Papain Concentration Research Articles

Usage of immobilized papain for enzymatic hydrolysis of proteins

In this presented work, magnetic poly(HEMA-GMA) nanoparticles were synthesized by using emulsion polymerization technique. These magnetic nanoparticles were then functionalized with the affinity dye ligand, Cibacron Blue F3GA. The dye attached magnetic nanoparticles were characterized by using FTIR, ESR, SEM, AFM and EDX analysis. Synthesized magnetic nanoparticles demonstrated magnetic behavior and their size was about 200nm. Dye loading on the nanoparticle was calculated as 173.96μmol/g polymer by using sulfur stoichiometry. Effects of medium pH, initial papain concentration and ionic strength on the papain adsorption onto dye attached magnetic nanoparticles were also investigated and maximum papain adsorption onto the nanoparticles was found to be 764.0mg/g polymer in pH 7.0 HEPES buffer. Kinetic constants of the free and immobilized papain were compared and their pH, thermal, operational and storage stabilities were investigated. It was demonstrated with these studies that, immobilized form of papain was much more stable than that of free form. At the end of the study, catalytic efficiency of immobilized papain was investigated toward the different type of proteins such as casein, BSA, IgG and cytochrome c and it was demonstrated that, highest catalytic efficiency was achieved with IgG.

A Potential Novel Anthelmintic? The Cysteine Proteases Show Potent Anthelmintic Activity Against Cyathostomins In Vitro

Reasons for performing studyAnthelmintic resistance is a global problem and constitutes a major threat to the welfare of equids worldwide. The cyathostomins are the most numerous and pathogenic gastrointestinal nematode (GIN) of equids in the developed world. Cyathostomins show widespread resistance to 2 out of 3 of the major classes of anthelmintic and recently there are reports of reduced efficacy to the potent macrocyclic lactones (MLs). None of the 3 novel classes of anthelmintic that have emerged in the last decade are licensed for use in equids. The cysteine proteases (CPs) are plant proteins that have shown potent activity against GINs in vivo in sheep and pigs.ObjectivesThis study aimed to evaluate the anthelmintic effect of the CP papain on cyathostomins in vitro using the egg hatch assay (EHA) and larval migration inhibition test (LMIT).MethodsSamples of cyathostomin eggs and third stage larvae were collected and cultured from a population of equids that have recently shown reduced ML efficacy in vivo. The EHA and LMIT were performed on repeated samples with increasing concentrations of papain. Dose–response curves were plotted and PROBIT analysis performed on the data to give EC‐50 values (concentration that gives 50% of the maximal response).ResultsPapain caused a dose dependent inhibition of both egg hatch and larval migration. The EC‐50 values were 2 μmol/l and 100 μmol/l in the EHA and LMIT respectively, indicating a more potent effect on egg hatch.ConclusionsThe CP papain shows potent anthelmintic activity against cyathostomins in vitro. Good evidence of anthelmintic effect against GINs in other host species is supportive of its potential use in equids. Further work is indicated to evaluate safety and in vivo efficacy.Ethical animal research: The study was approved by the University of Liverpool and Donkey Sanctuary Ethics Committee. Explicit owner informed consent for participation in this study was not stated. Sources of funding: The Donkey Sanctuary, University of Liverpool – Institute of Infection and Global Health. Competing interests: None.

Extraction optimization, preliminary characterization and immunological activity of polysaccharides from figs

Complex enzyme extraction (CEE), purification, characterization of fig polysaccharides (FPs) from dried figs were investigated. Orthogonal experiment was used to optimize the concentration of cellulose, pectinase and papain. Response surface methodology (RSM) was employed to optimize extraction conditions. The optimum extraction conditions were: enzyme concentration of 1.5%, 1.5%, 0.5% (wt%) of pectinase, papain, cellulose, ratio of water to raw material 40.3mL/g, extraction time 54.1min, temperature 34.15°C and pH 3.8. Under these conditions, the experimental yield was 7.98±0.17%. Two homogeneous heteropolysaccharides (FPs-1-1, FPs-2-1) were purified by DEAE-Sepharose and Sephadex G-200 chromatography, which were composed of rhamnose, arabinose, xylose, mannose, glucose and galactose with molecular weight of 1.52×106 and 4.75×105Da, respectively. The bioactivity assay showed that FPs-1-1 and FPs-2-1 could more significantly enhance splenic lymphocyte proliferation, phagocytosis and NO production of macrophages, could be explored as potential immunopotentiating agent for use in functional food or medicine.

