Protease Pretreatment Research Articles

EFFECT OF PROTEASE PRETREATMENT ON THE FUNCTIONAL PROPERTIES OF PROTEIN CONCENTRATE FROM DEFATTED PEANUT FLOUR

ABSTRACTPeanut protein concentrate (PPC) was extracted from commercial defatted peanut flour (DPF) after protease pretreatment. The effect of proteolysis by Alcalase 2.4 L, papain and Protamex at various degrees of hydrolysis (DHs; 1–8%) on the yield and functional properties of PPC were investigated. Proteolysis remarkably increased the nitrogen solubility of DPF in acidic and basic conditions. Enzymatic degradation of denatured and insoluble proteins in DPF was mainly responsible for the increase of nitrogen solubility index. Compared with the control, the yield of PPC was significantly increased by enzymatic pretreatment. In terms of the functional properties, PPCs prepared by papain and Protamex at 4% or 8% DH had high emulsifying activity index. For all three enzymatic treatments, foaming capacity and foaming stability were significantly improved at various DH (P < 0.05). This study demonstrated that protease pretreatment was a highly effective way to extract PPC with good functional properties from DPF.PRACTICAL APPLICATIONSPeanut is an important oilseed crop and a well‐accepted food. After oil production through thermal treatment, the defatted peanut flour is the main by‐product, which possesses large amount of proteins. However, due to the low extraction yield and poor functional properties of these proteins, they are not well utilised in industry till now. In this work, protease was used to pretreat defatted peanut flour. The results indicated that this pretreatment could improve the yield and functional properties of peanut proteins. Functional properties of food proteins are important in food processing and food product formulation. Peanut protein with good emulsifying and foaming properties would have a potential application in some food system, such as salad dressing, sausages, bologna, soups, confectionery, frozen desserts and cakes and so on. Therefore, this work is helpful for extensive utilisation of peanut proteins.

Effect of Acetic Acid and Commercial Protease Pretreatment on Salting and Characteristics of Salted Duck Egg

Salting of duck egg pretreated with 5% acetic acid and different commercial proteases (flavourzyme, protamex, alcalase, and neutrase) was studied. After 2 weeks of salting, duck eggs soaked in 5% acetic acid for 30 min, followed by soaking in 5% (w/v) flavourzyme and neutrase had the highest hardening ratio (90.14 ± 2.43%, 90.25 ± 1.23%) with the coincidental increase in salt content in egg white and decrease in moisture content of yolk, compared with those from other treatments (p 0.05). After 1 week of salting, protamex showed the similar effect on hardening ratio to other proteases (p > 0.05). When eggs were pretreated with neutrase at different concentrations (0.25%, 0.5%, and 0.75%, w/v) for different times (30, 60, and 90 min), those pretreated with 0.25% (w/v) neutrase for 90 min had the shorter salting time, while soaking time did not have the impact on hardening ratio for egg treated with 0.5% and 0.75% neutrase (P > 0.05). The oil exudation of egg treated with 0.25% neutrase had the higher oil exudates than the control at week 2 and 3 of salting (P 0.05). Microstructure study revealed that shell of salted egg pretreated with acetic acid had rough and porous surfaces when compared with control, whereas no changes in microstructure and FTIR spectra of shell membrane were found. Therefore, pretreatment of egg with 5% acetic acid for 30 min, followed by 0.25% neutrase for 90 min prior to salting could expedite the salting process and yielded egg yolk with hardening ratio and oil exudates, comparable to the traditionally salted egg.

Prognostic significance of circumferential cell surface immunoreactivity of glypican‐3 in hepatocellular carcinoma

