Effects of Laccase and Transglutaminase on the Physicochemical and Functional Properties of Hybrid Lupin and Whey Protein Powder.

Plant-based protein is considered a sustainable protein source and has increased in demand recently. However, products containing plant-based proteins require further modification to achieve the desired functionalities akin to those present in animal protein products. This study aimed to investigate the effects of enzymes as cross-linking reagents on the physicochemical and functional properties of hybrid plant- and animal-based proteins in which lupin and whey proteins were chosen as representatives, respectively. They were hybridised through enzymatic cross-linking using two laccases (laccase R, derived from Rhus vernicifera and laccase T, derived from Trametes versicolor) and transglutaminase (TG). The cross-linking experiments were conducted by mixing aqueous solutions of lupin flour and whey protein concentrate powder in a ratio of 1:1 of protein content under the conditions of pH 7, 40 °C for 20 h and in the presence of laccase T, laccase R, or TG. The cross-linked mixtures were freeze-dried, and the powders obtained were assessed for their cross-linking pattern, colour, charge distribution (ζ-potential), particle size, thermal stability, morphology, solubility, foaming and emulsifying properties, and total amino acid content. The findings showed that cross-linking with laccase R significantly improved the protein solubility, emulsion stability and foaming ability of the mixture, whereas these functionalities were lower in the TG-treated mixture due to extensive cross-linking. Furthermore, the mixture treated with laccase T turned brownish in colour and showed a decrease in total amino acid content which could be due to the enzyme's oxidative cross-linking mechanism. Also, the occurrence of cross-linking in the lupin and whey mixture was indicated by changes in other investigated parameters such as particle size, ζ-potential, etc., as compared to the control samples. The obtained results suggested that enzymatic cross-linking, depending on the type of enzyme used, could impact the physicochemical and functional properties of hybrid plant- and animal-based proteins, potentially influencing their applications in food.

Structure of an RNA G-quadruplex from the West Nile virus genome.

Potential G-quadruplex sites have been identified in the genomes of DNA and RNA viruses and proposed as regulatory elements. The genus Orthoflavivirus contains arthropod-transmitted, positive-sense, single-stranded RNA viruses that cause significant human disease globally. Computational studies have identified multiple potential G-quadruplex sites that are conserved across members of this genus. Subsequent biophysical studies established that some G-quadruplexes predicted in Zika and tickborne encephalitis virus genomes can form and known quadruplex binders reduced viral yields from cells infected with these viruses. The susceptibility of RNA to degradation and the variability of loop regions have made structure determination challenging. Despite these difficulties, we report a high-resolution structure of the NS5-B quadruplex from the West Nile virus genome. Analysis reveals two stacked tetrads that are further stabilized by a stacked triad and transient noncanonical base pairing. This structure expands the landscape of solved RNA quadruplex structures and demonstrates the diversity and complexity of biological quadruplexes. We anticipate that the availability of this structure will assist in solving further viral RNA quadruplexes and provides a model for a conserved antiviral target in Orthoflavivirus genomes.

Storage Stability of Plant-Based Drinks Related to Proteolysis and Generation of Free Amino Acids.

The market for plant-based drinks (PBDs) is experiencing a surge in consumer demand, especially in Western societies. PBDs are a highly processed food product, and little is known about this relatively new food product category when compared to bovine milk. In the present study, the storage stability, proteolysis and generation of free amino acids were investigated in commercially available PBDs over the course of a one-year storage period. Generally, pH, color and protein solubility were found to be stable in the PBDs during storage, except for the pea-based product, which showed less protein solubility after storage. The pea-based drinks also had higher initial levels of free N-terminals prior to storage compared with levels for the other plant-based drinks, as well as significantly increasing levels of total free, and especially bitter free, amino acids. The development of free amino acids in the oat-based drink indicated that the released amino acids could be involved in various reactions such as the Maillard reaction during the storage period.