Radiation synthesized protein-based nanoparticles: A technique overview

abstract Seeking for alternative routes for protein engineering a novel technique – radiation induced synthesis ofprotein nanoparticles – to achieve size controlled particles with preserved bioactivity has been recentlyreported. This work aimed to evaluate different process conditions to optimize and provide an overviewof the technique using γ -irradiation. Papainwas used as model protease and the samples were irradiatedin a gamma cell irradiator in phosphate buffer (pH¼7.0) containing ethanol (0–35%). The dose effect wasevaluated by exposure to distinct γ -irradiation doses (2.5, 5, 7.5 and 10 kGy) and scale up experimentsinvolving distinct protein concentrations (12.5–50 mg mL 1 ) were also performed. Characterizationinvolved size monitoring using dynamic light scattering. Bityrosine detection was performed usingfluorescence measurements in order to provide experimental evidence of the mechanism involved. Bestdose effects were achieved at 10 kGy with regard to size and no relevant changes were observed as afunction of papain concentration, highlighting very broad operational concentration range. Bityrosinechanges were identified for the samples as a function of the process confirming that such linkages playan important role in the nanoparticle formation.& 2014 Elsevier Ltd. All rights reserved.

Saxitoxins induce cytotoxicity, genotoxicity and oxidative stress in teleost neurons in vitro

The aim of this study was establish a protocol for isolation and primary culture of neurons from tropical freshwater fish species Hoplias malabaricus for assessment of the effects of neurotoxic substances as saxitoxins (STXs). Cells from brain of H. malabaricus were treated with different concentrations of trypsin, dispase and papain for tissue dissociation. Cells type was separated by cellular gradient and basic fibroblast growth factor (bFGF) supplement nutrition media were added. The dissociated cells were plated with medium and different STXs concentrations and the toxic cellular effects such as oxidative stress, neurotoxicity, and genotoxicity and apoptosis process were evaluated. Cultures treated with bFGF showed the greatest adherence, survival and cellular development. STXs increased specific activity of glutathione peroxidase and lipoperoxidation levels, were cytotoxic and genotoxic indicated by the comet assay. Although the STXs effects due the blockage of sodium channels is reported to be reversible, the time exposure and concentration of STXs suggested cellular injuries which can lead to neuropathology. The establishment of primary neuronal culture protocol enables new applications for neurotoxicological assessments.

A scanning electron microscope study on the effect of an experimental irrigation solution on smear layer removal.

The aim of this in vitro study was to evaluate the effect of an experimental irrigation solution, containing two different concentrations of papain, Tween 80, 2% chlorhexidine and EDTA, on removal of the smear layer. Thirty-six single-rooted teeth were divided into two experimental groups (n=12) and two positive and negative control groups of six. The canals were prepared with BioRaCe instruments up to BR7 (60/0.02). In group 1, canals were irrigated with a combination of 1% papain, 17% EDTA, Tween 80 and 2% CHX; in group 2, canals were irrigated with a combination of 0.1% papain, 17% EDTA, Tween 80 and 2% CHX. In group 3 (the negative control), the canal was irrigated with 2.5% NaOCl during instrumentation and at the end of preparation with 1 mL of 17% EDTA was used; in group 4 (positive control), normal saline was used for irrigation. The amount of the remaining smear layer was quantified according to Hulsmann method using scanning electron microscopy (SEM). Data was analyzed by the Kruskal-Wallis and Mann-Whitney tests. Two-by-two comparisons of the groups revealed no significant differences in terms of smear layer removal at different canal sections between the negative control group (standard regiment for smear layer removal) and 1% papain groups (P<0.05). Under the limitations of the present study, combination of 1% papain, EDTA, 2% chlorhexidine and Tween 80 can effectively remove smear layer from canal walls.

Rice Bran Stabilization using Papain Enzyme

Attempts were made to utilize papain enzyme for the stabilization of rice bran. Rice bran was treated with different concentrations of papain. Free fatty acid concentration (FFA), an indicator of hydrolytic rancidity, was measured at different durations of storage. The effect of three factors viz. concenntration of enzyme (mg per 100g of rice bran), duration of enzymatic treatment (min.) and duration of storage (days) was studied on four responses viz. % FFA content, % oil content, % fibre and % protein content of rice bran. Experiments were conducted using Response Surface Methodology. Based on optimization, the recommended concentration was 50 mg papain enzyme per 100 g bran, with 60 min treatment duration and 15 days of storage at 4°C. The predicted value for FFA at 15th day was 9.27%, that of total fat, protein and fibre levels were 18.46, 16.27 and 8.21% respectively.