GC33 is a recently developed monoclonal antibody against human glypican-3 (GPC3), which is significantly upregulated in hepatocellular carcinoma (HCC). GC33 recognizes a GPC3 ectodomain and shows significant antitumour activity in vivo. Thus, humanized GC33 antibody may be a promising tool for treating HCC having cell surface GPC3 expression. This study aims to determine the specificity, subcellular localization and prognostic impact of GPC3 immunoreactivity detected by GC33 in HCC clinical specimens. Immunohistochemical analysis was performed for 194 cases of resected HCC and prognostic analysis was performed for 185 eligible cases. Two antigen retrieval methods (autoclave and protease pretreatments) were used for immunohistochemistry and compared. The immunoscore system reflecting circumferential membranous GPC3 immunoreactivity was developed using either the autoclave or protease methods. The GPC3 mRNA level was analysed by quantitative real-time reverse transcription-polymerase chain reaction. GC33 immunostaining after autoclave is a sensitive method and revealed the GPC3 expression (≥20% of tumour cells) in the majority (77%) of HCC samples tested. Alternatively, protease pretreatment showed lower sensitivity, but was superior for evaluating the intensity and subcellular localization of GPC3. Correlation between immunoscores and the GPC3 mRNA level was also confirmed. Subsequent clinicopathological analysis revealed worse prognoses in HCC patients with circumferential membranous GPC3 immunoreactivity. For HCC patients with hepatitis C virus (HCV) infection in particular, the high membranous GPC3 immunoreactivity was an independent prognostic factor for disease-free survival. Circumferential membranous GPC3 immunoreactivity in HCC indicates poorer prognosis particularly in patients with HCV infection.

Extraction and dissolution of starch from rice and sorghum grains for accurate structural analysis

A complete dissolution without degradation and that removes non-starch components is important for accurate characterisation of starch molecular structure. Current milling and dissolution methods have limitations including incomplete dissolution and molecular degradation. An improved multi-step extraction/dissolution method was devised and tested, involving cryo-grinding, protease pre-treatment, dissolution in dimethyl sulfoxide solution containing 0.5% (w/w) LiBr (DMSO/LiBr) at 80 °C, centrifugation, ethanol precipitation, and, finally, re-dissolution in DMSO/LiBr at 80 °C. Cryo-grinding and dissolution in DMSO/LiBr were found to have negligible effects on the size distribution of rice starch molecules measured by size-exclusion chromatography (SEC). The peaks of non-starch components were removed or separated from the amylose and amylopectin peaks in the size distributions of rice and sorghum starches. The amylopectin component had a larger hydrodynamic radius than that obtained by conventional wet-milling and also that obtained without protease pre-treatment, suggesting that molecular degradation and aggregation were reduced with the new method. This new extraction/dissolution method allows a more accurate structural analysis of starch molecules from grains than conventional treatments.

Association of Lactobacillus acidophilus with mice Peyer's patches

Objective To clarify the adhesion mechanism of Lactobacillus acidophilus to Peyer's patches. Methods Adhesion of L. acidophilus FN001 to mice Peyer's patches was studied in vitro using a fluorescent quantization method. The nature of adhesion mediator was studied by the differing effects of physical, chemical, and enzymatic pre-treatments of the bacteria and the inhibitory effects of sugars on the adhesion. The presence of lectin-like proteins on the cell surface was determined by hemagglutination assay. The effect of L. acidophilus FN001 on the inhibition of adhesion of pathogens to Peyer's patches was also studied. Results The adhesion of L. acidophilus FN001 was strongly inhibited in the presence of D-mannose and methyl-α-D-mannoside. Pretreatment of L. acidophilus FN001 with pepsin and trypsin decreased the adhesive capacity indicating that some cell surface proteins might be involved in the adhesion. L. acidophilus FN001 showed agglutinating activity toward the rabbit red cells in a mannose specific manner, which was decreased after protease pretreatment, suggesting possible occurrence of mannose specific lectin(s) on the L. acidophilus FN001 surface. In adhesion inhibition assay, L. acidophilus NF001, when applied to Peyer's patches first or at the same time with pathogen, significantly inhibited adhesion of Escherichia coli ATCC25922 to Peyer's patches. Conclusion L. acidophilus FN001 contains some mannose-specific protein(s) on its surface that mediates its adhesion to the Peyer's patches. FN001 inhibits the adhesion of E. coli, which also contains mannose specific lectin.

Role of Different Pre-Treatments on Composition and Rheology of Synovial Fluids

Different pre-treatments applied to synovial fluids (SF) before their analyses are tested to characterize SF after storage under different conditions and to investigate their evolution along a viscosupplementation treatment. The main techniques proposed involve steric exclusion chromatography with triple detection (SEC) and viscometry; it is the first time that such a study is developed. SEC gives the molecular weight distribution and concentration of hyaluronan (HA) and proteins separately; the steady state viscosity is always non-Newtonian and not directly related to SF composition. Pre-treatment of SF (storage in cold, filtration, centrifugation) allows us to conclude that, in order to store SF, it is best to freeze it, even if in some cases, viscosity is modified but not the composition. All the data obtained (including protease pre-treatment) allow us to conclude that a small fraction of HA-protein complex forms a loose 3D-network and controls the rheology.

