Comparative digestomics of Tropomyosin of vertebrates and invertebrates in real food matrix

Shellfish, is a highly nutritive food resource in the world, but also among the eight allergic food groups accounting for approximately 90% of all immunoglobulin E food allergies worldwide [1]. This work focuses on the only well-recognized major allergen muscle protein tropomyosin(TM) that is responsible for cross reactivity between shellfish and other invertebrates [2]. By contrary, TM of vertebrates (chicken, pig, cow) is not a prominent allergen. The stability of food allergens to digestion is an important factor contributing to their allergenicity. Most in vitro digestibility studies are based on the protein extract rather than whole food matrix thus overlooking its effect on TM stability [3]. Our objective was to primarily test the pepsin digestibility of invertebrates and vertebrates (raw and thermally treated based on their real life consumption modes) mimicking the gastric digestion under standardized conditions. To closely observe and compare the vertebrates’ and invertebrates’ TM stability, we aimed to perform the specific antibody based western blot analysis with two primary antibodies; ❶Rabbit anti shrimp TM antibody (invertebrates), and ❷ Rabbit anti human TM antibody (species reactivity to vertebrates). Methods: Thermal treatment of selected samples to compare TM heat stability, Standardized static in vitro methods of simulated gastric digestion[4] for the evaluation and comparison of TM resistance to pepsin, Sodium Dodecyl Sulfate-Polyacryl amide Gel Electrophoresis (SDS-PAGE) of digesta supernatant under reducing and non-reducing conditions to quantify proteins and compare thermally treated invertebrates and vertebrates protein profiles focusing on TM, specific antibody based semi dry Western blot analysis. Results and discussions: SDS-PAGE analysis of vertebrates and invertebrates’ samples showed a range of proteins in varied amounts between 10-250 kDa. Depending upon samples, varied numbers of prominent protein bands were observed including the distinct bands corresponding with the molecular weights of TM(37-39kDa). In agreement with publications, TM was, indeed, resistant against pepsin digestion as well as thermal treatment prominently in case of invertebrates. This was confirmed upon Ab based Western blot analysis. Our results show that, upon thermal treatment, TM is partially degraded as is observed in case of raw and cooked beef electrophoretic profile as well as WB analysis. Significantly, upon pepsin digestion, TM (allergen) is completely degraded in vertebrates in contrast to the invertebrates’ TM (which is pepsin resistant and heat stable). This result provides an insight on the differences in digestibility of allergenic versus non-allergenic TM in real food matrix and upon thermal treatments of solid food samples. Methods: Thermal treatment of selected samples to compare TM heat stability, Standardized static in vitro methods of simulated gastric digestion[4] for the evaluation and comparison of TM resistance to pepsin, Sodium Dodecyl Sulfate-Polyacryl amide Gel Electrophoresis (SDS-PAGE) of digesta supernatant under reducing and non-reducing conditions to quantify proteins and compare thermally treated invertebrates and vertebrates protein profiles focusing on TM, specific antibody based semi dry Western blot analysis. Results and discussions: SDS-PAGE analysis of vertebrates and invertebrates’ samples showed a range of proteins in varied amounts between 10-250 kDa. Depending upon samples, varied numbers of prominent protein bands were observed including the distinct bands corresponding with the molecular weights of TM(37-39kDa). In agreement with publications, TM was, indeed, resistant against pepsin digestion as well as thermal treatment prominently in case of invertebrates. This was confirmed upon Ab based Western blot analysis. Our results show that, upon thermal treatment, TM is partially degraded as is observed in case of raw and cooked beef electrophoretic profile as well as WB analysis. Significantly, upon pepsin digestion, TM (allergen) is completely degraded in vertebrates in contrast to the invertebrates’ TM (which is pepsin resistant and heat stable). This result provides an insight on the differences in digestibility of allergenic versus non-allergenic TM in real food matrix and upon thermal treatments of solid food samples

Comparative digestomics of Tropomyosin of vertebrates and invertebrates in real food matrix