Papain enzyme supported on magnetic nanoparticles: Preparation, characterization and application in the fruit juice clarification

The preparation, characterization, and application of silica-coated magnetic nanoparticles for papain immobilization is reported. Papain was covalently attached onto the (3-chloropropyl) trimethoxysilane-modified silica-coated magnetic nanoparticles. The enzyme-immobilized nanoparticles were characterized by Fourier transform infrared spectroscopy, X-ray powder diffraction, scanning electron microscopy, and vibrating sample magnetometry techniques. Response surface methodology combined with statistical analyses using Minitab were employed to evaluate optimum operating conditions to immobilize papain on the magnetic nanoparticles. The optimum conditions were: temperature = 27.3 °C, pH of the enzyme solution = 7.1, concentration of papain = 3.3 mg/mL, and immobilization time = 10 h. Compared with the free papain, the immobilized papain displayed enhanced enzyme activity, better tolerance to variations in the medium pH and temperature, improved storage stability, and good reusability. Both the free and immobilized enzymes were effective for the clarification of pomegranate juice.

Degumming of Mulberry Silk with Papain in Non-Aqueous Systems

The nonionic reverse micelles used for degumming of mulberry silk were prepared with a non-ionic surfactant Triton X-100 (TX-100) by injecting small amount of papain aqueous solution. The weight loss of mulberry silk degummed at different concentration of papain in TX-100 reverse micelles was investigated. The effect of the amount of solubilized water was also discussed. The polarizing optic microscope was employed to observe the surface morphology of the mulberry silk. The results indicated that the weight loss of the mulberry silk was increased with increasing concentration of papain, but the W values exhibited the reverse trend. The good degumming of mulberry silk with papain was obtained in TX-100 reverse micelles. The strength of mulberry silk decreased with increasing concentration of papain.

Papain-directed synthesis of luminescent gold nanoclusters and the sensitive detection of Cu2+

Highly fluorescent papain stabilized gold nanoclusters (NCs) have been synthesized through a simple wet chemical route. Papain was used for the first time as an effective capping and reducing agent for these clusters. The optimal conditions for the synthesis of the gold nanoclusters, including the concentrations of papain and NaOH, reaction time and temperature, were investigated. The as-prepared Au clusters show intense red emission at ∼660nm (QY ∼4.3%) and are uniform in size. The clusters are quite stable and the intense red emission remained unchanged at a buffer pH range of 6–12. The fluorescent Au NCs were then used as a label-free probe for the sensitive detection of Cu2+. A limit of detection of 3nM was obtained. The sensing strategy is also highly selective against the various potential interference ions.

Extraction of starch from hulled and hull-less barley with papain and aqueous sodium hydroxide.

Starch was isolated from hulled (VJM 201) and hull-less (BL 134) barley with papain and aqueous sodium hydroxide treatments. For enzyme-assisted extraction, barley was steeped in water containing 0.2% SO2 + 0.55% lactic acid at 50° ± 2°C for 4-5h. The slurry was mixed with 0.4-2.0g papain/kg barley and incubated at 50° ± 2°C for 1-5h. Aqueous sodium hydroxide (0.01-0.05M) was added to the finely ground barley meal. The alkaline slurry was incubated at ambient temperature (25° ± 2°C) for 15-60min. The starch and grain fractions were isolated by screening and centrifugation. Increases in the time of treatment significantly affected the fiber, centrifugation and non-starch residue losses. Concentration of papain and sodium hydroxide had negligible effect on extraction losses. The enzyme-assisted extraction efficiency of starch was higher (80.7-84.6%) than the alkaline method (70.9-83.7%). The hulled barley showed higher extraction efficiency than the hull-less barley. The slurry treated with 0.4g papain/kg barley for 5h and 0.03M sodium hydroxide for 60min produced maximal yield of starch. Barley starch showed desirably high pasting temperature, water binding capacity and hold viscosity; and low final and setback viscosity compared with the commercial corn starch. The alkaline extracted hull-less barley starch showed exceptionally high peak and hold viscosities.