Highly sensitive ELISA for determining serum keratan sulphate levels in the diagnosis of OA

There have been a large number of reports on alterations in the serum level of keratan sulphate (KS), a potential marker of articular cartilage degeneration in patients with arthropathy. Such studies have commonly employed ELISA using the anti-KS monoclonal antibody 1/20/5D4 (5D4-ELISA) to determine KS levels. Recently, a highly sensitive KS ELISA (HS-ELISA) kit has been developed, allowing determination of serum KS levels even in small animals, which were formerly undetectable with 5D4-ELISA. However, the effectiveness of this kit in humans has not been demonstrated. The objective of this study was to assess the usefulness of the HS-ELISA for the analysis of human serum samples. Serum samples were collected from 28 patients with knee OA and 23 healthy volunteers. KS was determined by 5D4-ELISA and HS-ELISA, and measurements were compared with those obtained by HPLC. KS levels in serum samples with protease pretreatment were also determined by HS-ELISA. KS levels determined by HS-ELISA exhibited a better correlation with those determined by HPLC, and a higher diagnostic sensitivity for OA compared with 5D4-ELISA. Protease pretreatment of serum further improved the correlation between the values obtained by HS-ELISA and HPLC, as well as the diagnostic sensitivity of HS-ELISA for OA. HS-ELISA proved useful for determining KS level in serum and the diagnosis of OA. Pretreatment of serum samples with a protease further improved the performance of HS-ELISA.

Identification and Characterization of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase from Bacillus megaterium

A gene that codes for a novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase, designated PhaZ1, has been identified in the genome of Bacillus megaterium. A native PHB (nPHB) granule-binding assay showed that purified soluble PhaZ1 had strong affinity for nPHB granules. Turbidimetric analyses revealed that PhaZ1 could rapidly degrade nPHB granules in vitro without the need for protease pretreatment of the granules to remove surface proteins. Notably, almost all the final hydrolytic products produced from the in vitro degradation of nPHB granules by PhaZ1 were 3-hydroxybutyric acid (3HB) monomers. Unexpectedly, PhaZ1 could also hydrolyze denatured semicrystalline PHB, with the generation of 3HB monomers. The disruption of the phaZ1 gene significantly affected intracellular PHB mobilization during the PHB-degrading stage in B. megaterium, as demonstrated by transmission electron microscopy and the measurement of the PHB content. These results indicate that PhaZ1 is functional in intracellular PHB mobilization in vivo. Some of these features, which are in striking contrast with those of other known nPHB granule-degrading PhaZs, may provide an advantage for B. megaterium PhaZ1 in fermentative production of the biotechnologically valuable chiral compound (R)-3HB.

Study on adhesion of Lactobacillus acidophilus to mice Peyer's patches and inhibition of pathogen invasion

Objective To investigate the adhesion mechanism of Lactobacillus acidophilus FN001外to Peyer's patches. Methods Adhesion of L. acidophilus FN001 to mice Peyer's patches was studied in vitro using a fluorescent quantization method. The nature of adhesion mediator was studied by the effects of physical, chemical and enzymatic pre-treatments of the bacteria on their adhesion and effect of sugars on in- hibition of adhesion. The presence of lectin-like proteins in the cell surface was determined by hemagglutina- tion. Effect of L. acidophilus FN001 on inhibition of adhesion of pathogens to Peyer's patches was also stud- ied. Results The adhesion of L. acidophilus FN001 was strongly inhibited in the presence of D-mannose and methyl-ct-D-mannoside. Pretreatment of L. acidophilus FN001 with pepsin and trypsin decreased the ad- hesive capacity indicating that cell surface proteins are involved in adhesion to Peyer's patches. L. acidophi- lus FN001 could agglutinate rabbit red cell in mannose specific manner and protease pretreatment could de-crease hemagglutinin, suggesting that L. acidophilus FN001 has mannose specific lectin (s). In adherence inhibition assay, L. acidophilus NF001 could significantly inhibit adhesion of E. coli ATCC25922 to Peyer's patches when L. acidophilus NF001 were applied to Peyer's patches first or at the same time with pathogen. Conclusion It was concluded that a mannose-specific protein mediated adhesion of L. acidophilus FN001 to the Peyer's patches, and L. acidophilus FN001 could inhibit adhesion of pathogen with similar lectins speci- ficity to Peyer's patches. Key words: Lactobacillus; Peyer's patches; Mannose-specific adhesion; Adherence inhibition