Shellfish, is a highly nutritive food resource in the world, but also among the eight allergic food groups accounting for approximately 90% of all immunoglobulin E food allergies worldwide [1]. This work focuses on the only well-recognized major allergen muscle protein tropomyosin(TM) that is responsible for cross reactivity between shellfish and other invertebrates [2]. By contrary, TM of vertebrates (chicken, pig, cow) is not a prominent allergen. The stability of food allergens to digestion is an important factor contributing to their allergenicity. Most in vitro digestibility studies are based on the protein extract rather than whole food matrix thus overlooking its effect on TM stability [3]. Our objective was to primarily test the pepsin digestibility of invertebrates and vertebrates (raw and thermally treated based on their real life consumption modes) mimicking the gastric digestion under standardized conditions. To closely observe and compare the vertebrates’ and invertebrates’ TM stability, we aimed to perform the specific antibody based western blot analysis with two primary antibodies; ❶Rabbit anti shrimp TM antibody (invertebrates), and ❷ Rabbit anti human TM antibody (species reactivity to vertebrates). Methods: Thermal treatment of selected samples to compare TM heat stability, Standardized static in vitro methods of simulated gastric digestion[4] for the evaluation and comparison of TM resistance to pepsin, Sodium Dodecyl Sulfate-Polyacryl amide Gel Electrophoresis (SDS-PAGE) of digesta supernatant under reducing and non-reducing conditions to quantify proteins and compare thermally treated invertebrates and vertebrates protein profiles focusing on TM, specific antibody based semi dry Western blot analysis. Results and discussions: SDS-PAGE analysis of vertebrates and invertebrates’ samples showed a range of proteins in varied amounts between 10-250 kDa. Depending upon samples, varied numbers of prominent protein bands were observed including the distinct bands corresponding with the molecular weights of TM(37-39kDa). In agreement with publications, TM was, indeed, resistant against pepsin digestion as well as thermal treatment prominently in case of invertebrates. This was confirmed upon Ab based Western blot analysis. Our results show that, upon thermal treatment, TM is partially degraded as is observed in case of raw and cooked beef electrophoretic profile as well as WB analysis. Significantly, upon pepsin digestion, TM (allergen) is completely degraded in vertebrates in contrast to the invertebrates’ TM (which is pepsin resistant and heat stable). This result provides an insight on the differences in digestibility of allergenic versus non-allergenic TM in real food matrix and upon thermal treatments of solid food samples. Methods: Thermal treatment of selected samples to compare TM heat stability, Standardized static in vitro methods of simulated gastric digestion[4] for the evaluation and comparison of TM resistance to pepsin, Sodium Dodecyl Sulfate-Polyacryl amide Gel Electrophoresis (SDS-PAGE) of digesta supernatant under reducing and non-reducing conditions to quantify proteins and compare thermally treated invertebrates and vertebrates protein profiles focusing on TM, specific antibody based semi dry Western blot analysis. Results and discussions: SDS-PAGE analysis of vertebrates and invertebrates’ samples showed a range of proteins in varied amounts between 10-250 kDa. Depending upon samples, varied numbers of prominent protein bands were observed including the distinct bands corresponding with the molecular weights of TM(37-39kDa). In agreement with publications, TM was, indeed, resistant against pepsin digestion as well as thermal treatment prominently in case of invertebrates. This was confirmed upon Ab based Western blot analysis. Our results show that, upon thermal treatment, TM is partially degraded as is observed in case of raw and cooked beef electrophoretic profile as well as WB analysis. Significantly, upon pepsin digestion, TM (allergen) is completely degraded in vertebrates in contrast to the invertebrates’ TM (which is pepsin resistant and heat stable). This result provides an insight on the differences in digestibility of allergenic versus non-allergenic TM in real food matrix and upon thermal treatments of solid food samples

Aggregability and digestibility study of fruit juice fortified camel milk powder proteins