Papain-induced in vitro disc degeneration model for the study of injectable nucleus pulposus therapy

Background contextProteolytic enzyme digestion of the intervertebral disc (IVD) offers a method to simulate a condition of disc degeneration for the study of cell-scaffold constructs in the degenerated disc. PurposeTo characterize an in vitro disc degeneration model (DDM) of different severities of glycosaminoglycans (GAG) and water loss by using papain, and to determine the initial response of the human mesenchymal stem cells (MSCs) introduced into this DDM. Study designDisc degeneration model of a bovine disc explant with an end plate was induced by the injection of papain at various concentrations. Labeled MSCs were later introduced in this model. MethodsPhosphate-buffered saline (PBS control) or papain in various concentrations (3, 15, 30, 60, and 150 U/mL) were injected into the bovine caudal IVD explants. Ten days after the injection, GAG content of the discs was evaluated by dimethylmethylene blue assay and cell viability was determined by live/dead staining together with confocal microscopy. Overall matrix composition was evaluated by histology, and water content was visualized by magnetic resonance imaging. Compressive and torsional stiffness of the DDM were also recorded. In the second part, MSCs were labeled with a fluorescence cell membrane tracker and injected into the nucleus of the DDM or a PBS control. Mesenchymal stem cell viability and distribution were evaluated by confocal microscopy. ResultsA large drop of GAG and water content of the bovine disc were obtained by injecting >30 U/mL papain. Magnetic resonance imaging showed Grade II, III, and IV disc degeneration by injecting 30, 60, and 150 U/mL papain. A cavity in the center of the disc could facilitate later injection of the nucleus pulposus tissue engineering construct while retaining an intact annulus fibrosus. The remaining disc cell viability was not affected. Mesenchymal stem cells injected into the protease-treated DDM disc showed significantly higher cell viability than when injected into the PBS-injected control disc. ConclusionsBy varying the concentration of papain for injection, an increasing amount of GAG and water loss could be induced to simulate the different severities of disc degeneration. MSC suspension introduced into the disc has a very low short-term survival. However, it should be clear that this bovine IVD DDM does not reflect a clinical situation but offers exciting possibilities to test novel tissue engineering protocols.

Bactericidal Effects of Natural Tenderizing Enzymes on Escherichia Coli and Listeria monocytogenes

&lt;p&gt;The objective of this study was to determine the antimicrobial activity of proteolytic, meat-tenderizing enzymes (papain and bromelain) against &lt;em&gt;E. coli&lt;/em&gt; and &lt;em&gt;L. monocytogenes&lt;/em&gt; at three different temperatures (5, 25 and 35°C). Two overnight cultures of &lt;em&gt;E. coli&lt;/em&gt; JM109 and &lt;em&gt;L. monocytogenes&lt;/em&gt; were separately suspended in 1% peptone water and exposed to a proteolytic enzyme (papain or bromelain) at three different temperatures. Bromelain concentrations (4 mg/ml) and (1 mg/ml) tested at 25°C against &lt;em&gt;E. coli&lt;/em&gt; and &lt;em&gt;L. monocytogenes,&lt;/em&gt; respectively, were the most effective concentrations tested reducing populations by 3.37 and 5.7 log CFU/ml after 48 h, respectively. Papain levels of (0.0625 mg/ml) and (0.5 mg/ml) were the most effective concentration tested at 25°C against &lt;em&gt;E. coli&lt;/em&gt; and &lt;em&gt;L. monocytogenes,&lt;/em&gt; respectively, reducing populations by 4.94 and 6.58log CFU/ml after 48h, respectively. Interestingly, the lower papain concentration tested (0.0625 mg/ml) was more effective than the higher concentration (0.5 mg/ml) against &lt;em&gt;E. coli&lt;/em&gt; at all three temperatures. As expected, the temperature was directly related to enzyme efficacy against both &lt;em&gt;E. coli&lt;/em&gt; and &lt;em&gt;L. monocytogenes.&lt;/em&gt;&lt;/p&gt;

