High Molecular Weight Polypeptides Research Articles

Sorghum proteins: Effect of extraction pH on physicochemical properties and emulsion formation

Extraction methods suitable for food applications should be studied to obtain new plant protein isolates. This study aimed to evaluate the effect of pH (8.0, 10.0, and 12.0) and temperature (20 and 40 °C) during alkaline extraction on the yield and functional and physicochemical characteristics of sorghum protein isolates and to test their ability to stabilize emulsions. The best extraction yield (58.52 %) was at pH 12.0 and 40 °C. The obtained isolates had protein concentration superior to 4-fold as compared to the raw material. Electrophoresis showed mainly prolamins, with some globulin, glutelin, and higher molecular weight polypeptides (>250 kDa). Sorghum protein isolate obtained at pH 12.0 had improved solubility (64.77 %) compared to the isolates obtained at pH 8.0 (14.50 %) and pH 10.0 (17.68 %). Emulsions with 70 % (w/w) of oil showed no phase separation during storage, and those produced with the isolate obtained at pH 12.0 were more stable, with smaller droplet sizes after the 7-day storage (25.17–44.77 μm), lower creaming index, higher emulsion stability index (ESI), and less flocculation. The study demonstrated the functional ability of sorghum protein isolates obtained by alkaline extraction to form concentrated oil-in-water emulsions, avoiding using organic solvents.

Insights into the growth parameters, mineral composition, protein and UHPLC–QTOF–LC/MS‐based non‐targeted metabolomic fingerprints of wheatgrass as a function of photoperiod and temperature

SummaryWheatgrass juice powder (WJP) obtained from four Indian wheat varieties grown under different photoperiod and temperature conditions were examined for growth parameters, chlorophyll content, mineral composition, protein and metabolomic profile. The percentage yield and total chlorophyll content of WJP varied from 2.5% to 9.12% and 1.08 to 4.76 mg g−1, respectively. The WJP obtained from PBW‐752 showed the highest yield and total chlorophyll content. The mineral composition (Na, K, Ca and Mg) of WJP also showed variation with different photoperiod and temperature conditions. The electrophoretic pattern of total soluble proteins of WJP showed variability in the accumulation of high and low molecular weight polypeptides against different photoperiod and temperature conditions. The comprehensive metabolites profile of WJP was analysed by UHPLC–QTOF–liquid chromatography/mass spectrometry. Based on the retention times and m/z values, 1012 feature peaks in positive ionisation mode were detected. The metabolomic profile of WJP revealed 180 different classes of primary and secondary metabolites.

Suitability of high-molecular-weight tissue-derived elastin polypeptides and their particles as cosmetic biomaterials.

We aimed to determine the coacervation properties of high-molecular-weight (HMW) tissue-derived elastin (TDE) and to examine the potential use of TDE particles as a cosmetic biomaterial. TDE solutions were filtered and divided into three fractions (1-3) according to the molecular weight of the elastin. The turbidity of fraction 2, which contained a large portion (58%) of HMW elastin polypeptides (>100 kDa), was measured under several pH values (3.0-11.0) and NaCl concentrations (0-1000 mM) to examine its coacervation ability. HMW TDE exhibited coacervation under the physiological conditions (temperature, pH, and NaCl concentration) of the skin surface. We performed inclusion and release experiments using three model chemicals with different molecular weights and measured the size and zeta potential of the fraction 3 particles to investigate the suitability of HMW elastin polypeptides. Fraction 3, which contained a larger portion (64%) of HMW elastin polypeptides, displayed a strong coacervation property at a phase transition temperature of 19.8 ± 0.1°C. The inclusion ratio of the model chemical Biebrich Scarlet (BS) with a molecular weight of <600 was approximately 92.1 ± 0.7%. The release profiles of BS from the particles linearly increased and reached a plateau after 15 days. Moreover, the average size of the particles with BS was 474.2 ± 24.6 nm. The low-molecular-weight (LMW) elastin peptides have moisturizing and whitening functions for the skin. We concluded that TDE, as a mixture of HMW polypeptides and LMW peptides, can potentially serve as a multifunctional and effective cosmetic biomaterial.