In this work, we observed the effect of grape juice (% concentrated juice/% concentrated camel milk: GJ20/80, GJ50/50) and pomegranate juice (PJ20/80, PJ40/60) fortification on camel milk (CM) protein solubility and digestibility. Proteins were dissolved in sodium phosphate buffer to 50 mg/ml and defatted prior Bradford assay of protein concentration, then analyzed by Size Exclusion-Ultra High-Performance Liquid chromatography (SE-UHPLC). The CM protein aggregation and their stability were further monitored at different pH 2.0, 4.0, and 7.5 via sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Freeze dried CM (FDCM) was the reference sample and our results showed that GJ50/50 and PJ40/60 with the highest fruit juice ratio had the lowest protein content in the supernatant, hence the decreased solubility. SE-UHPLC of supernatants showed a slight decrease in retention times of 11 kDa and 62 kDa proteins for GJ50/50 and PJ40/60 suggesting a possibility of adduct formation due to fortification leading to higher molecular weight. The simulated static in vitro gastrointestinal digestion of samples revealed that most soluble proteins were readily digested by pepsin, trypsin and chymotrypsin enzymes leading to small peptides. However, the SDS PAGE of pellets showed the partial resistance of casein and α-lactalbumin against peptic digestion.Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/4747

Digestomics of Japanese abalone in real food matrix

Objective: Haliotis discus (Japanese abalone), mollusks among various shellfish, is a highly nutritive food resource in the world, but also among the eight allergic food groups accounting for approximately 90% of all immunoglobulin E food allergies worldwide. The general objective of our research is to comprehensively investigate stability and structures of pepsin-resistant allergens, of their larger fragments, and of short digestion resistant peptides (SDRPs) released by pepsin digestion of whole raw and extract of shellfish, under standardized and physiologically relevant gastric conditions. Materials and Methods: Extract of raw whole shellfish (eRSS) and whole raw shellfish (wRSS), were pepsin digested according to standardized static digestion protocol. Controls were treated in a same manner without adding pepsin. Supernatant of samples and its counterpart controls were precipitated with TCA/acetone. Obtained proteins were assessed by 2D SDS PAGE and 1D SDS- PAGE, under reducing and non-reducing conditions. 1D SDS-PAGE of RSS were analyzed by ncLC-MS/MS (Orbitrap LTQ) shot-gun proteomics. Relative quantification was performed by LFQ algorithm within Peaks 8.5 software package Bioinformatics Solutions Inc. (BSI), Waterloo, Canada. Results and Conclusion: 1D SDS-PAGE analysis of eRSS and wRSS, and its controls showed a range of proteins in varied concentrations between 10-250 kDa. In extracted and whole raw shellfish, approximately 22 prominent protein bands were observed including the distinct bands corresponding with the molecular weights of recognized shellfish allergen, tropomyosin (37- 39kDa). Fewer high molecular weight proteins were observed followed by protein smearing, specifically around the low molecular weight protein bands. The smearing could possibly be due to the breakdown products and the glycation. There were slight differences between the protein profiles under reducing and non-reducing conditions as well. Nevertheless, there was the retention of a band in the 37kDa molecular weight marker in all 4 samples, likely consistent with heat stable tropomyosin (TM). Mass spectrometry showed allergens that are characterized (Hal d 1 and Hal di 1), with 90% of sequence homology with main tropomyosin allergens from seafood. Scientific impact and relevance: The results will highlight effects of food matrix on shellfish allergens digestibility proving its relevancy in molecular allergology. Moreover, an insight will be obtained on the differences in digestibility of allergenic versus non-allergenic tropomyosins in the real food matrix.Book of Abstract

Protein modifications screening of raw and thermally treated meat gastrointestinal digesta