Metabolite profiling identifies chemical markers associated with the cytotoxic properties of roasted fermented avocado seeds.

Studies have demonstrated avocado seeds are a good source of bioactive compounds. This study investigated the effects of roasting on the metabolites and anticancer activities of fermented avocado seeds. All three anti-cancer activities of fermented avocado seeds were higher at lower roasting temperature and time. The best inhibition effect was found against Hep G2 followed by the MDA-MB-231 and MCF-7 cancer cell lines. Untargeted metabolite profiling using gas chromatography-mass spectrometry resulted in identification of 208 metabolites. In total, 41 metabolites identified had VIP values more than 1 using PLS-R that were related to anticancer activities. All amino acids and most sugars were higher at lower roasting temperature and positively correlated to anticancer activity. The roasting conditions for optimal antioxidant and anticancer activities were determined to be 121 °C for 9 min. Findings showed that fermented avocado seed powder has the potential to become a functional food ingredient with beneficial bioctive properties.

Development of high affinity broadly reactive aptamers for spike protein of multiple SARS-CoV-2 variants.

We have developed broadly reactive aptamers against multiple variants by alternating the target between spike proteins from different SARS-CoV-2 variants during the selection process. In this process we have developed aptamers which can recognise all variants, from the original wild-type 'Wuhan' strain to Omicron, with high affinity (Kd values in the pM range).

Seroepidemiology and Carriage of Diphtheria in Epidemic-Prone Area and Implications for Vaccination Policy, Vietnam.

In 2019, a community-based, cross-sectional carriage survey and a seroprevalence survey of 1,216 persons 1-55 years of age were conducted in rural Vietnam to investigate the mechanism of diphtheria outbreaks. Seroprevalence was further compared with that of an urban area that had no cases reported for the past decade. Carriage prevalence was 1.4%. The highest prevalence, 4.5%, was observed for children 1-5 years of age. Twenty-seven asymptomatic Coerynebacterium diphtheriae carriers were identified; 9 carriers had tox gene-bearing strains, and 3 had nontoxigenic tox gene-bearing strains. Child malnutrition was associated with low levels of diphtheria toxoid IgG, which might have subsequently increased child carriage prevalence. Different immunity patterns in the 2 populations suggested that the low immunity among children caused by low vaccination coverage increased transmission, resulting in symptomatic infections at school-going age, when vaccine-induced immunity waned most. A school-entry booster dose and improved infant vaccination coverage are recommended to control transmissions.

Enhancing the Biological Activities of Food Protein-Derived Peptides Using Non-Thermal Technologies: A Review.

Bioactive peptides (BPs) derived from animal and plant proteins are important food functional ingredients with many promising health-promoting properties. In the food industry, enzymatic hydrolysis is the most common technique employed for the liberation of BPs from proteins in which conventional heat treatment is used as pre-treatment to enhance hydrolytic action. In recent years, application of non-thermal food processing technologies such as ultrasound (US), high-pressure processing (HPP), and pulsed electric field (PEF) as pre-treatment methods has gained considerable research attention owing to the enhancement in yield and bioactivity of resulting peptides. This review provides an overview of bioactivities of peptides obtained from animal and plant proteins and an insight into the impact of US, HPP, and PEF as non-thermal treatment prior to enzymolysis on the generation of food-derived BPs and resulting bioactivities. US, HPP, and PEF were reported to improve antioxidant, angiotensin-converting enzyme (ACE)-inhibitory, antimicrobial, and antidiabetic properties of the food-derived BPs. The primary modes of action are due to conformational changes of food proteins caused by US, HPP, and PEF, improving the susceptibility of proteins to protease cleavage and subsequent proteolysis. However, the use of other non-thermal techniques such as cold plasma, radiofrequency electric field, dense phase carbon dioxide, and oscillating magnetic fields has not been examined in the generation of BPs from food proteins.

Evaluation of the Major Seed Storage Proteins, the Conglutins, Across Genetically Diverse Narrow-Leafed Lupin Varieties.