Metabolism Reactivation in Erythrocytes Cryopreserved with Polyethylene Glycol: Protein State and Reactive Oxygen Species Production

Production of reactive oxygen species (ROS) and protein state of membrane-cytoskeleton complex (MCC) in the erythrocytes, subjected to freeze-thawing with polyethylene glycol (PEG) and subsequently transferred into physiological conditions in vitro after cryoprotective agent removal were examined. Results of SDS-PAGE with β-mercaptoethanol showed a decrease in spectrin content in the MCC profile of cryopreserved erythrocytes. The using of diamide revealed an increase in the accessibility of –SH groups of spectrin for the reagent and an enhanced level of high molecular weight polypeptide complexes relative to the control. A decrease in the content of protein band 8 (peroxiredoxin) in the MCC of cryopreserved erythrocytes revealed by SDS-PAGE with β-mercaptoethanol is apparently associated with a change in its localization caused by an increased ROS production at the previous stages of cryopreservation. At the same time, the absence of significant changes in the ROS production relative to the control upon the metabolism reactivation in erythrocytes indicated the ability of cryopreserved cells to control the oxidative processes during short-term incubation.

Bifunctional Fluoroalcohol Catalysts Enabled Sustainable Synthesis of Poly(amino acid)s†

Main observation and conclusionA series of novel bifunctional single‐molecule hydrogen‐bonding organocatalysts based on fluorinated tertiary alcohol were synthesized to mediate the metal‐free polymerization of α‐amino acid N‐carboxyanhydride (NCA) monomer. Through the screening of catalysts, the polymerization catalyzed by the preferred bifunctional fluoroalcohol catalyst proceeded in a fast and controlled manner, affording high molecular weight polypeptide up to 35.2 × 104 Da with relatively narrow molecular weight distribution (Đ &lt; 1.20). A phenomenon of self‐accelerating effect of polymerization induced by α‐helical structure of polypeptides was observed, and a possible polymerization pathway has been discussed.

Modifications of proteins of membrane-cytoskeleton complex and production of reactive oxygen species in erythrocytes cryopreserved with polyethylene glycol

Protein modifications in the membrane-cytoskeleton complex (MCC) of human erythrocytes, as well as changes in the intensity of reactive oxygen species (ROS) production upon cell cryopreservation with polyethylene glycol (PEG) were investigated. The protein profile of ghosts of erythrocytes frozen with PEG has common features with both the control and cells frozen without cryoprotectant. PEG makes it possible to restrict the structural rearrangements of the main MCC proteins under the effect of extreme factors and to restrain the amount of high molecular weight polypeptide complexes induced by the protein-cross-linking reagent diamide at the control level, in contrast to cells frozen without a cryoprotectant. However, changes related to the protein peroxiredoxin 2 in ghosts of erythrocytes cryopreserved with PEG are also attributed to cells frozen without a cryoprotectant that may be associated with the activation of oxidative processes. This is evidenced by a 10-fold increase in ROS formation in erythrocytes frozen under PEG protection. Thus, upon cryopreservation of erythrocytes with PEG, certain disorders in MCC proteins may be associated with increased formation of ROS, which may contribute to the disorganization of the structural components of MCC and disrupt the stability of cryopreserved cells under physiological conditions.