Effects of Papain Hydrolysis on the Pasting Properties of Wheat Flour

As one of the most effective enzymatic modification methods of protein, papain hydrolysis is applied widely in food production, accompanying starch pasting frequently in order to improve industrial quality. Effects of the papain hydrolysis on flour pasting properties were investigated in five papain/flour concentrations and five time-treatments. The structure of starch and protein networks in slurry was investigated under microscope before and after pasting. Results showed that papain hydrolysis influenced the pasting properties of wheat flour significantly through affecting structural characteristics, amylase activity and exothermic transition, especially during the early stage of hydrolysis. Peak viscosity, trough, final, integral area, and setback significantly decreased along with the increasing concentration of papain. Both hydrolysis time and concentration of papain had obviously effect on the breakdown. Pasting temperature and pasting time increased significantly with the enhancement of papain concentration. Hydrolysis time exerted minor effect on the pasting temperature and pasting time. The average peak time was slightly prolonged by lower concentration of papain, otherwise slightly shortened by higher concentration.

Simple and sensitive determination of papain by resonance light-scattering with CdSe quantum dots

In this contribution, a simple and sensitive method for papain detection was established based on the increment of the resonance light-scattering (RLS) intensity of mercaptoacetic acid-capped CdSe quantum dots (MAA-QDs) in aqueous solution. Meanwhile, the RLS characteristics and the optimal conditions were investigated in detail. Under the optimized conditions, the linear range of MAA-QDs RLS intensity versus the concentration of papain was 1.0×10−8M to 6.0×10−7M, with a correlation coefficient (R2) of 0.9977 and a limit of detection (3σ black) of 5.1×10−9M. The relative standard deviation (R.S.D.) for 1.6×10−7M papain was 1.0% (n=5). There was almost no interference to coexisting foreign substances including common ions, proteins and 20 amino acids. The proposed method possessed the advantages of simplicity, rapidity and sensitivity. Three synthetic samples were analyzed by the methodology and the results were satisfying. The interaction between MAA-QDs and papain was also investigated systematically by using visual method, transmission electron microscopy and time-resolved fluorescence spectra, and the results indicated that the main force between MAA-QDs and papain was electrostatic interaction.

The effect of various concentrations of papain on the properties and hydrolytic rates of β-casein layers

Recently, much attention has been focused on the investigation of the surface biocatalysis of proteases. In this study, papain, a proteolytic enzyme was used to hydrolyze a bovine β-casein (β-CN) layer, which was monitored by a quartz crystal microbalance with dissipation (QCM-D). The changes of the β-CN layers before and after hydrolysis were characterized by atomic force microscopy (AFM) imaging, grazing angle infrared spectroscopy (GA-FTIR) spectra, and contact angle measurement. Our results demonstrated that the proteolytic reaction was enzyme concentration-dependent and started with the hydrophobic C-terminal sequence of the β-CN. The remaining β-CN layer became thinner, smoother, stiffer, and hydrophilic after hydrolysis. These results are conducive to the further understanding of the catalysis of papain on β-CN layers in liquid–solid interfaces and the promotion of biocatalytic applications.

A Reproducible Papain-Induced Disk Degeneration Model of Reduced GAG and Hydration