Meat samples were subjected to thermal processing combined with simulated INFOGEST in vitro gastrointestinal (GI) digestion. Protein modifications (PMs) were screened with commercially available PM-specific antibodies. Specific proteins at 20, 37, 50, and 65 kDa react to more than 3 PM-specific antibodies among meat proteins. Lysine methylation and methionine oxidation were the most prominent PMs in WB. Mass spectrometry confirmed bands at ≈20 kDa as allergenic proteins: sarcoplasmic calcium-binding protein in oyster, 37 kDa as tropomyosin in shrimp, oyster, and abalone. MS-identified PMs of shellfish allergens were aligned to the IgE binding epitopes. GI digestion-resistant peptides of shellfish proteins were identified as paramyosins in oyster and abalone and SBP in shrimp. Our results point to the high susceptibility of immunodominant epitopes of major shellfish allergens to PMs. In TPM, saturation of oxidative modification increases with thermal processing resulting in higher susceptibility of TPM to gastric digestion

Role of resveratrol in prevention and control of cardiovascular disorders and cardiovascular complications related to COVID-19 disease: Mode of action and approaches explored to increase its bioavailability

Resveratrol is a phytoalexin produced by many plants as a defense mechanism against stress-inducing conditions. The richest dietary sources of resveratrol are berries and grapes, their juices and wines. Good bioavailability of resveratrol is not reflected in its high biological activity in vivo because of resveratrol isomerization and its poor solubility in aqueous solutions. Proteins, cyclodextrins and nanomaterials have been explored as innovative delivery vehicles for resveratrol to overcome this limitation. Numerous in vitro and in vivo studies demonstrated beneficial effects of resveratrol in cardiovascular diseases (CVD). Main beneficial effects of resveratrol intake are cardioprotective, anti-hypertensive, vasodilatory, anti-diabetic, and improvement of lipid status. As resveratrol can alleviate the numerous factors associated with CVD, it has potential as a functional supplement to reduce COVID-19 illness severity in patients displaying poor prognosis due to cardio-vascular complications. Resveratrol was shown to mitigate the major pathways involved in the pathogenesis of SARS-CoV-2 including regulation of the renin-angiotensin system and expression of angiotensin-converting enzyme 2, stimulation of immune system and downregulation of pro-inflammatory cytokine release. Therefore, several studies already have anticipated potential implementation of resveratrol in COVID-19 treatment. Regular intake of a resveratrol rich diet, or resveratrol-based complementary medicaments, may contribute to a healthier cardio-vascular system, prevention and control of CVD, including COVID-19 disease related complications of CVD

Detection and quantification of tropomyosin in differentially treated clams from Korea

Tropomyosin (TM) is known to be a major shrimp allergen (e.g., Pen m 1) and considered a cross-reacting panallergen among shellfish/invertebrates. The clam TM is also considered its major allergen but has not been widely studied. The food processing techniques can alter the TM allergenicity. Hence, the objective of this research is to detect and quantify TM in fresh and differentially treated clams collected in Korea via in-house developed sandwich ELISA protocol to evaluate the effect of various real-life processing techniques on TM stability. Freshly bought live clams (FC), 4 groups of randomly selected equal number of similarly sized clams were differentially treated. Fresh and packaged (FPC), fresh and frozen at -20◦C (FroC). The fresh clams boiled (BC) in boiling water and the marinated clams (MC) suspended in marinade solution for 5 days; soluble protein extracted overnight from 5 samples in PBS buffer with protease inhibitor; BCA assay determined the protein content; capture-detection-enzyme linked secondary antibody in-house ELISA. ELISA was validated with specific antibody based Western blot (WB). The total soluble protein content of raw clams (FC, FPC, FroC) was between 2.8-4.9 mg/ml. The cooked clams (BC, MC) lost total protein during the cooking and was determined FroC (290) >FC (75) >MC. It (and WB) showed that boiling has no effect on heat stable TM IgG binding, BW contained considerable amount of TM (with pronounced IgG binding). MC, however showed no TM epitope recognition in WB (no band in SDS PAGE) and was not quantified by ELISA nor in MS (<LLOQ). Marination might degrade the TM to significant extent possibly altering the allergenicity