Influence of high molecular weight polypeptides on the mouthfeel of commercial beer

Low malt beers have high sales volumes in Japan, but improving their mouthfeel, including softness, smoothness and decreasing astringency, is challenging because the compounds responsible remain unclear. In this study, beer was fractionated by preparative size-exclusion chromatography, with the polypeptide and maltodextrin fractions purified using solid-phase extraction and ion-exchange resin. Sensory data from a spike test showed that the mouthfeel (softness, smoothness, and reduced astringency) of low malt beer was improved both by the degree of polymerisation (DP) of maltodextrins (DP of 2-10; at increased concentration of 40 to 60%; P < 0.01) and by 10 - 20 kilodalton (kDa) high molecular weight (HMW) polypeptide and 2-3 kDa low molecular weight polypeptide fractions (at a 50% increase in concentration; P < 0.01). Furthermore, highly purified 10 to 20 kDa HMW polypeptides improved the softness and smoothness and decreased the astringency (at a 25% increase in concentration). This report is the first to provide experimental sensory data indicating that HMW polypeptides improve the mouthfeel of beer. Based on these findings, a new low malt beer was developed that showed significantly higher levels of the 10-20 kDa HMW polypeptides with an overall improved mouthfeel. Mass spectrometric analysis of the 10 to 20 kDa proteins identified several unique foam positive proteins, including barley dimeric alpha-amylase inhibitor-1 and non-specific lipid-transfer protein 1. These 10-20 kDa HMW proteins are likely to be responsible for the improved mouthfeel of beer. © 2020 Kirin Holdings Kabushik Kaisha Co. Ltd. Journal of the Institute of Brewing published by John Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling

IgE, IgG1, and IgG4 Reactivity to Dermatophagoides pteronyssinus Glycosylated Extract in Allergic Patients.

Background House dust mites are important allergen sources and some of these allergenic proteins may contain carbohydrate moieties, which are able to be isolated using lectins, as Concanavalin A (ConA). This study aimed to investigate allergenicity (IgE) and antigenicity (IgG1 and IgG4) of ConA-unbound and ConA-bound Dermatophagoides pteronyssinus (Dpt) crude extracts using sera of mite-allergic patients as well as inhibition capacity of antibody binding. Material and Methods We obtained mannose-enriched and mannose-depleted fractions from Dpt by ConA affinity chromatography. Both ConA-bound and ConA-unbound fractions were evaluated by ELISA and Western Blotting for specific IgE, IgG1, and IgG4 reactivity with sera obtained from 95 mite-allergic patients (DP+) and 92 nonallergic (NA) subjects. Inhibition ELISA was used to assess cross-reactivity between Dpt extract and its fractions. Results Among the DP+ patients, no difference was found between ConA-unbound and ConA-bound fractions regarding the levels of specific IgE, IgG1, and IgG4. Nonallergic subjects had the same levels of specific IgG1 to both ConA-unbound and ConA-bound fractions, although for specific IgG4, values were higher for ConA-bound. A positive correlation was found among specific IgE, IgG1, and IgG4 levels when Dpt was compared to ConA-unbound and ConA-bound fractions. Recognition of crude Dpt by IgE, IgG1, and IgG4 was highly inhibited by ConA-unbound and ConA-bound fractions. Western Blotting revealed a broad spectrum of bands ranging from 14 to 116 kDa recognized by specific IgE and IgG4. However, IgG1 reached higher frequency values on high molecular weight polypeptides. Conclusion ConA-unbound and ConA-bound fractions derived from D. pteronyssinus crude extract revealed important components involved in the IgE recognition in allergic patients as well as IgG1 and/or IgG4 in allergic and healthy subjects.

Physicochemical and Biological Examination of Two Glatiramer Acetate Products.