Lupin seeds have an excellent nutritional profile, including a high proportion of protein and dietary fiber. These qualities make lupin seeds an ideal candidate to help meet the growing global demand for complementary sources of protein. Of consequence to this application, there are nutritional and antinutritional properties assigned to the major lupin seed storage proteins-referred to as α-, ß-, δ- and γ-conglutins The variation in the abundance of these protein families can impact the nutritional and bioactive properties of different lupin varieties. Hence, exploring the conglutin protein profiles across a diverse range of lupin varieties will yield knowledge that can facilitate the selection of superior genotypes for food applications or lupin crop improvement. To support this knowledge generation, discovery proteomics was applied for the identification of the 16 known conglutin subfamilies from 46 domestic and wild narrow-leafed lupin (NLL) genotypes. Consequently, the diversity of abundance of these proteins was evaluated using liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS). This comparative study revealed a larger variability for the ß- and δ-conglutin content across the lines under study. The absence/lower abundance of the ß2- to ß6-conglutin subfamilies in a subset of the domesticated cultivars led to substantially lower overall levels of the allergenic ß-conglutin content in these NLLs, for which the elevation of the other conglutin families were observed. The diversity of the conglutin profiles revealed through this study-and the identification of potential hypoallergenic genotypes-will have great significance for lupin allergic consumers, food manufactures as well as grain breeders through the future development of lupin varieties with higher levels of desirable bioactive proteins and lower allergen content.

Evaluation of protein extraction methods for in-depth proteome analysis of narrow-leafed lupin (Lupinus angustifolius) seeds.

Lupin is slated as a potential contributor towards future food security. Lupin possesses several nutritional and nutraceutical attributes, many linked to seed proteins. For in-depth characterisation of the lupin proteome, liquid chromatography-tandem mass spectrometry was used to evaluate four protein extraction procedures. The proteomes of three narrow-leafed lupin were qualitatively evaluated using protein/peptide identifications and further quantitatively assessed by data-independent proteome measurement. Each extraction buffer led to unique protein identifications; altogether yielding 2,760 protein identifications from lupin varieties. The analysis of protein abundance data highlighted distinct differences between Tris-HCl and urea extracted proteomes, while also revealing variation amongst the cultivar proteomes with the wild accession (P27255) distinctly different from the domesticated cultivars (Tanjil, Unicrop). The extraction buffer used influenced the proteome coverage, downstream functional annotation results and consequently the biological interpretation demonstrating the need to optimise and understand the impact of protein extraction conditions.

Factors driving the compositional diversity of Apis mellifera bee venom from a Corymbia calophylla (marri) ecosystem, Southwestern Australia.

Bee venom (BV) is the most valuable product harvested from honeybees ($30 - $300 USD per gram) but marginally produced in apiculture. Though widely studied and used in alternative medicine, recent efforts in BV research have focused on its therapeutic and cosmetic applications, for the treatment of degenerative and infectious diseases. The protein and peptide composition of BV is integral to its bioactivity, yet little research has investigated the ecological factors influencing the qualitative and quantitative variations in the BV composition. Bee venom from Apis mellifera ligustica (Apidae), collected over one flowering season of Corymbia calophylla (Myrtaceae; marri) was characterized to test if the protein composition and amount of BV variation between sites is influenced by i) ecological factors (temperature, relative humidity, flowering index and stage, nectar production); ii) management (nutritional supply and movement of hives); and/or iii) behavioural factors. BV samples from 25 hives across a 200 km-latitudinal range in Southwestern Australia were collected using stimulatory devices. We studied the protein composition of BV by mass spectrometry, using a bottom-up proteomics approach. Peptide identification utilised sequence homology to the A. mellifera reference genome, assembling a BV peptide profile representative of 99 proteins, including a number of previously uncharacterised BV proteins. Among ecological factors, BV weight and protein diversity varied by temperature and marri flowering stage but not by index, this latter suggesting that inter and intra-year flowering index should be further explored to better appreciate this influence. Site influenced BV protein diversity and weight difference in two sites. Bee behavioural response to the stimulator device impacted both the protein profile and weight, whereas management factors did not. Continued research using a combination of proteomics, and bio-ecological approaches is recommended to further understand causes of BV variation in order to standardise and improve the harvest practice and product quality attributes.