Introduction Nucleus pulposus (NP) digestion using chymopapain is a clinical procedure to release pressure of a herniated disk. It reduces matrix content and hence offers a method to simulate a condition of disk degeneration with reduced glycosaminoglycans (GAG) and water content.1 The aims of the study is to characterize a set of in vitro disk degeneration model (DDM) of different severity of GAG and water loss by using various concentration of papain and second to determine the initial response of mesenchymal stem cells (MSC) introduced in these papain-induced DDM. Materials and Methods Disk Degeneration Model. Bovine caudal IVDs (diameter 16 to 22 mm) with endplates were isolated as previously described which is readily for in vitro culture in a multiloading bioreactor.2 Various concentration of papain (3, 15, 30, 60, 150 U/mL, n = 4 for each group) (Sigma-Aldrich, Switzerland) or phosphate buffer saline (PBS control) were injected into the center of the disk using a 25 G. Ten days after the injection of papain, cell viability was assessed by staining the tissue with Live/Dead stain (Fluka, Sigma-Aldrich, Switzerland) and visualized by confocal microscopy.3 The other half of the tissue was evaluated by histology for overall matrix organization. Nondecalcified tissues were embedded in methylmethacrylate (MMA) for histology sectioning. Overall matrix composition was evaluated by staining the 6 µm section with safranin O and fast green, and hematoxylin and eosin. Disk hydration of the bovine tail disk injected with papain and PBS after 2 days was assessed by magnetic resonance imaging (MRI) using T2* protocol. MSC Injection. Human MSCs were isolated from bone marrow obtained from spinal surgery. MSC were labeled with a fluorescence cell membrane tracker DiO (Invitrogen, Basel, Switzerland) and then suspended in culture medium and injected into the nucleus of the DDM (30 U/mL) or a PBS control disk. MSC distribution and viability were evaluated after 2 days of free swelling culture by staining the tissue with DAPI and calcein AM and imaging with confocal microscopy. The presence of live injected cells were visualized as colocalization of orange DiL dye and calcein AM live cell stain. Results Disk Degeneration Model. Cell viability of the papain-digested NP was maintained at ∼75% after 10 days in all the injected papain concentration. In the mild DDM (3 U/mL), small amount of GAG was maintained in the center of the disk. In the medium DDM (30 to 60 U/mL), GAG was completely lost in the entire disk with a small cavity in the center while AF maintained intact. In the DDM (150 U/mL), GAG was completely lost in the entire disk with a large cavity present in the center of the disk and the AF was collapsed. MRI result showed that there was a mild decrease in disk hydration by injecting 3 U/mL papain, and half of the disk hydration was lost when injecting 150 U/mL papain. ( Fig. 1 ) MSC Injection. At day 2, MSC injected into a control disk (injected with PBS) formed a cluster and largely decreased the MSC viability. While MSC injected in the DDM dispread inside the NP and a significantly higher cell viability was resulted when MSC were injected into this papain-induced DDM (40.64 ± 10.81%) than in the PBS control (14.50 ± 6.08%) ( n = 4, p = 0.0329). [Figure: see text] Conclusion A reproducible DDM of various severities of reduced GAG and hydration can be achieved by injecting various dosage of papain without compromising the disk cell viability. This model is ready for the testing of injectable tissue engineering strategies inside a controlled loading and culture environment of a bioreactor. MSCs injected as cell suspension have a poor-chance of survival in a healthy or degenerated disk. This indicated that matrix content of the disk affect the initial survival of the introduced cells and revealed that the use of cell carrier/scaffold will be needed for cell therapy. Acknowledgements This project was supported by the Swiss National Science Foundation (SNF # 310030–127586/1). I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Roberts et al. BMC Musculoskelet Disord 2008;9:24 Chan S et al. JoVE, In press, 2011 Gantenbein-Ritter B et al. Tissue Engineering Part C 2008;14:353–358

A Reproducible Papain-Induced Disk Degeneration Model of Reduced GAG and Hydration