Herein we compared 40 mg/mL lots of the active ingredient, glatiramer acetate, manufactured by Mylan/Natco to the active ingredient, glatiramer acetate in Copaxone (Teva Pharmaceuticals, Ltd., Netanya Israel) using physicochemical (PCC) methods and biological assays. No differences were seen between the Mylan/Natco and Teva lots with some low resolution release PCC assays (amino acid analysis, molecular weight distribution, interaction with Coomassie Brilliant Blue G-250). Changes in polydispersity between Mylan/Natco and Copaxone lots were found using size exclusion chromatography and the high resolution PCC method, known as Viscotek, and suggestive of a disparity in the homogeneity of mixture, with a shift towards high molecular weight polypeptides. Using RPLC-2D MALLS, 5 out of 8 Mylan/Natco lots fell outside the Copaxone range, containing a high molecular weight and high hydrophobicity subpopulation of polypeptides not found in Copaxone lots. Cation exchange chromatography showed differences in the surface charge distribution between the Copaxone and Mylan/Natco lots. The Mylan/Natco lots were found to be within Copaxone specifications for the EAE model, monoclonal and polyclonal binding assays and the in vitro cytotoxicity assay, however higher IL-2 secretion was shown for three Mylan/Natco lots in a potency assay. These observations provide data to inform the ongoing scientific discussion about the comparability of glatiramer acetate in Copaxone and follow-on products.

Synthesis of polypeptides via bioinspired polymerization of in situ purified N-carboxyanhydrides

Ribozymes synthesize proteins in a highly regulated local environment to minimize side reactions caused by various competing species. In contrast, it is challenging to prepare synthetic polypeptides from the polymerization of N-carboxyanhydrides (NCAs) in the presence of water and impurities, which induce monomer degradations and chain terminations, respectively. Inspired by natural protein synthesis, we herein report the preparation of well-defined polypeptides in the presence of competing species, by using a water/dichloromethane biphasic system with macroinitiators anchored at the interface. The impurities are extracted into the aqueous phase in situ, and the localized macroinitiators allow for NCA polymerization at a rate which outpaces water-induced side reactions. Our polymerization strategy streamlines the process from amino acids toward high molecular weight polypeptides with low dispersity by circumventing the tedious NCA purification and the demands for air-free conditions, enabling low-cost, large-scale production of polypeptides that has potential to change the paradigm of polypeptide-based biomaterials.

Alcalase-assisted production of fish skin gelatin rich in high molecular weight (HMW) polypeptide chains and their characterization for film forming capacity

Gelatin rich in high molecular weight (HMW) polypeptide chains exhibit superior film forming capacity. Alcalase enzyme was used to produce gelatin rich in HMW polypeptide chains from shark (spiny dogfish) skin. The optimum conditions were established as 1.15 U/g alcalase pretreatment for 2.1 h followed by water extraction at 50.4 °C for 4.6 h and produced gelatin with 33% HMW polypeptide chains of total protein. There was significant interaction (p < 0.1) between alcalase treatment and water extraction to control the HMW polypeptide chain content in gelatin. Optimized gelatin had 31.6% of glycine, 10% of proline and 7.9% of hydroxyproline. Gelatin rich in HMW polypeptide chains had strong capacity to form films from solutions with low gelatin concentrations (0.5–4%, w/v). Films formed by 3% gelatin solution (GF3) had low opacity and showed no difference with films formed by 4% gelatin solution (GF4), in terms of water solubility, water vapor permeability, as well as tensile strength and elongation at break (p > 0.05). FTIR spectrum of the GF3 film disclosed five characteristic amide bands, and the TGA trace showed GF3 film was stable up to 225 °C. Microstructure of GF3 using SEM displayed a smooth and compact film network.

A new temperature-dependent strategy to modulate the epidermal growth factor receptor