Effect of enzyme pre-dehulling treatments on dehulling and cooking properties of legumes

Xylanase and protease pre-treatments were used to evaluate the dehulling properties of green gram, black gram, red gram and horse gram. Xylanase-mediated degradation of non starch polysaccharides (NSP) has facilitated the easy dehulling of green gram, black gram and horse gram. Xylanase pre-treatment of horse gram resulted in 84.4% dehulled kernels, whereas, 78.4% and 75.7% dehulled kernels were produced in green gram and black gram, respectively. However, protease pre-treatment was more efficient in improving the dehulling properties of green gram and black gram in addition to red gram with higher amount of dehulled kernels (>78%) and lower amount of fines. Selective improvements in the degree of dehulling, dehulling index and dehulling efficiency were observed in enzyme treatments compared to buffer- and oil-treated controls. Enzyme pre-treatments did not alter the cooking properties of dehulled legumes. These results indicate that, partial degradation of NSP and/or proteins of mucilage, which is present in between hulls and cotyledon by enzymes has facilitated the improvement in the dehulling properties of legumes.

Enhancement of the natural dyestuff dyeability of wool fabrics with protease pretreatment

Enhancement of the natural dyestuff dyeability of wool fabrics with protease pretreatment

Expansion properties and ultrastructure of legumes: Effect of chemical and enzyme pre-treatments

Expanded cereals and legumes are commonly used as ready-to-eat breakfast foods or as ingredients in food formulations. The use of horse gram and pigeon pea in food formulations and in legume composite flours is limited due to the presence of high levels of antinutritional factors, poor functional and expansion properties. Sodium bicarbonate and protease pre-treatments altered the cell wall structure of these legumes leading to the development of expanded grains. Expansion processing of pre-treated legumes resulted in statistically significant ( P < 0.05) increases in grain size and expansion volume compared to control grains. Highest yield of expanded grains were obtained with sodium bicarbonate pre-treatment (80 grains/100grains in pigeon pea and 96 grains/100 grains in horse gram), whereas, protease treatment yielded 68 and 94 expanded grains per 100 grains of pigeon pea and horse gram, respectively. Pre-treated expanded grains had lower bulk densities in the range of 480–510 g/L compared to untreated controls (about 760 g/L). Increased porosity and decreased cell wall thickness in expanded grains resulted in the collapse of cell walls and the appearance of large void spaces within the intercellular matrix. These results suggest the potential utility of under-utilized expanded pigeon pea and horse gram grains or their flours as ingredients in food processing or in legume composite flours.

Quantitative Glycomics of Human Whole Serum Glycoproteins Based on the Standardized Protocol for Liberating N-Glycans

Global glycomics of human whole serum glycoproteins appears to be an innovative and comprehensive approach to identify surrogate non-invasive biomarkers for various diseases. Despite the fact that quantitative glycomics is premised on highly efficient and reproducible oligosaccharide liberation from human serum glycoproteins, it should be noted that there is no validated protocol for which deglycosylation efficiency is proven to be quantitative. To establish a standard procedure to evaluate N-glycan release from whole human serum glycoproteins by peptide-N-glycosidase F (PNGase F) treatment, we determined the efficiencies of major N-glycan liberation from serum glycoproteins in the presence of reducing agents, surfactants, protease treatment, or combinations of pretreatments prior to PNGase F digestion. We show that de-N-glycosylation efficiency differed significantly depending on the condition used, indicative of the importance of a standardized protocol for the accumulation and comparison of glycomics data. Maximal de-N-glycosylation was achieved when serum was subjected to reductive alkylation in the presence of 2-hydroxyl-3-sulfopropyl dodecanoate, a surfactant used for solubilizing proteins, or related analogues, followed by tryptic digestion prior to PNGase F treatment. An optimized de-N-glycosylation protocol permitted relative and absolute quantitation of up to 34 major N-glycans present in serum glycoproteins of normal subjects for the first time. Moreover PNGase F-catalyzed de-N-glycosylation of whole serum glycoproteins was characterized kinetically, allowing accurate simulation of PNGase F-catalyzed de-N-glycosylation required for clinical glycomics using human serum samples. The results of the current study may provide a firm basis to identify new diagnostic markers based on serum glycomics analysis.

