Inhibition of bacteriophage m13 replication with esterified milk proteins

Esterified milk proteins [methylated (Met) or ethylated (Et) alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), and beta-casein (BCN)], unmodified native milk proteins, and native basic proteins (calf thymus histone and hen egg white lysozyme) were tested for their antiviral activity against the bacteriophage M13 and for their influence on its replication (except BCN). All esterified milk proteins showed an antiviral activity against the bacteriophage M13, proportional to the extent of esterification and, hence, to the increased basicity of the modified proteins. Antiviral activity of 100% Met-BLG disappeared after its pepsinolysis but not after its trypsinolysis. The antiviral activity of Met-BLG was much higher than that of native basic proteins (histone and lysozyme). One hundred percent Met-BLG and 73% Et-BLG inhibited the replication of bacteriophage M13 completely, whereas 60% Met-ALA inhibited phage replication partially. Calf thymus histone inhibited the replication of bacteriophage M13 at a lower extent (20%) than Met- and Et-BLG (100% inhibition). Protein concentration, pH, and concentration of the Escherichia coli culture in the preincubation medium of the virus were other factors influencing antiviral activity. Interactions of esterified proteins with the phage DNA (phenol extracted) followed the same pattern as observed during studies of the inhibition of the phage replication: Met-BLG > Et-BLG > or = Met-ALA.     

Antiviral activity of esterified alpha-lactalbumin and beta-lactoglobulin against herpes simplex virus type 1. Comparison with the effect of acyclovir and L-polylysines

The antiviral activity of methylated alpha-lactalbumin (Met-ALA), methylated and ethylated beta-lactoglobulins (Met- and Et-BLG) was evaluated against acyclovir (ACV)-sensitive and -resistant strains of herpes simplex virus type 1 (HSV-1) and compared to that of ACV and L-polylysines (4-15 kDa) using fixed or suspended Vero cell lines. Esterified whey proteins and their peptic hydrolyzates displayed protective action against HSV-1, which was relatively lower than that induced by ACV or L-polylysines. The higher activity of L-polylysines was maintained against an ACV-resistant strain of HSV-1, whereas ACV lost much of its activity. The mean 50% inhibitory concentration (IC50) was about 0.8-0.9 microg/mL for L-polylysines against ACV-sensitive and -resistant strains of HSV-1 when using two concentrations of virus (50% and 100% cytopathic effect, CPE). The IC50 values of ACV against the sensitive strain of HSV-1 were 3 and 15 microg/mL when using the low and high concentrations of virus, respectively. When using 50% CPE, IC50 values for esterified whey proteins ranged from 20 to 95 microg/mL, depending on the nature of the ester group, the degree of esterification, and the nature of the protein. Using the real-time PCR technique, it was shown that Met-ALA inhibited HSV-1 replication.     

Radical scavenging capacity and antimutagenic properties of purified proteins from Solanum betaceum fruits and Solanum tuberosum tubers

In this study, antioxidant activities in free-radical-mediated oxidative systems and the genotoxic/antigenotoxic effects of two proteins with molecular mass around 17 kDa, purified from Solanum betaceum fruits (cyphomine) and Solanum tuberosum tubers (solamarine), were investigated. Both proteins inhibited uric acid formation with IC(50) values between 55 and 60 μg/mL, and both proteins were able to reduce oxidative damage by scavenging hydroxyl radicals and superoxide anion in a dose-dependent manner. Furthermore, the DPPH? reduction assay showed SC(50) values of 55-73 μg/mL. Cyphomine and solamarine were able to retain their antioxidant activity after heat treatment at 80 °C for 15 min. Allium cepa and Salmonella /microsome assays showed no genotoxic and mutagenic effects. Solamarine showed an antimutagenic effect against a direct mutagen (4-nitro-o-phenylenediamine). Consequently, the present study showed that the investigated proteins are promising ingredients for the development of functional foods with a beneficial impact on human health and an important source for the production of bioactive peptides.     