IntroductionNucleus pulposus (NP) digestion using chymopapain is a clinical procedure to release pressure of a herniated disk. It reduces matrix content and hence offers a method to simulate a condition of disk degeneration with reduced glycosaminoglycans (GAG) and water content.1 The aims of the study is to characterize a set of in vitro disk degeneration model (DDM) of different severity of GAG and water loss by using various concentration of papain and second to determine the initial response of mesenchymal stem cells (MSC) introduced in these papain-induced DDM. Materials and MethodsDisk Degeneration Model. Bovine caudal IVDs (diameter 16 to 22 mm) with endplates were isolated as previously described which is readily for in vitro culture in a multiloading bioreactor.2 Various concentration of papain (3, 15, 30, 60, 150 U/mL, n = 4 for each group) (Sigma-Aldrich, Switzerland) or phosphate buffer saline (PBS control) were injected into the center of the disk using a 25 G. Ten days after the injection of papain, cell viability was assessed by staining the tissue with Live/Dead stain (Fluka, Sigma-Aldrich, Switzerland) and visualized by confocal microscopy.3 The other half of the tissue was evaluated by histology for overall matrix organization. Nondecalcified tissues were embedded in methylmethacrylate (MMA) for histology sectioning. Overall matrix composition was evaluated by staining the 6 µm section with safranin O and fast green, and hematoxylin and eosin. Disk hydration of the bovine tail disk injected with papain and PBS after 2 days was assessed by magnetic resonance imaging (MRI) using T2* protocol.MSC Injection. Human MSCs were isolated from bone marrow obtained from spinal surgery. MSC were labeled with a fluorescence cell membrane tracker DiO (Invitrogen, Basel, Switzerland) and then suspended in culture medium and injected into the nucleus of the DDM (30 U/mL) or a PBS control disk. MSC distribution and viability were evaluated after 2 days of free swelling culture by staining the tissue with DAPI and calcein AM and imaging with confocal microscopy. The presence of live injected cells were visualized as colocalization of orange DiL dye and calcein AM live cell stain.Results Disk Degeneration Model. Cell viability of the papain-digested NP was maintained at ∼75% after 10 days in all the injected papain concentration. In the mild DDM (3 U/mL), small amount of GAG was maintained in the center of the disk. In the medium DDM (30 to 60 U/mL), GAG was completely lost in the entire disk with a small cavity in the center while AF maintained intact. In the DDM (150 U/mL), GAG was completely lost in the entire disk with a large cavity present in the center of the disk and the AF was collapsed. MRI result showed that there was a mild decrease in disk hydration by injecting 3 U/mL papain, and half of the disk hydration was lost when injecting 150 U/mL papain. (Fig. 1)MSC Injection. At day 2, MSC injected into a control disk (injected with PBS) formed a cluster and largely decreased the MSC viability. While MSC injected in the DDM dispread inside the NP and a significantly higher cell viability was resulted when MSC were injected into this papain-induced DDM (40.64 ± 10.81%) than in the PBS control (14.50 ± 6.08%) (n = 4, p = 0.0329).Figure 1 MRI image of the bovine tail DDM as caused by injecting increasing dosage of papain. ConclusionA reproducible DDM of various severities of reduced GAG and hydration can be achieved by injecting various dosage of papain without compromising the disk cell viability. This model is ready for the testing of injectable tissue engineering strategies inside a controlled loading and culture environment of a bioreactor. MSCs injected as cell suspension have a poor-chance of survival in a healthy or degenerated disk. This indicated that matrix content of the disk affect the initial survival of the introduced cells and revealed that the use of cell carrier/scaffold will be needed for cell therapy.Acknowledgements This project was supported by the Swiss National Science Foundation (SNF # 310030–127586/1).I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredRoberts et al. BMC Musculoskelet Disord 2008;9:24Chan S et al. JoVE, In press, 2011Gantenbein-Ritter B et al. Tissue Engineering Part C 2008;14:353–358

Effects of Enzymatic Hydrolysis of Protein on the Pasting Properties of Different Types of Wheat Flour

As one of the most effective methods to modify proteins, enzymatic hydrolysis is used widely in the preparation of wheat products in the food industry. During the same process, starch pasting occurs frequently. The effects of wheat protein hydrolysis with papain, pepsin, and trypsin on the pasting properties of 3 different kinds of flour were investigated in 5 concentrations. Results showed that the peak viscosity, trough, final, and integral area of pasting curve of these flours decreased with increasing enzymatic hydrolysis of protein, and decreased significantly with the increasing enzyme concentrations. Medium-gluten flour was the least sensitive to enzymatic activity and weak-gluten the most sensitive. Downtrends appeared with increasing papain and trypsin concentrations in the form of breakdown. Enzymes had no significant different effect on the peak times of strong- and medium-gluten flour, but prolonged peak time slightly in weak-gluten flour. The pasting time and temperature of strong- and medium-gluten flour were significantly increased in a concentration-dependent manner. However, there were no significant effects on the pasting times of weak-gluten flour. These results could supply a basis for utilization of enzymatic hydrolysis of wheat protein in food industry and for further studies into the interactions between hydrolyzed protein and starch in food or processing industries.

Effects of protein hydrolysis on pasting properties of wheat flour

AbstractProtein hydrolysis is used widely in the food industry for the purpose of improvement of food or industrial quality, and the processing exists concurrently or subsequently with starch pasting. The effects of protein hydrolysis using papain, pepsin, trypsin, and dithiothreitol (DTT) on flour pasting properties were here investigated at five set concentrations. There were notable decreases in the peak, the trough viscosity, and the integral area of the viscosity curve as the concentrations of papain, trypsin, and DTT increased. DDT and pepsin concentration did not have any effect on final viscosity. Both breakdown and setback showed significant downtrends as the concentration of papain increased but no change as DDT concentration varied. Low concentrations of papain and pepsin prolonged peak time slightly, but higher concentrations shortened it significantly. For trypsin and DTT, low concentrations increased peak time notably, but high concentrations decreased it significantly. Pasting time and temperature increased in the presence of papain, pepsin, and trypsin, more so at higher concentrations. These results supply a basis for the utilization of hydrolyzed protein in the food industry and for the further studies on the interactions between protein and starch during processing.