Regional-specific meniscal extracellular matrix hydrogels and their effects on cell–matrix interactions of fibrochondrocytes

Decellularized meniscal extracellular matrix (ECM) material holds great potential for meniscus repair and regeneration. Particularly, injectable ECM hydrogel is highly desirable for the minimally invasive treatment of irregularly shaped defects. Although regional-specific variations of the meniscus are well documented, no ECM hydrogel has been reported to simulate zonally specific microenvironments of the native meniscus. To fill the gap, different (outer, middle, and inner) zones of porcine menisci were separately decellularized. Then the regionally decellularized meniscal ECMs were solubilized by pepsin digestion, neutralized, and then form injectable hydrogels. The hydrogels were characterized in gelation behaviors and mechanical properties and seeded with bovine fibrochondrocytes to evaluate the regionally biochemical effects on the cell–matrix interactions. Our results showed that the decellularized inner meniscal ECM (IM) contained the greatest glycosaminoglycan (GAG) content and the least collagen content compared with the decellularized outer meniscal ECM (OM) and middle meniscal ECM (MM). The IM hydrogel showed lower compressive strength than the OM hydrogel. When encapsulated with fibrochondrocytes, the IM hydrogel accumulated more GAG, contracted to a greater extent and reached higher compressive strength than that of the OM hydrogel at 28 days. Our findings demonstrate that the regionally specific meniscal ECMs present biochemical variation and show various effects on the cell behaviors, thus providing information on how meniscal ECM hydrogels may be utilized to reconstruct the microenvironments of the native meniscus.

Automated Quantitative Image Evaluation of Antigen Retrieval Methods for 17 Antibodies in Placentation and Implantation Diagnostic and Research.

Immunostaining in clinical routine and research highly depends on standardized staining methods and quantitative image analyses. We qualitatively and quantitatively compared antigen retrieval methods (no pretreatment, pretreatment with pepsin, and heat-induced pretreatment with pH 6 or pH 9) for 17 antibodies relevant for placenta and implantation diagnostics and research. Using our newly established, comprehensive automated quantitative image analysis approach, fluorescent signal intensities were evaluated. Automated quantitative image analysis found that 9 out of 17 antibodies needed antigen retrieval to show positive staining. Heat induction proved to be the most efficient form of antigen retrieval. Eight markers stained positive after pepsin digestion, with β-hCG and vWF showing enhanced staining intensities. To avoid the misinterpretation of quantitative image data, the qualitative aspect should always be considered. Results from native placental tissue were compared with sections of a placental invasion model based on thermo-sensitive scaffolds. Immunostaining on placentas in vitro leads to new insights into fetal development and maternal pathophysiological pathways, as pregnant women are justifiably excluded from clinical studies. Thus, there is a clear need for the assessment of reliable immunofluorescent staining and pretreatment methods. Our evaluation offers a powerful tool for antibody and pretreatment selection in placental research providing objective and precise results.

Salt-Mediated Organic Solvent Precipitation for Enhanced Recovery of Peptides Generated by Pepsin Digestion.

Conventional solvent-based precipitation makes it challenging to obtain a high recovery of low mass peptides. However, we previously demonstrated that the inclusion of salt ions, specifically ZnSO4, together with high concentrations of acetone, maximizes the recovery of peptides generated from trypsin digestion. We herein generalized this protocol to the rapid (5 min) precipitation of pepsin-digested peptides recovered from acidic matrices. The precipitation protocol extended to other organic solvents (acetonitrile), with high recovery from dilute peptide samples permitting preconcentration and purification. Mass spectrometry profiling of pepsin-generated peptides demonstrated that the protocol captured peptides as small as 800 u, although with a preferential bias towards recovering larger and more hydrophobic peptides. The precipitation protocol was applied to rapidly quench, concentrate, and purify pepsin-digested samples ahead of MS. Complex mixtures of yeast and plasma proteome extracts were successfully precipitated following digestion, with over 95% of MS-identified peptides observed in the pellet fraction. The full precipitation workflow—including the digestion step—can be completed in under 10 min, with direct MS analysis of the recovered peptide pellets showing exceptional protein sequence coverage.