Quantitative LC-MS/MS analysis of high-value milk proteins in Danish Holstein cows.

High-value milk proteins, which can be obtained by optimized fractionation procedures, are ideal ingredients in many food applications. Thus, a simple and robust analytical method is required for the identification and quantification of these individual milk proteins. Here, we present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) to simultaneously detect and measure target peptides of two major milk proteins, α-lactalbumin (α-LA) and ß-casein (ß-CN), in raw milk samples from 662 Danish Holstein cows. The MRM quantification of α-LA and ß-CN was achieved with limit of detection (LOD) of 0.14 and 0.16 g/L, respectively and reproducibility of the assay <15%. By this newly established MRM-based method, the concentration of α-LA and ß-CN in an individual cow's milk ranged from 0.5 to 1.9 (average 1.1) g/L, and from 7.5 to 23.4 (average 15) g/L, respectively. There was no significant effect of parity, whereas significantly increasing concentrations of α-LA and ß-CN were observed through lactation (P < 0.001). This shows a considerable biological variation of these two ingredient milk proteins, providing potential varying outputs of fractionation in the dairy streams.

Diphtheria Outbreaks in Schools in Central Highland Districts, Vietnam, 2015-2018.

During 2015-2018, seven schools in rural Vietnam experienced diphtheria outbreaks. Multilocus sequence types were the same within schools but differed between schools. Low vaccine coverage and crowded dormitories might have contributed to the outbreaks. Authorities should consider administering routine vaccinations and booster doses for students entering the school system.

A highly stable RNA aptamer probe for the retinoblastoma protein in live cells.

Although RNA aptamers can show comparable or better specificity and affinity to antibodies and have the advantage of being able to access different live cell compartments, they are often much less stable in vivo. We report here the first aptamer that binds human retinoblastoma protein (RB) and is stable in live cells. RB is both a key protein in cell cycle control and also a tumour suppressor. The aptamer was selected from an RNA library against a unique 12-residue helical peptide derived from RB rather than the whole protein molecule. It binds RB with high affinity (K d = 5.1 ± 0.1 nM) and is a putative RNA G-quadruplex structure formed by an 18-nucleotide sequence (18E16 - GGA GGG UGG AGG GAA GGG), which may account for its high stability. Confocal fluorescence microscopy of live cells transfected with the aptamer shows it is stable intracellularly and efficient in entering the nucleus where an analogous antibody was inaccessible. The findings demonstrate this aptamer is an advanced probe for RB in live cell applications.

Interplay between Residual Protease Activity in Commercial Lactases and the Subsequent Digestibility of ß-Casein in a Model System.

One of the conventional ways to produce lactose-hydrolyzed (LH) milk is via the addition of commercial lactases into heat-treated milk in which lactose is hydrolyzed throughout storage. This post-hydrolysis method can induce proteolysis in milk proteins due to protease impurities remaining in commercial lactase preparations. In this work, the interplay between lactose hydrolysis, proteolysis, and glycation was studied in a model system of purified ß-casein (ß-CN), lactose, and lactases using peptidomic methods. With a lactase presence, the proteolysis of ß-CN was found to be increased during storage. The protease side-activities mainly acted on the hydrophobic C-terminus of ß-CN at Ala, Pro, Ile, Phe, Leu, Lys, Gln, and Tyr positions, resulting in the formation of peptides, some of which were N-terminal glycated or potentially bitter. The proteolysis in ß-CN incubated with a lactase was shown to act as a kind of "pre-digestion", thus increasing the subsequent in vitro digestibility of ß-CN and drastically changing the peptide profiles of the in vitro digests. This model study provides a better understanding of how the residual proteases in commercial lactase preparations affect the quality and nutritional aspects of ß-CN itself and could be related to its behavior in LH milk.