Peptides derived from atlantic salmon skin gelatin as dipeptidyl-peptidase IV inhibitors

The dipeptidyl-peptidase IV (DPP-IV)-inhibitory activity of peptides derived from Atlantic salmon skin gelatin hydrolyzed by alcalase (ALA), bromelain (BRO), and Flavourzyme (FLA) was determined. The FLA hydrolysate with the enzyme/substrate ratio of 6% showed the greatest DPP-IV-inhibitory activity. The hydrolysate was fractionated by ultrafiltration with 1 and 2.5 kDa cutoff membranes, and the <1 kDa fraction had the highest DPP-IV-inhibitory activity with an IC(50) value of 1.35 mg/mL. The F-1 fraction further isolated by HPLC showed the IC(50) value against DPP-IV of 57.3 μg/mL, and the peptide sequences were identified as Gly-Pro-Ala-Glu (372.4 Da) and Gly-Pro-Gly-Ala (300.4 Da). The synthetic peptides showed dose-dependent inhibition effects on DPP-IV with IC(50) values of 49.6 and 41.9 μM, respectively. The results suggest that the peptides derived from Atlantic salmon skin gelatin would be beneficial ingredients for functional foods or pharmaceuticals against type 2 diabetes.     

Scavenging of free radicals, antimicrobial, and cytotoxic activities of the Maillard reaction products of beta-lactoglobulin glycated with several sugars

The Maillard reaction occurs during many industrial and domestic thermal treatments of foods. It is widely used because of its role in creating colors, flavors, textures, and other functional properties in foodstuffs. Proteins glycated without the use of conventional chemical reagents have improved technofunctional properties such as heat stability, emulsifying, and foaming properties. The present study was carried out to determine the extent to which this reaction can convey antioxidant, antimicrobial, or cytotoxic activities to beta-lactoglobulin (BLG) and to its tryptic and peptic hydrolysates. BLG was modified with six different sugars in solution at 60 degrees C. Antiradical properties were estimated using a radical scavenging activity test. Antimicrobial activities against different bacterial strains were studied with a diffusion disk method. Cytotoxic tests were performed using two cell lines and the 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) rapid colorimetric assay. Glycation induced a radical scavenging activity to BLG, the intensity of which depended on the sugar used for modification. Proteins modified with ribose and arabinose showed the highest radical scavenging activities depicted by about 80 and 60% of 2,2-diphenyl-1-picrylhydrazyl (DPPH) absorption decrease at 515 nm. No antimicrobial effect of any glycated form of BLG against Escherichia coli, Bacillus subtilis, Listeria innocua, and Streptococcus mutans was observed. The MTT test showed no enhancement of cytotoxicity by modified proteins and peptides against COS-7 and HL-60 cells. Thus, glycated proteins could be used in formulated food as functional ingredients with a radical scavenging activity able to delay deterioration due to oxidation. This use could be even more advisable considering the lack of toxicity to eukaryotic and prokaryotic cell cultures demonstrated in this work.     

Effects of hydration,lipids, and temperature on the binding of the volatile aroma terpenes by beta-lactoglobulin powders

The binding properties of dry proteins are relatively poorly known. Many proteins are present in emulsions and suspensions and also in dry forms. This is particularly true of dairy proteins, which are often stored and sold in powdered form. In the present work, the binding of three terpenes (alpha-terpinene, gamma-terpinene, and terpinolene), which belong to the basic aroma components, and of decane by powdered beta-lactoglobulin (BLG) was studied at different hydration levels (0.05-0.40 g of H(2)O/g of protein) and temperatures (298 and 309.5 K), in the presence or absence of lipids and small concentrations of ethanol. Vapor sorption isotherms were determined for these systems by a static method of headspace gas chromatographic analysis. A cooperative effect of hydrophobic hydration was observed for the binding of aroma terpenes and decane by the solid BLG. The temperature increase from 298 to 309.5 K reduced the observed hydration threshold of BLG by 0.05-0.08 g of H(2)O/g of protein. Lipids (1.2% w/w) in hydrated BLG gave at least a 2-fold increase in its binding affinity for the hydrocarbons studied, and synergic effects of the hydration and lipid on this affinity were observed.     