Effect of processing method on quality characteristics of harit soybean (glycine max): in vitro protein digestibility, hplc, ftir analysis

PurposeSoybean is potentially a rich source of protein and lipids along with isoflavone. However, the use of soybean is limited due to presence of anti-nutritional factors such as trypsin inhibitor, phytic acid, etc. The present study aimed at evaluation of Harit soya seeds of different treatments such as soaking, dehulling, germination, soaking+roasting and roasting in order to enhance its quality.Design/methodology/approachHarit soya seeds were utilized were soaked overnight and subjected for dehulling, germination for 3 days as well as roasting followed by drying to obtain fine powder for quality evaluation.FindingsDehulling and germination produce significant (p < 0.01) increase in protein, crude fiber, total chlorophyll, ascorbic acid, total phenolics, flavonoids and isoflavones while fat was decreased non-significantly. The highest value of 12.4 mg/100g, 5.3 mgGAE/g and 6.6 mg/g for ascorbic acid, total phenolics and isoflavones was found in germinated seeds. Also, the effect of soaking, roasting and their combination lead to significant decrease in the functional properties of the seeds. Anti-nutritional factors were significantly (p < 0.01) reduced in all forms of treated seeds while pepsin and trysin digestibility was enhanced during soaking, dehulling and germination with maximum value of 72.6 and 25.8%, respectively in germinated seeds.Originality/valueImprovement in quality of evergreen variety of soybean using cost effective traditional methods

KFP-1, a Novel Calcium-Binding Peptide Isolated from Kefir, Promotes Calcium Influx Through TRPV6 Channels.

Kefir is an acidic and alcoholic fermented milk product with multiple health-promoting benefits. A previous study demonstrated that kefir enhanced calcium absorption in intestinal Caco-2 cells. In this study, kefir-fermented peptide-1 (KFP-1) is isolated from the kefir peptide fraction, and its function as a calcium-binding peptide is characterized. KFP-1 was identified as a 17-residue peptide with a sequence identical to that of κ-casein (residues 138-154) in milk protein. KFP-1 is demonstrated to promote calcium influx in Caco-2 and IEC-6 small intestinal cells in a concentration-dependent manner. TRPV6, but not L-type voltage-gated calcium channels, is associated with the calcium influx induced by KFP-1. An in vitro calcium binding assay indicates that the full-length KFP-1 peptide has a higher calcium-binding capacity than the two truncated KFP-1 peptides, KFP-1∆C5 and KFP-1C5. Alexa Fluor 594 labeling shows that KFP-1 is taken up by Caco-2 cells and interacts with calcium ions and TRPV6 protein. Moreover, KFP-1 is found moderately resistant to pepsin and pancreatin digestions and enhanced calcium uptake by intestinal enterocytes in vivo. These data suggest that KFP-1, a novel calcium-binding peptide, binds extracellular calcium ions and enters Caco-2 and IEC-6 cells, and promotes calcium uptake through TRPV6 calcium channels. The present study is of great importance for developing kefir-derived metal ion-binding peptides as functional nutraceutical additives.

First Report of Sarcocystis Masoni in a Captive Alpaca (Vicugna Pacos) From China.

Background: Sarcocystosis is a parasitic disease caused by intracellular protozoan parasite of the genus Sarcocystis. Tissue samples of alpacas (n = 4) from Henan province (China) were screened for Sarcocystis spp. infection by histological examination, pepsin digestion, and molecular assays.Results: Sarcocystis spp. was detected in heart, liver, spleen, lung, and kidney of an alpaca by molecular assays. Many sarcocysts with inflammation responses were observed in this alpaca myocardium, and they showed a high similarity to Sarcocystis masoni by sequence analysis.Conclusion: This study is the first to demonstrate Sarcocystis spp. infection in alpaca from China. The higher parasite load in the alpaca myocardium indicated that it had contact with an environment contaminated with sporocysts, and that the alpaca was susceptible to Sarcocystis spp.

Prevalence of Toxoplasma Gondii in Retail Fresh Meat Products from Free-range Chickens in Spain.