Pharmacokinetic characterization of kalata B1 and related therapeutics built on the cyclotide scaffold.

Oral activity has been described for cyclotide-containing traditional medicines, and demonstrated for reengineered cyclotides bearing grafted therapeutic epitopes, highlighting their potential for translation to the clinic. Here we report preclinical pharmacokinetic parameters for the prototypic cyclotide kalata B1 (kB1) and two orally active grafted analogues, ckb-KAL and ckb-KIN, to provide the first in vivo dose-exposure metrics for cyclotides. Native and grafted kB1 molecules exhibited multiple compartment kinetics and measurable but limited oral bioavailability of similar magnitude to several orally administered peptide drugs in the clinic. Cyclotides are mostly associated with the central compartment, and display small (0.07-0.13 L kg-1 for kB1 and ckb-KIN) to moderate (1 L kg-1 for ckb-KAL) steady state volumes of distribution. This study provides new data essential to the evaluation of cyclotides as therapeutics, validating them as a viable drug design scaffold with tunable pharmacokinetic properties.

Digestibility of glycated milk proteins and the peptidomics of their in vitro digests.

BACKGROUND: Milk proteins are widely used in food production and are often glycated by reducing sugar. Although many studies have reported the digestibility of glycated milk protein, most have focused on measuring degree of hydrolysis (DH), showing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) image of digests. Detailed information on the changes in peptide composition of digests has seldom been revealed. Therefore, in addition to measuring the DH and showing the SGS-PAGE images of digests, we also analyzed the peptidomics in digests using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and Mascot database in this work to further reveal the influence of glycation on protein nutrition. RESULTS: Compared with ß-lactoglobulin and bovine serum albumin (BSA), DH of ß-casein was suppressed to a lesser extent by glycation in both gastric and intestinal stages. Aggregates of glycated BSA were less sensitive to the action of digestive enzymes throughout gastrointestinal digestion according to SDS-PAGE images. Changes in the peptide composition of digests induced by glycation were distinctly displayed, showing both absence of peptides and occurrence of new peptides, based on the results obtained from LC-ESI-MS/MS. CONCLUSIONS: Glycation can greatly change the peptide composition in digests of milk protein. The nutritional impact of the change in the peptide composition requires further investigation, and the impact of MRPs in unabsorbed digests on the gut flora should be an interesting field for further studies. © 2018 Society of Chemical Industry.

Development and validation of a simple LC-MS/MS method for the simultaneous quantitative determination of trimethylamine-N-oxide and branched chain amino acids in human serum.

Serum branched chain amino acids and trimethylamine-N-oxide are monitored as potential indicators of diabetes and cardiovascular health respectively. A rapid method for their simultaneous determination using liquid chromatography and tandem mass spectrometry is described here. Branched chain amino acids and trimethylamine-N-oxide were quantified based on their specific MS/MS fragments using a selected reaction monitoring approach. A number of columns were tested for their ability to separate the analytes. A C18-PFP column separated the analytes in just 4 minutes, and resulted in excellent peak shape and retention time repeatability, and was therefore chosen as the optimal column. A second column, the Intrada Amino Acid column, was chosen for comparison and validation experiments as it provided an orthogonal separation mechanism. The intra-day and inter-day precision and accuracy were less than 12% for trimethylamine-N-oxide and less than 6% for the branched chain amino acids. Recoveries, where serum was spiked with three different concentrations of the analytes, ranged from 97 to 113%. The LODs and LOQs for trimethylamine-N-oxide were 1 and 6 ng/mL, for leucine and isoleucine were 4 and 8 ng/mL, and for valine were 5 and 15 ng/mL, respectively. The C18-PFP column method was validated using the Intrada Amino Acid column method and percentage agreement for all four analytes was within 10%. Sample preparation was minimal, and use of labelled internal standards accounted for matrix effects. The method was successfully applied to human plasma samples. Graphical abstract ᅟ.