Characterization of yam bean (Pachyrhizus erosus) proteins

Seed proteins from Mexican yam bean seeds (Pachyrhizus erosus L.) were sequentially extracted according to the Osborne classification. Albumins were the major fraction (52.1-31.0%), followed by globulins (30.7-27.5%). The minor protein fraction was prolamins (0.8%). Defatting with chloroform/methanol remarkably affected the distribution of protein solubility classes; albumins were the most affected fraction (4.3-17.5%). Electrophoretic patterns of albumins showed bands at 55, 40, 35, and 31 kDa. After reduction of the globulin fraction exhibited two triplets, one from 35 to 31 kDa and the second from 19 to 21 kDa, these could be compared to the acid and basic polypeptides of 11S-like proteins. Prolamins showed one band at 31 kDa, and glutelins after reduction showed three main bands at 52, 27, and 14 kDa. Trypsin inhibitors were assayed in saline extracts; the values found (1232-2608 IU/g of meal) were lower than those of other legumes. In general, yam bean seed proteins showed an excellent balance of all essential amino acids; albumins contain the highest amount of essential amino acids.     

Involvement of proteins in cloud instability of shamouti orange [Citrus sinensis (L.) Osbeck] juice.

The present study provides evidence for the involvement of protein in cloud instability of natural orange juice. No heat-coagulable proteins were found in the serum. Insoluble cloud matter (ICM) was heat-flocculated following enzymatic pectin degradation (EPD). The degree of flocculation depended on temperature (from approximately 50 to 75 degrees C) and was highest at pH 3.5. The fresh juice contained about 6.5 and 1.8 mg mL(-1) of ICM and alcohol-insoluble solids of the serum (AISS), respectively. The ICM and the AISS contained, respectively, proteins (182+/-14 and 119+/-3 microg mg(-1)), galacturonic acid (37+/-6.6 and 175+/-1 microg mg(-1)), and neutral sugars (350+/-44 and 338+/-22 microg mg(-1)). EPD resulted in removal of a marked portion of the pectin and was accompanied by partial removal of neutral sugars (mainly glucose and galactose) and some proteins from the pectic polymer in both AISS and ICM. Under electrophoresis, proteins of the AISS included bands in the range of 20-52 kDa and 10-14 kDa and those of the ICM at 22 and 50 kDa.     

Active recombinant thioredoxin h protein with antioxidant activities from sweet potato (Ipomoea batatas [L.] Lam Tainong 57) storage roots

Recombinant thioredoxin h (Trx2) overproduced in Escherichia coli (M15) was purified by Ni2+-chelated affinity chromatography. The molecular mass of Trx2 is approximately 1.4 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Total antioxidant status, 1,1-diphenyl-2-picrylhydrazyl (DPPH) staining, reducing power method, Fe2+-chelating ability, ferric thiocyanate (FTC) method, and protection of calf thymus DNA against hydroxyl radical-induced damage were studied. The thioredoxin h protein with a concentration of 12.5 mg/mL exhibited the highest activity (expressed as 0.37 +/- 0.012 mM ABTS* radical cation being cleared) in a total antioxidant status test. In the DPPH staining thioredoxin h appeared as white spots when it was diluted to 50 mg/mL (a final amount of 15 microg). Like the total antioxidant status, the reducing power, Fe2+-chelating ability, FTC activity, and protection against hydroxyl radical-induced calf thymus DNA damage were found with the thioredoxin h protein. It was suggested that thioredoxin h might contribute to its antioxidant activities against hydroxyl and peroxyl radicals.     

Effect of pulsed-light treatment on milk proteins and lipids

Pulsed-light treatment offers the food industry a new technology for food preservation. It allows the inactivation of numerous micro-organisms including most infectious foodborne pathogens. In addition to microbial destruction, one can also question whether pulsed-light treatment induced conformational changes in food components. To investigate this question, the influence of pulsed-light treatment on protein components of milk was evaluated by using UV spectroscopy, spectrofluorometry, electrophoresis, and determination of amino acid composition. Pulsed-light treatment resulted in an increase of UV absorbance at 280 nm. The intrinsic tryptophan fluorescence of beta-lactoglobulin (BLG) showed a 7 nm red shift after 10 pulses. SDS-PAGE showed the formation of dimers after treatment of BLG by 5 pulses and more. No significant changes in the amino acid composition of proteins and lipid oxidation were observed after pulsed-light treatment. The obtained results indicated changes in the polarity of the tryptophanyl residue microenvironment of BLG solutions or changes in the tryptophan indole structure and some aggregation of studied proteins. Hence, pulsed-light treatment did not lead to very significant changes in protein components; consequently, it could be applied to process protein foods for their better preservation.     