IntroductionToxoplasma gondii is one of the most prevalent zoonotic protozoan parasites worldwide and affects the vast majority of warm-blooded animal species, including humans. Postnatal infection in humans occurs through the ingestion of sporulated T. gondii oocysts or via the oral intake of parasite tissue cysts during the consumption of raw or undercooked meat. In this regard, given their high exposure to oocysts, chickens (Gallus domesticus) raised on the ground constitute a potential source of T. gondii.Material and MethodsFor the first time in Spain, a survey was undertaken in commercial retail free-range poultry. A total of 50 thighs from different animals were analysed. The samples were homogenised and an acid pepsin digestion procedure was applied prior to molecular analysis. Toxoplasma gondii DNA was isolated from meat by qPCR. Two sets of primers were used for DNA amplification targeting the specific sequence of a 529 bp repeat element and another set of primers was utilised for the surface antigen protein-1 gene.ResultsDNA extracted from 5 out of 50 tissue samples was positive for both genes by qPCR amplification.ConclusionThe 10% prevalence of Toxoplasma infection found in commercial free-range chickens raises public health issues.

Effects of radiation dose and nitrogen purge on collagen scaffold properties.

Sterilization of medical devices is commonly performed using radiation methods. However, collagen materials can be damaged when using standard radiation doses (25kGy). Small increases of radiation dose can allow for increases in the acceptable initial bioburden load of aseptically manufactured devices while maintaining required sterility assurance levels, which is often critical in early stage translational settings. In this study, we hypothesized that small increases in radiation dose from 15 to 20kGy would result in significant changes to several key characteristics of collagen scaffolds. Scaffolds were manufactured by lyophilizing the pepsin digest of dense bovine connective tissue in cylindrical molds and were irradiated at either 0, 15, 17.5, or 20kGy with an additional group packaged in nitrogen and irradiated at 17.5 kGy. Groups were evaluated for changes to the soluble collagen and glycosaminoglycan mass fractions, protein banding patterns in electrophoresis, a collagen fragmentation assay, and resistance to enzymatic degradation. All parameters were statistically analyzed using one-way analysis of variance with Tukey's correction for multiple comparisons. The soluble collagen mass fraction was significantly decreased in the 20kGy group; however, there was no significant effect of radiation dose or a nitrogen-rich environment on the other measured parameters, including protein banding patterns, fragmented collagen content, and resistance to enzymatic degradation.Statement of Clinical Significance: Collagen scaffolds have proven useful in clinical applications but can be damaged by standard radiation doses. Low-dose sterilization may be a viable alternative that minimally impacts key properties of these scaffolds.

Trichinella pseudospiralisinfectionin red-eared slider,Trachemys scripta,influenced by environmental temperature.

Experimental infection with Trichinella pseudospiralis larvae in red-eared slider Trachemys scripta was carried out. Ten turtles were divided into 3 groups and kept at different temperature (38, 32 and 28°C). The turtles were fed mice muscles which were infected with T. pseudospiralis larvae (444 - 23,013 larvae/mouse), kept in the aforementioned temperature and necropsied at day 30 after infection. At necropsy, the tongue, forelimbs, hindlimbs and spinal column muscles of turtles were removed separately for pepsin digestion, and the larvae counted. Larvae were found from all 3 turtles kept at 38°C. Larvae per gram of muscle were highest in the tongue. No larvae were recovered from turtles kept at 32 and 28°C. The result suggested that T. pseudospiralis is able to infect the red-eared slider in a high temperature environment. We speculated that environmental temperature play an important role in altering the physiological condition of the turtle to facilitate the infection of T. pseudospiralis.

Phycocyanobilin-modified β-lactoglobulin exhibits increased antioxidant properties and stability to digestion and heating

β-lactoglobulin (BLG) is a major whey protein with numerous techno-functional properties desirable for the food industry. Phycocyanobilin (PCB), a bioactive pigment of Arthrospira platensis with health-promoting effects, covalently binds to BLG at physiological pH. This study investigated the effects of this covalent modification on BLG functional properties. The BLG–PCB adduct possesses enhanced antioxidant properties, and bound PCB protects BLG against free radical-induced oxidation. Despite the similar thermal stabilities of BLG and BLG–PCB, BLG–PCB is less susceptible to covalent and noncovalent aggregation under moderate heat treatment (63 °C, 30 min). Blocked thiol group and reduced hydrophobicity due to hindering of hydrophobic residues by bound PCB, as well as the heat-induced transition of β-sheet to α-helix, contributed to the low susceptibility of BLG–PCB to aggregation. BLG–PCB has a higher resistance to pepsin and pancreatin digestion than BLG and unaltered IgE-binding properties. The improved functional properties of BLG–PCB make it a useful ingredient in the food industry.