Modification of Host Erythrocyte Membranes by Trypsin and Chymotrypsin Treatments and Effects on the In Vitro Growth of Bovine and Equine Babesia Parasites

In the present study, we investigated the effects of protease pretreatments of host erythrocytes (RBC) on the in vitro growth of bovine Babesia parasites (Babesia bovis and B. bigemina) and equine Babesia parasites (B. equi and B. caballi). The selected proteases, trypsin and chymotrypsin, clearly modified several membrane proteins of both bovine and equine RBC, as demonstrated by SDS-PAGE analysis; however, the protease treatments also modified the sialic acid content exclusively in bovine RBC, as demonstrated by lectin blot analysis. An in vitro growth assay using the protease-treated RBC showed that the trypsin-treated bovine RBC, but not the chymotrypsin-treated ones, significantly reduced the growth of B. bovis and B. bigemina as compared to the control. In contrast, the growth of B. equi and B. caballi was not affected by any of these proteases. Thus, the bovine, but not the equine, Babesia parasites require the trypsin-sensitive membrane (sialoglyco) proteins to infect the RBC.

The optimization of sample treatment for spectral karyotyping with applications for human tumour cells

Spectral karyotyping (SKY) represents an important tool for the investigation of the complex chromosomal rearrangements (CCRs) in many human malignancies which may be difficult to characterize by conventional banding techniques. The main goal of our work was to optimize the most important steps in the preparation of molecular cytogenetic slides for a SKY protocol. This approach consisted of optimization of both the aging procedure and protease pretreatment of the slides, with special regard given to the preservation of chromosome structure and shape, as well as to the intensity of hybridization signals. The best results were obtained with a chemical aging procedure using SSC or ethanol in combination with trypsin pretreatment applied at a higher concentration for a shorter period of pretreatment. A resulting protocol for SKY also applicable to human solid tumour cells was subsequently proposed. The practical potential of the SKY technique was demonstrated on examples of two types of human embryonal tumours – neuroblastoma and Wilms’ tumour, in which some kinds of chromosomal aberrations were not detectable by means of classic cytogenetic methods.

High‐molecular intestinal alkaline phosphatase by agarose gel electrophoresis

The presence of high-molecular intestinal ALP (HIALP) overlapping with bone ALP in the alpha(2)beta region has been demonstrated. In this study we evaluated a method of separating HIALP after its conversion into ALP(5) by the action of protease. Serum samples from patients were mixed with protease at a ratio of 5:1 and left at room temperature for more than 30 min. The protease-treated and nontreated samples were both subjected to agarose gel electrophoresis. Patients who showed a decrease in ALP(3) in the alpha2beta region and an increase in ALP(5) in the beta region were regarded as HIALP-positive. HIALP was observed in 26.7-33.1% of patients with liver diseases, collagen diseases, and diabetes mellitus. Renal disease was ABO blood group-dependent and showed high positive rates for blood groups B and O. The HIALP-positive rate was low (7.1-15.5%) in patients with cardiovascular diseases, malignant tumors, and other disorders. ALP(5) was also observed in 98.4% of HIALP-positive patients with liver diseases. In patients with collagen diseases or diabetes mellitus, the positive rate of ALP(5) was 40.4-66.7%. In conclusion, this method, in which HIALP is converted into ALP(5) by protease pretreatment and is separated from bone ALP, allows HIALP to be identified while other fractions remain unaffected.