Principal component similarity analysis of Raman spectra to study the effects of pH,heating, and kappa-carrageenan on whey protein structure

Raman spectroscopy was used to elucidate structural changes of beta-lactoglobulin (BLG), whey protein isolate (WPI), and bovine serum albumin (BSA), at 15% concentration, as a function of pH (5.0, 7.0, and 9.0), heating (80 degrees C, 30 min), and presence of 0.24% kappa-carrageenan. Three data-processing techniques were used to assist in identifying significant changes in Raman spectral data. Analysis of variance showed that of 12 characteristics examined in the Raman spectra, only a few were significantly affected by pH, heating, kappa-carrageenan, and their interactions. These included amide I (1658 cm(-1)) for WPI and BLG, alpha-helix for BLG and BSA, beta-sheet for BSA, CH stretching (2880 cm(-1)) for BLG and BSA, and CH stretching (2930 cm(-1)) for BSA. Principal component analysis reduced dimensionality of the characteristics. Heating and its interaction with kappa-carrageenan were identified as the most influential in overall structure of the whey proteins, using principal component similarity analysis.     

Detection of proteins related to starch synthase activity in the developing mungbean (Vigna radiata L.)

Proteins associated with starch synthase (SS) activities were identified in immature mungbeans (Vigna radiata L. cv KPS1). Seed soluble extract was separated by native-PAGE and subjected to in situ activity staining. The gel zymogram located starch-enzyme complex bands. The soluble extract was also partitioned by preparative-IEF and screened for SS activity using radioactive assay. IEF fractions eluted within pH 4-6 revealed enriched SS activity of 145-fold. Parallel comparison of the protein profiles among the activity stained enzyme complex and the active isoelectric focused fractions on SDS-PAGE depicted three SS-activity-related proteins with molecular size of 32, 53, and 85 kDa. The 85 kDa protein, however, was identified to be methionine synthase by MALDI-TOF analysis and should be a protein physically associated with the active SS. Polyclonal antibodies raised from eluted native enzyme complex neutralized up to 90% activity and antigenically recognize the other 53 and 32 kDa proteins on Western blot. Antibodies raised from the two individual denatured proteins were able to neutralize SS activities near 60% separately, indicating that the 53 kDa and 32 proteins associated with SS activity are potentially involved in starch biosynthesis during mungbean seed development.     

Monoclonal antibodies specific to thermostable proteins in animal blood

Bovine plasma proteins are used as high-quality protein supplements in animal feed and as binders or colorants in food for human consumption. Religious observance, as well as recent fears of epidemic bovine spongiform encephalopathy, highlights the need for methods to detect bovine blood in processed food and animal feed for regulatory purposes, as the currently available methods are neither species-specific, blood-specific, nor valid for excessively heat-processed samples. This paper reports the development of monoclonal antibodies (MAbs) raised against bovine thermostable plasma proteins that display a unique species specificity pattern for plasma proteins. Immunoblotting revealed several thermostable antigenic proteins (10, 25, 40, and 60 kDa) in bovine plasma sterilized at 121 degrees C for 15 min. These MAbs can be employed individually or combined in immunoassays for analytical purposes and investigations of the chemical and biological properties of the thermostable plasma proteins identified here.     