High-Resolution Bioprinting of Recombinant Human Collagen Type III.

The development of commercial collagen inks for extrusion-based bioprinting has increased the amount of research on pure collagen bioprinting, i.e., collagen inks not mixed with gelatin, alginate, or other more common biomaterial inks. New printing techniques have also improved the resolution achievable with pure collagen bioprinting. However, the resultant collagen constructs still appear too weak to replicate dense collagenous tissues, such as the cornea. This work aims to demonstrate the first reported case of bioprinted recombinant collagen films with suitable optical and mechanical properties for corneal tissue engineering. The printing technology used, aerosol jet® printing (AJP), is a high-resolution printing method normally used to deposit conductive inks for electronic printing. In this work, AJP was employed to deposit recombinant human collagen type III (RHCIII) in overlapping continuous lines of 60 µm to form thin layers. Layers were repeated up to 764 times to result in a construct that was considered a few hundred microns thick when swollen. Samples were subsequently neutralised and crosslinked using EDC:NHS crosslinking. Nanoindentation and absorbance measurements were conducted, and the results show that the AJP-deposited RHCIII samples possess suitable mechanical and optical properties for corneal tissue engineering: an average effective elastic modulus of 506 ± 173 kPa and transparency ≥87% at all visible wavelengths. Circular dichroism showed that there was some loss of helicity of the collagen due to aerosolisation. SDS-PAGE and pepsin digestion were used to show that while some collagen is degraded due to aerosolisation, it remains an inaccessible substrate for pepsin cleavage.

Physical-chemical properties and in vitro digestibility of phosphorylated and glycosylated soy protein isolate

Increased demand for highly functional soy protein isolate (SPI) in the food industry draws extra attention to SPI modification. The structural properties and in vitro digestibility of phosphorylated and glycosylated SPI were investigated in this study. The phosphorylation was carried out with sodium tripolyphosphate addition at 45 °C for 4 h. Glucose and dextran were used for glycosylation at 100 °C for 1 h. Apparent molecular weight distribution was determined by size exclusion-high performance liquid chromatography. Thiol content and surface hydrophobicity were also determined. The apparent molecular weight and thiol content of phosphorylated SPI increased with the increase of phosphorylation degree. Meanwhile, phosphorylation negatively affected gastrointestinal digestibility, especially gastric digestibility. The fraction of glycosylated SPI was prone to be low molecular weight, suggesting glycosylation inhibited SPI aggregation, which resulted from the attenuated surface hydrophobicity. The digestibility of SPI was increased by glycosylation. Glucose-based glycosylation contributed to lower intestinal digestion compared with dextran-based glycosylated SPI. This work highlighted that the phosphorylation and glycosylation to SPI primarily affected the in vitro pepsin or pancreatin digestion, inspiring to produce modified SPI with balanced functional and nutritional properties for the food industry.

A loop-mediated isothermal amplification assay for Schistosoma mansoni detection in Biomphalaria spp. from schistosomiasis-endemic areas in Minas Gerais, Brazil

BackgroundSchistosomiasis a neglected tropical disease endemic in Brazil. It is caused by the trematode Schistosoma mansoni, which is transmitted by snails of the genus Biomphalaria. Among measures used to control and eliminate schistosomiasis, accurate mapping and monitoring of snail breeding sites are recommended. Despite the limitations of parasitological methods, they are still used to identify infected snails. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cost-effective diagnostic method for the identification of infected snails. In the work reported here, we aimed to validate the use of LAMP for the detection of S. mansoni in snails of the genus Biomphalaria.MethodsSnails were collected in five municipalities of the Mucuri Valley and Jequitinhonha Valley regions in the state of Minas Gerais, Brazil. Snails were pooled according to collection site and then squeezed for the detection of S. mansoni and other trematode larvae. Pooled snails were subjected to pepsin digestion and DNA extraction. Molecular assays were performed for species-specific identification and characterization of the samples. A previously described LAMP assay was adapted, evaluated, and validated using laboratory and field samples.ResultsUsing the parasitological method described here, S. mansoni cercariae were detected in snails from two collection sites, and cercariae of the family Spirorchiidae were found in snails from one site. The snails were identified by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP). Biomphalaria glabrata, the main snail host of S. mansoni in Brazil, was detected in 72.2% of the collection sites. Biomphalaria kuhniana, which is resistant to S. mansoni infection, was found in the remaining sites. Multiplex, low stringency (LS), and conventional PCR allowed the detection of positive snails in four additional sites. Trematodes belonging to the families Strigeidae and Echinostomatidae were detected by multiplex PCR in two sites. The LAMP assay was effective in detecting the presence of S. mansoni infection in laboratory (7 days post-infection) and field samples with no cross-reactivity for other trematodes. When compared to LS and conventional PCR, LAMP showed 100% specificity, 85.7% sensitivity, and a κ index of 0.88.ConclusionsOur findings suggest that LAMP is a good alternative method for the detection and monitoring of transmission foci of S. mansoni, as it was three times as effective as the parasitological examination used here for the detection of infection, and is more directly applicable in the field than other molecular techniques.Graphical abstract