Improvement of shrink-resistance and tensile strength of wool fabric treated with a novel microbial transglutaminase from Streptomyces hygroscopicus

In this study, a novel microbial transglutaminase (MTG) from Streptomyces hygroscopicus WSH03-13 was applied in the processing of wool fabrics. The results indicated that MTG treatment could improve felting properties and decrease tensile strength loss of wool fabrics. For the wool fabrics used in this study, MTG treatment following chemical and protease pretreatment led to a 2.32% of area shrinkage and about 16% recovery in tensile strength based on the samples without MTG treatment. Moreover, a traditional resin treatment was compared with the role of MTG. Although the tensile strength of wool fabrics treated by MTG was lower than that treated by resin treatment, the fabrics had similar anti-felting properties, and the chemical oxygen demand of wastewater was only half of the latter.

Fluorescence in Situ Hybridization Analysis of Immunoglobulin Heavy Chain Translocations in Plasma Cell Myeloma Using Intact Paraffin Sections and Simultaneous CD138 Immunofluorescence

Fluorescence in situ hybridization (FISH) studies are much more sensitive than classical cytogenetics for identification of karyotypic abnormalities in plasma cell myeloma. However, FISH analysis of bone marrow samples is often challenging because of a large number of admixed non-neoplastic hematopoietic elements. In this report, we describe a novel method using FISH analysis of intact paraffin sections of formalin-fixed, bone marrow clot preparations with simultaneous CD138 tyramine signal amplification (TSA)-mediated immunofluorescence. We studied 22 cases of plasma cell myeloma for translocations involving the immunoglobulin heavy chain locus that are of known diagnostic and/or prognostic significance. All cases were analyzed using dual color, break-apart immunoglobulin heavy chain probe and dual color, dual fusion probes for t(11;14)(q13;q32) and t(4;14)(p16;q32). TSA-mediated fluorochrome deposition in CD138+ cells was unaltered by protease pretreatment. Translocations were identified in 10 cases, including five with t(11;14)(q13;q32) and three with t(4;14)(p16.3;q32). When present, abnormalities were identified in a large percentage of CD138+ cells (47 to 93%, median 84%). This technique allows for efficient molecular cytogenetic analysis of plasma cell myeloma using routinely archived paraffin-embedded material.

Use of a Plackett-Burman experimental design to examine the impact of extraction parameters on yields and compositions of pectins extracted from chicory roots (Chicorium intybus L.).

Chicory root pectin was isolated by acid extraction followed by alcohol precipitation. Because the extraction conditions have important effects on the features of pectins, an experimental design was used to study the influence of 17 different extraction parameters on yield and composition of pectin: pH, temperature, time of extraction, solid/liquid ratio, and different pretreatments of the pulps before extraction. Twenty extractions were conducted and examined for their significance on yield and sugar content using the Plackett-Burman factorial design. The acid extraction of chicory roots resulted in an average yield of 11% containing 86% of sugars. It was found that extraction temperature, time, protease pretreatment, water purity, and water washing of pulps significantly affected yield and pectin composition with an increase of yield and purity of pectin in harsher extraction conditions.

Binding and Functions of ADP-ribosylation Factor on Mammalian and Yeast Peroxisomes

We have analyzed in vitro the binding characteristics of members of the ADP-ribosylation factor (ARF) family of proteins to a highly purified rat liver peroxisome preparation void of Golgi membranes and studied in vivo a role these proteins play in the proliferation of yeast peroxisomes. Although both ARF1 and ARF6 were found on peroxisomes, coatomer recruitment only depended on ARF1-GTP. Recruitment of ARF1 and coatomer to peroxisomes was significantly affected both by pretreating the animals with peroxisome proliferators and by ATP and a cytosolic fraction designated the intermediate pool fraction depleted of ARF and coatomer. In the presence of ATP, the concentrations of ARF1 and coatomer on peroxisomes were reduced, whereas intermediate pool fraction led to a concentration-dependent decrease in ARF and increase in coatomer. Brefeldin A, a fungal toxin that is known to reduce ARF1 binding to Golgi membranes, did not affect ARF1 binding to peroxisomes. In Saccharomyces cerevisiae, both ScARF1 and ScARF3, the yeast orthologs of mammalian ARF1 and ARF6, were implicated in the control of peroxisome proliferation. ScARF1 regulated this process in a positive manner, and ScARF3 regulated it in a negative manner.