Digestibility of Bovine Serum Albumin and Peptidomics of the Digests: Effect of Glycation Derived from α-Dicarbonyl Compounds.

α-Dicarbonyl compounds, which are widely generated during sugar fragmentation and oil oxidation, are important precursors of advanced glycation end products (AGEs). In this study, the effect of glycation derived from glyoxal (GO), methylglyoxal (MGO) and diacetyl (DA) on the in vitro digestibility of bovine serum albumin (BSA) was investigated. Glycation from α-dicarbonyl compounds reduced digestibility of BSA in both gastric and intestinal stage of digestion according to measurement of degree of hydrolysis. Changes in peptide composition of digests induced by glycation were displayed, showing absence of peptides, occurrence of new peptides and formation of peptide-AGEs, based on the results obtained using liquid chromatography electron-spray-ionization tandem mass spectrometry (LC-ESI-MS/MS). Crosslinked glycation structures derived from DA largely reduced the sensitivity of glycated BSA towards digestive proteases based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results. Network structures were found to remain in the digests of glycated samples by transmission electron microscope (TEM), thus the impact of AGEs in unabsorbed digests on the gut flora should be an interest for further studies.

Effect of glycation derived from α-dicarbonyl compounds on the in vitro digestibility of ß-casein and ß-lactoglobulin: A model study with glyoxal, methylglyoxal and butanedione.

α-Dicarbonyl compounds, which are widely found in common consumed food, are one of the precursors of advanced glycation end products (AGEs). In this study, the effect of glycation derived from glyoxal (GO), methylglyoxal (MGO) or butanedione (BU) on the in vitro digestibility of ß-casein (ß-CN) and ß-lactoglobulin (ß-Lg) was investigated. Glycation from α-dicarbonyl compounds reduced the in vitro digestibility of studied proteins in both gastric and intestinal stage. In addition, glycation substantially altered the peptides released through gastric and gastrointestinal digestion, as detected by liquid chromatography electrospray-ionization tandem mass spectrometry (LC-ESI-MS/MS). Crosslinked glycation structures derived from BU considerably reduced the sensitivity of glycated ß-Lg towards digestive proteases, albeit to a lesser degree in glycated ß-CN due to its intrinsic unordered structure. By contrast, non-crosslinked AGEs that formed adjacent to enzymatic cleavage sites did not block the enzymatic reaction in several cases, as evidenced by the corresponding digested peptides modified with glycation structures. These findings expand our understanding of the nutritional influence of α-dicarbonyl compounds and health impact of relevant dietary AGEs.

Effect of Storage on Lactase-Treated ß-Casein and ß-Lactoglobulin with Respect to Bitter Peptide Formation and Subsequent in Vitro Digestibility.

Using active lactose to hydrolyze lactose during storage is a common process to produce lactose-hydrolyzed (LH) milk. Proteolysis induced by residual proteases in commercial lactase was studied in a system using purified ß-casein or ß-lactoglobulin during a 60-day storage period at 22 or 38 °C. The proteolysis of ß-casein by residual proteases occurred more extensively than that of ß-lactoglobulin. Peptidomic analysis by LC-ESI-MS/MS revealed that Ile, Leu, Tyr, and Phe residues near the C-terminus of ß-casein were the main sites of cleavage by the residual proteases, generating assumed bitter peptides. In the subsequent in vitro digestion study, proteolysis during storage was shown to greatly affect the subsequent digestibility of ß-casein, leading to an elevated degree of hydrolysis and the formation of new digested peptides. This study highlights the potential influence of residual proteases in commercial lactase on the storage quality and digestibility of LH milk containing active lactase.