Characterization of methanol-soluble and methanol-insoluble proteins from developing peanut seed

The 85% methanol-soluble proteins are known to specifically contribute to the production of flavor of roasted peanut. To determine the nature of the 85% methanol-soluble proteins, they were isolated from the peanut seed, and the 85% methanol-soluble (MS) and 85% methanol-insoluble (MIS) fractions were characterized using polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis. The results showed that the 85% MS fraction contained lower amounts (9-10%) of protein than the MIS fraction (15-33%). Protein content of the MIS fraction increased more significantly during seed maturation than it did in the MS fraction. Unlike the protein, free amino acids and soluble sugars levels of the MS fraction decreased significantly during seed maturation. The 85% MS fraction contained predominantly low molecular weight (<20 kDa) proteins/polypeptides, whereas the MIS fraction contained a mixture of polypeptides with molecular weight between 14 kDa and 90 kDa. SDS-PAGE showed no major changes in the polypeptide composition of the MS fraction during seed maturation. Capillary electrophoretic analysis revealed major qualitative and quantitative changes in the protein and polypeptide composition of the MS and MIS fractions during seed maturation. Fatty acid analysis of these fractions indicated that the MS fraction is lipoprotein in nature and rich in oleic and linoleic acids.     

Insecticidal activity of Arum maculatum tuber lectin and its binding to the glycosylated insect gut receptors

Mannose binding approximately 50 kDa homotetrameric lectin, purified from edible Arum maculatum tuber, was analyzed through SDS-PAGE and studied for its agglutination property using rabbit erythrocytes. Cross reactivity of the purified lectin was verified through western blot using Colocasia esculantum(Family, Araceae) tuber lectin antibody. The insecticidal activity of Arum maculatum tuber lectin (ATL) was tested against two economically important sucking pests, Lipaphis erysimi and Aphis craccivora, in an artificial diet. The LC(50) values for L. erysimi and A. craccivora were determined to be 21 microg/mL and 16 microg/mL, respectively. Addition of alpha-d-mannose in ATL-supplemented diet reduced the aphid mortality. Two major receptor proteins of ATL (approximately 40 kDa and approximately 35 kDa) were detected from the brush border membrane vesicle (BBMV) protein of L. erysimi and A. craccivora guts, respectively, using ligand-binding assay. Alpha-d-Mannose was found to be a deterrent to such binding of ATL to the BBMV receptors.     

Antioxidant activities of trypsin inhibitor, a 33 KDa root storage protein of sweet potato (Ipomoea batatas (L.) Lam cv. Tainong 57).

Trypsin inhibitors (TIs), root storage proteins, were purified from sweet potato (Ipomoea batatas[L.] Lam cv. Tainong 57) roots by trypsin affinity column according to the methods of Hou and Lin (Plant Sci. 1997, 126, 11-19 and Plant Sci. 1997, 128, 151-158). A single band of 33 kDa TI was obtained by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. This purified 33 kDa TI had scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. There was positive correlation between scavenging effects against DPPH (2 to 22%) and amounts of 33 kDa TI (1.92 to 46 pmol). The scavenging activities of 33 kDa TI against DPPH were calculated from linear regression to be about one-third of those of glutathione between 5 and 80 pmol. Using electron paramagnetic resonance (EPR) spectrometry for hydroxyl radical detection, it was found that 33 kDa TI could capture hydroxyl radical, and the intensities of EPR signal were significantly decreased from 1.5 to 6 pmol of 33 kDa TI compared to those of the controls. It is suggested that 33 kDa TI, one of the sweet potato root storage proteins, may play a role as an antioxidant in roots and may be beneficial to health when it is consumed.     

Cloning and characterization of an antifungal class III chitinase from suspension-cultured bamboo ( Bambusa oldhamii ) cells