The dynamic manipulation of kinases remains a major obstacle to unraveling cell-signaling networks responsible for the activation of biological systems. For example, epidermal growth factor (EGF) stimulates the epidermal growth factor receptor (EGFR/ErbB1); however, EGF also recruits other kinases (HER2/ErbB2) involved with various signaling pathways. To better study EGFR we report a new strategy to selectively activate receptor tyrosine kinases fused to elastin-like polypeptides (ELPs), which can be visualized inside mammalian cells using fixed and live-cell fluorescence microscopy. ELPs are high molecular weight polypeptides that phase separate abruptly upon heating. When an EGFR-ELP fusion is heated, it clusters, initiates receptor internalization, phosphorylates, initiates downstream kinase signaling, and undergoes retrograde transport towards the cell body. Unlike other strategies to block EGFR (small molecule inhibitors, RNAi, or transcriptional regulators), EGFR-ELP clustering can be specifically switched on or off within minutes. Live-cell imaging suggests that EGFR-ELPs assemble in most cells with only a 3 °C increase in temperature. This strategy was found reversible and able to dynamically control the downstream phosphorylation/activation of the ERK1/2 pathway. For the first time, this strategy enables the rational engineering of specific temperature-sensitive receptors that may have broad applications in the study and manipulation of biological processes.

Plastein reaction enhanced bile-acid binding capacity of soybean protein hydrolysates and whey protein hydrolysates

Plastein reaction is a modification reaction that can improve the functional properties of protein hydrolysate. The product of the reaction is a thixotropic aggregation of peptides. This study investigated the formation condition of soybean-whey plastein and bile acid binding capacity of plastein. Soy protein and whey protein were hydrolyzed by pepsin. The mixture (1:1, w/w) of two hydrolysates was modified by pepsin again. After the reaction, the decrease in free amino groups and the turbidity of the modified hydrolysate were measured to obtain appropriate reaction condition. Results showed that the concentration of hydrolysates 40% (w/v), enzyme ratio of 2.0KU/g protein, pH 5.0, 37°C, reaction time of 3.0h respectively, were showed maximum changes in protein hydrolysates. Tricine SDS-PAGE analysis under denaturing conditions revealed that whey protein was more sensitive to pepsin and yielded different polypeptides (PPs) of molecular weight ranged from 3.5-17 kDa. However, a high molecular weight PP was completely hydrolyzed while PPs of 14.2-26 kDa were partially digested after pepsin treatment. Native page analysis further revealed the presence of a high-molecular weight PP in crude and purified plastein product. The bile acid binding capacity was improved by the plastein reaction. The amount of binding sodium deoxycholate, sodium taurocholate, and sodium cholate were 0.75, 2.0 and 1.87μmol/100mg respectively.

Improved collagen extraction from jellyfish (Acromitus hardenbergi) with increased physical-induced solubilization processes

Efficiency and effectiveness of collagen extraction process contribute to huge impacts to the quality, supply and cost of the collagen produced. Jellyfish is a potential sustainable source of collagen where their applications are not limited by religious constraints and threats of transmittable diseases. The present study compared the extraction yield, physico-chemical properties and toxicology in vitro of collagens obtained by the conventional acid-assisted and pepsin-assisted extraction to an improved physical-aided extraction process. By increasing physical intervention, the production yield increased significantly compared to the conventional extraction processes (p < .05). Collagen extracted using the improved process was found to possess similar proximate and amino acids composition to those extracted using pepsin (p > .05) while retaining high molecular weight distributions and polypeptide profiles similar to those extracted using only acid. Moreover, they exhibited better appearance, instrumental colour and were found to be non-toxic in vitro and free of heavy metal contamination.

Influence of extraction pH on the foaming, emulsification, oil-binding and visco-elastic properties of marama protein.

Marama bean protein, as extracted previously at pH 8, forms a viscous, adhesive and extensible dough. To obtain a protein isolate with optimum functional properties, protein extraction under slightly acidic conditions (pH 6) was investigated. Two-dimensional electrophoresis showed that pH 6 extracted marama protein lacked some basic 11S legumin polypeptides, present in pH 8 extracted protein. However, it additionally contained acidic high molecular weight polypeptides (∼180 kDa), which were disulfide crosslinked into larger proteins. pH 6 extracted marama proteins had similar emulsification properties to soy protein isolate and several times higher foaming capacity than pH 8 extracted protein, egg white and soy protein isolate. pH 6 extracted protein dough was more elastic than pH 8 extracted protein, approaching the elasticity of wheat gluten. Marama protein extracted at pH 6 has excellent food-type functional properties, probably because it lacks some 11S polypeptides but has additional high molecular weight proteins. © 2017 Society of Chemical Industry.