Molecular Mechanisms of Possible Action of Phenolic Compounds in COVID-19 Protection and Prevention

The worldwide outbreak of COVID-19 was caused by a pathogenic virus called Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Therapies against SARS-CoV-2 target the virus or human cells or the immune system. However, therapies based on specific antibodies, such as vaccines and monoclonal antibodies, may become inefficient enough when the virus changes its antigenicity due to mutations. Polyphenols are the major class of bioactive compounds in nature, exerting diverse health effects based on their direct antioxidant activity and their effects in the modulation of intracellular signaling. There are currently numerous clinical trials investigating the effects of polyphenols in prophylaxis and the treatment of COVID-19, from symptomatic, via moderate and severe COVID-19 treatment, to anti-fibrotic treatment in discharged COVID-19 patients. Antiviral activities of polyphenols and their impact on immune system modulation could serve as a solid basis for developing polyphenol-based natural approaches for preventing and treating COVID-19

Comparative digestion of thermally treated vertebrates and invertebrates allergen pairs in real food matrix

The digestion stability of allergen pairs, tropomyosin, TM (fish and seafood allergen), and myosin light chain, MLC (chicken meat allergen) is compared among vertebrates and invertebrates in raw and cooked food matrix under standardized simulated in vitro gastrointestinal (GI) digestion. SDS-PAGE followed by multiple TM and MLC-specific antibodies in semidry WB revealed pepsin resistance of invertebrate TMs (abalone, oyster, shrimp) under diet-relevant conditions (raw, cooked). Vertebrate TMs (chicken, pork, beef) were less stable to digestion except that the raw chicken TM was observed pepsin resistant (not diet-relevant). Vertebrate (chicken) MLC was thermally stable. A new 28 kDa protein bound to anti-MLC antibody in cooked chicken and pork; could be the aggregates of MLC. Raw shrimp MLC showed pepsin resistance among invertebrates. A good correlation was observed between combined resistance of TM and MLC to GI digestion following the diet-relevant thermal treatment and reported protein allergenicity among vertebrates and invertebrates

Insights into oxidative protein modifications in shellfish allergens: Impact of GI digestion following thermal treatment

Protein modifications (PMs) are covalent changes occurring on amino acid (AA) side chains of proteins via enzymatic action or spontaneously. They increase the protein structure complexity from the level of the genome to the proteome, and can determine their activity and, interactions1. It has been shown, on myofibrillar fish proteins that oxidative modifications depending on their saturation can decrease or enhance protein digestability2. Thus in this study shellfish meat samples were subjected to cooking followed by simulated INFOGEST in vitro gastrointestinal digestion protocol. Mass Spectrometry and in gel based proteomics, were used to detect PMs and digestion resistan peptides of raw and cooked shellfish meat samples with a foucs on major shellfish allergens: sarcoplasmic calcium-binding protein (SBP) and tropomyosin (TPM). Lastly, identified PMs were marked on TPM alignment from shrimp, oyster and abalone species, to gain insight into their pattern on the known IgE binding epitopes. Focusing on oxidative PMs, mostly on oxidation of Met, it was confirmed that, in all shellfish samples, PMs on TPM and SBP were more predominant after cooking. For instance in abalone, double oxidation of Met was only detected in cooked samples (sulfone M68 and M81). Another indicator of oxidative stress, 4-hydroxynonenal (HNE), was identified on shrimp TPM and, notably HNE was 21 times more abundant in the cooked sample. Study highlights the susceptibility of immunodominant epitopes in major shellfish allergens to oxidative PMs, which could impact interactions with the immune system in sensitive individuals. Significant resistance to digestion was demonstrated by paramyosin fragments in abalone and oyster suggesting the need for further analysis of their allergenicity. SBP of raw shrimp was strikingly resistant to both gastric and intestinal digestion. Moreover, our results indicate that oxidative modifications may decrease the stability of proteins like shrimp TPM, making them more digestible in thermally treated samples, highlighting a potential strategy for reducing allergenicity