A class III chitinase cDNA (BoChi3-1) was cloned using a cDNA library from suspension-cultured bamboo ( Bambusa oldhamii ) cells and then transformed into yeast ( Pichia pastoris X-33) for expression. Two recombinant chitinases with molecular masses of 28.3 and 35.7 kDa, respectively, were purified from the yeast's culture broth to electrophoretic homogeneity using sequential ammonium sulfate fractionation, Phenyl-Sepharose hydrophobic interaction chromatography, and Con A-Sepharose chromatography steps. N-Terminal sequencing and immunoblotting revealed that both recombinant chitinases were encoded by BoChi3-1, whereas SDS-PAGE and glycoprotein staining showed that the 35.7 kDa isoform (35.7 kDa BoCHI3-1) was glycosylated and the 28.3 kDa isoform (28.3 kDa BoCHI3-1) was not. For hydrolysis of ethylene glycol chitin (EGC), the optimal pH values were 3 and 4 for 35.7 and 28.3 kDa BoCHI3-1, respectively; the optimal temperatures were 80 and 70 degrees C, and the K(m) values were 1.35 and 0.65 mg/mL. The purified 35.7 kDa BoCHI3-1 hydrolyzed EGC more efficiently than the 28.3 kDa isoform, as compared with their specific activity and activation energy. Both recombinant BoCHI3-1 isoforms showed antifungal activity against Scolecobasidium longiphorum and displayed remarkable thermal (up to 70 degrees C) and storage (up to a year at 4 degrees C) stabilities.     

Characterization of haze-active proteins in apple juice

The nature of the haze-active protein (HAP) in apple juice was investigated. Heat treatment removed protein indiscriminately while polyvinylpolypyrrolidone (PVPP) treatment was fairly specific for proteins of 15 and 28 kDa. Presumably, the PVPP bound to polyphenols, which in turn were complexed with protein. Three candidate apple HAPs were isolated. Two were extracted from juice with acetone and fractionated by hydrophobic interaction chromatography and solid phase extraction with C18 (HAP I) or SAX (HAP II) material. Hydroxyproline-rich protein was extracted from apple tissue (HAP III). The order of haze formation with tannic acid was gliadin > HAP III > HAP II > HAP I > bovine serum albumin, which shows increasing haze formation with increasing proline content. The sizes of HAP I, II, and III were 28, 15, and 12 kDa; the first two corresponded to the sizes of proteins removed by PVPP treatment and are involved in juice haze formation.     

Involvement of proteins in cloud instability of valencia orange [Citrus sinensis (L.) Osbeck] juice.

The present study examined the involvement of proteins in cloud flocculation of Valencia orange juice. Marked differences in cloud instability were found between juices of different harvest dates. Heating of enzymatic pectin degraded juice from April and June harvests resulted in development of clumps and their precipitation. Although the juice from both harvesting dates remained hazy, the juice of April harvest was more turbid than that from June. Usually clarification increases as the temperature increases from ambient to 125 degrees C. Clarification occurred at pH 2.5-4.5 and was maximal at pH 3.5. The clarification of the April harvest juice was markedly lower than that of the June harvest. The fresh juice contained about 5.2 and 1.7 mg mL(-1) insoluble cloud matter (ICM) and alcohol-insoluble serum solids (AISS), respectively. The ICM and the AISS, respectively, contained: proteins (244.5+/-8.7 and 132+/-1.8 microg mg(-1)), galacturonic acid (40+/-0 and 120+/-0 microg mg(-1)) and neutral sugars (270+/-39 and 329+/-23 microg mg(-1)). Enzymatic pectin degradation resulted in removal of a marked portion of the pectin, and was accompanied by partial removal of neutral sugars (mainly glucose and galactose) and some proteins from the pectic polymer in both AISS and ICM. Proteins of the AISS included major bands at 10-14, 20, and 28 kDa and those of the ICM bands at 22, 24, 26, and 45 kDa.     

Jojoba seed meal proteins associated with proteolytic and protease inhibitory activities

The jojoba, Simmondsia chinensis, is a characteristic desert plant native to the Sonoran desert. The jojoba meal after oil extraction is rich in protein. The major jojoba proteins were albumins (79%) and globulins (21%), which have similar amino acid compositions and also showed a labile thrombin-inhibitory activity. SDS-PAGE showed two major proteins at 50 kDa and 25 kDa both in the albumins and in the globulins. The 25 kDa protein has trypsin- and chymotrypsin-inhibitory activities. In vitro digestibility of the globulins and albumins resembled that of casein and soybean protein concentrates and was increased after heat treatment. The increased digestibility achieved by boiling may be attributed to inactivation of the protease inhibitors and denaturation of proteins.