Fabrication, Physicochemical Characterization, and Performance Evaluation of Biodegradable Polymeric Microneedle Patch System for Enhanced Transcutaneous Flux of High Molecular Weight Therapeutics.

A revolutionary paradigm shift is being observed currently, towards the use of therapeutic biologics for disease management. The present research was focused on designing an efficient dosage form for transdermal delivery of α-choriogonadotropin (high molecular weight biologic), through biodegradable polymeric microneedles. Polyvinylpyrrolidone-based biodegradable microneedle arrays loaded with high molecular weight polypeptide, α-choriogonadotropin, were fabricated for its systemic delivery via transdermal route. Varied process and formulation parameters were optimized for fabricating microneedle array, which in turn was expected to temporally rupture the stratum corneum layer of the skin, acting as a major barrier to drug delivery through transdermal route. The developed polymeric microneedles were optimized on the basis of quality attributes like mechanical strength, axial strength, insertion ratio, and insertion force analysis. The optimized polymeric microneedle arrays were characterized for in vitro drug release studies, ex vivo drug permeation studies, skin resealing studies, and in vivo pharmacokinetic studies. Results depicted that fabricated polymeric microneedle arrays with mechanical strength of above 5N and good insertion ratio exhibited similar systemic bioavailability of α-choriogonadotropin in comparison to marketed subcutaneous injection formulation of α-choriogonadotropin. Thus, it was ultimately concluded that the designed drug delivery system can serve as an efficient tool for systemic delivery of therapeutic biologics, with an added benefit of overcoming the limitations of parenteral delivery, achieving better patient acceptability and compliance.

Tuning the conformation of synthetic co-polypeptides of serine and glutamic acid through control over polymer composition

Ring opening polymerization (ROP) of N-carboxy anhydride (NCA) amino acids presents a rapid way to synthesize high molecular weight polypeptides with different amino acid compositions. The compositional and functional versatility of polypeptides make these materials an attractive choice for biomaterials. The functional performance of polypeptide materials is equally linked to their conformation which is determined by the amino acid sequence in the polymer chains. Here, the interplay between composition and conformation of synthetic polypeptides obtained by NCA polymerization was explored. Various copolypeptides from Glu(Bzl) and Ser(Bzl) were prepared to investigate how polypeptide composition affected the conformation of the resulting copolymer. Polymerization kinetics indicated that the copolymerization of Glu(Bzl) and Ser(Bzl) preferentially yielded alternating copolymers. Both the polydispersity and the conformation of the polypeptides were dependent on the Ser(Bzl) content in the polymer, demonstrating that polypeptide functionalities could be tuned directly by altering the relative amounts of amino acids in the chain. This work presents the first step toward an improved understanding and control over polypeptide conformation through modulating the amino acid composition of the material. Understanding this sequence–functionality relationship is essential to advancing the use of ROP as a technique to design smart polypeptide based materials with specific functions. © 2016 The Authors. Journal of Polymer Science Part A: Polymer Chemistry Published by Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016, 54, 2331–2336

Cross antigenicity of immunodominant polypeptides of somatic antigen of Oesophagostomum columbianum with other helminths by western blotting.