Comparison of Direct and Indirect Toxoplasma gondii Detection and Genotyping in Game: Relationship and Challenges.

The importance of game as a source of Toxoplasma gondii (T. gondii) infection in humans is largely unknown. New data on the presence of T. gondii in game hunted in the Federal State of Brandenburg, Germany, were obtained by direct and indirect detection (ELISA). DNA extracted either directly (5 g heart or foreleg muscle, DE) or after acid pepsin digestion (50 g heart, PD) or enriched by magnetic capture (50 g heart, MC) was examined by real-time PCR (qPCR). ELISA revealed seroprevalences of 20% in wild boar (Sus scrofa), 11% in roe deer (Capreolus capreolus) and 6% in red deer (Cervus elaphus). T. gondii DNA was detected by at least one direct detection method in 12% of wild boar, 6% of roe deer, 2% of fallow deer (Dama dama) and 2% of red deer. In both, positive wild boar and roe deer, T. gondii type II specific alleles were the most prevalent, as assessed by PCR-restriction fragment length polymorphism. The highest proportion of positive animals was detected by MC qPCR, followed by PD qPCR with a similar proportion of positive findings. Investigation of 50 g of heart muscle revealed a significantly higher proportion of positive qPCR results than analysis of 5 g (p = 0.048). An association between seropositivity and direct detection was evident in wild boar and roe deer (p < 0.001). Infectivity of T. gondii DNA–positive samples was confirmed by bioassay (4/4), providing evidence that game could represent a relevant source of viable T. gondii posing a risk for human infection.

Front Cover: Pepsin Digest of Gliadin Forms Spontaneously Amyloid‐Like Nanostructures Influencing the Expression of Selected Pro‐Inflammatory, Chemoattractant, and Apoptotic Genes in Caco‐2 Cells: Implications for Gluten‐Related Disorders

Mol. Nutr. Food Res. 2021, 65, 202100200 DOI: 10.1002/mnfr.202100200 An integral physicochemical and functional approach demonstrated that pepsin gliadin digest forms spontaneously amyloid-type nanostructures whose interaction with the Caco-2 enterocyte cellular model induces the expression of selected mRNAs involved in the activation of immune cells. Therefore the pathogenic role of gliadin nanostructures may be relevant for biodemical research. This is reported by Veronica I. Dodero and co-workers in article number 2100200.

Optimization of tools for the detection and identification of Cryptocotyle metacercariae in fish: Digestion method and viability studies.

Some trematode metacercariae, including marine digeneans belonging to the genus Cryptocotyle, induce black spots in target tissues due to the attraction of fish host melanophores. To promote precise quantification of infection, the counting of black spots has to be confirmed by reliable quantification of metacercariae after tissue digestion. This process ensures the isolation of undamaged parasites for morphological and molecular identification. The aim of this work was to optimize the pepsin digestion protocol and to assess the duration of viability of Cryptocotyle metacercariae in fish post‐mortem (pm). Four digestion protocols were compared by measuring the viability rate of metacercariae. The present study shows that the orbital digestion method was the least destructive for metacercariae and allowed better quantification of Cryptocotyle infection. Moreover, morphological identification seemed reliable up to 8 days pm for Cryptocotyle infection.