Aim:Oesophagostomum columbianum in small ruminants in India is found as mixed infection commonly in sheep and goat. Haemonchus contortus, an abomasal nematode is found as concurrent infection with it. Eggs of Haemonchus and O. columbianum cannot be easily distinguished. Diagnosis of O. columbianum may only be possible if a non-cross antigenic polypeptide was available for immunodiagnosis.Materials and Methods:Somatic antigen (SoAg) of O. columbianum was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodominant polypeptides were identified by western blotting with homologous hyperimmune serum (HIS) and experimental sera of sheep or goat infected with other helminths.Results:SoAg of O. columbianum was immunoaffinity purified. Sharp polypeptide bands of 130, 72 and 68 KDa were observed along with several faint bands of lower molecular weight. Western blot of purified SoAg of O. columbianum with homologous HIS showed reaction with all the protein bands of 17, 28, 30, 32, 35, 38, 50, 68, 100, 130, 150, and 170 kDa. For identification of non-cross antigenic polypeptide, immunoaffinity purified SoAg of O. columbianum was reacted to heterologous HIS against H. contortus, Paramphistomum epiclitum, and Fasciola gigantica in western blotting utilizing completely dry method (i-blot). Among high molecular weight polypeptides 100 and 150 kDa were non-cross antigenic and among low molecular weight except 50 kDa polypeptide, 17, 30, 32, 35, and 38 kDa of O. columbianum were not cross antigenic with other helminths.Conclusions:Hence, polypeptides of 17, 30, 32, 35 and 38 kDa as well as 100 and 150 kDa polypeptides of O. columbianum may be exploited for immunodiagnosis of the infection in sheep and goat with extensive studies on cross antigenicity.

Characterization and cloning of an 11S globulin with hemagglutination activity from Murraya paniculata.

A ~56 kDa protein having hemagglutination activity was purified and characterized from the Murraya paniculata seeds. The gel electrophoresis studies demonstrated that protein is primarily of two different subunits, molecular weight ~ 35 and 21 kDa held together by disulfide-linkages and predominantly by secondary forces. The cloning and sequence analysis revealed that the protein exhibited a substantial sequence identity to seed storage 11S globulin family proteins. The sequence analysis of Murraya paniculata globulin (MPG) demonstrated higher and lower molecular weight polypeptides to be acidic (α) and basic (β) respectively. The sequence analysis further showed that it possesses a characteristic bi-cupin motif and a putative metal binding pocket. CD analysis revealed that the MPG was a β/α protein with a slightly higher content of the former. Conformational changes in protein have been studied by fluorescence spectrometry by using various chemical treatments. The results demonstrated that MPG belongs to 11S globulin family and exhibit's hemagglutination activity, which implicates it to be possessing lectin-like property.

Effect of high pressure processing on the immunoreactivity of almond milk

Influence of high pressure processing (HPP) at 450 and 600MPa, 30°C for various holding times (0, 30, 60,180, 300 and 600s) on almond milk amandin was investigated. The immunoreactivity of pressure treated almond milk was compared with raw and thermally processed (TP) almond milk (72, 85 and 99°C for 0 to 300s) using a sandwich enzyme-linked immunosorbent assay (ELISA), Western blot and dot blot. Monoclonal antibodies (mAbs) targeting linear (4F10) and conformational (4C10) epitopes on amandin were used to assess amandin immunoreactivity. To determine the aggregation of almond proteins, almond milk protein solubility was quantified after 300s of HPP (up to 600MPa, 30°C) and TP (at 72, 85 and 99°C, 0.1MPa). After HPP (for all holding times), amandin can no longer be detected by the anti-conformational mAb in ELISA while signal generated from the anti-linear epitopes mAb was reduced by half (P<0.05). On the other hand, most TP samples did not show significant reductions in immunoreactivity (P>0.05) unless processed at 85 and 99°C for 300s. Western blot and dot blot also confirmed the loss of immunoreactivity by both antibodies for HPP almond milk. The reduced band intensity of the 61 and 63kDa polypeptides and concomitant appearance of high molecular weight polypeptides in Western blot indicated that the observed decrease in immunoreactivity was partly due to the aggregation of amandin. The tested HPP and TP treatments respectively caused a maximum of ~70% and ~75% reduction in protein solubility. The study demonstrated that the loss of protein solubility, rather than the epitope destruction, may be responsible for the observed decrease in amandin immunoreactivity.