Mahmoud Sitohy

Degradability of different phosphorylated starches and thermoplastic films prepared from corn starch phosphomonoesters

Different starch types (corn, rice, potato, corn amylose and corn amylopectin) were phosphorylated to varying degrees of substitution (DS) and tested both for acid hydrolysis during 3 h in a boiling bath and for enzymatic hydrolysis with a thermostable bacterial α-amylase (Bacillus licheniformis) for 30 min at 95 °C. Generally, phosphorylated starches showed a reduced degree of acid hydrolysis during the entire time of hydrolysis (3 h) as well as reduced susceptibility to α-amyIase hydrolysis. The enzyme action was inhibited by the presence of phosphate groups in the modified starch molecules and the extent of inhibition increased with increasing degree of phosphate substitution, regardless of the starch type. Thermoplastic films were fabricated by blending modified corn starches of different DS with polyacrylate, urea and water at a ratio of 4:5:1:50, heating for 30 min at 95 °C before casting and allowing to cool, stand and dry at room temperature. The plastic films prepared from phosphorylated corn starch showed both higher disintegration rate and a greater degradability by thermostable bacterial α-amylase than the ones prepared from non-phosphorylated starch. These new acquired properties can meet the increasing demand for biodegradable disposable plastic bags.

Proteolysis by Lactobacillus fermentum IFO3956 isolated from Egyptian milk products decreases immuno-reactivity of αS1-casein.

Proteinase activity of Lactobacillus fermentum IFO3956 cells was higher when they were grown on milk-based media than on 10% reconstituted skim milk. The lowest protease activity was observed when cells were grown on milk-free media. The extraction of milk-induced cell-bound proteases from Lb. fermentum IFO3956 was most efficient using 1% Tween 80 while the use of 1% SDS inhibited all proteolytic activity. Two bands of ∼35 and >100 kDa were observed by zymogram, indicating that proteolytic activity corresponded to the presence of at least two types of enzymes or two molecular forms of one enzyme. Mass spectrometry analyses of αS1-casein hydrolysates detected 24 peptides with sizes ranging from 5 to 36 amino acids, including 9 phosphorylated peptides, resulting from the fermentation of Lb. fermentum IFO3956 of αS1-casein. Most of the identified peptides originated from the N-terminal portion of αS1-casein. The studied bacterial strain could hydrolyze αS1-casein in many sites including the epitopes triggering the allergic reactions against αS1-casein e.g. at the positions 23, 30, 41, 71, 91, 98, 126, 179. After hydrolysis of αS1-casein with Lb. fermentum IFO3956 the recognition and the binding of this casein to IgE from the pooled sera of 18 patients with cows milk allergy was significantly reduced.

Anticytomegaloviral Activity of Esterified Milk Proteins and L-Polylysines

MRC-5 fibroblasts infected with human cytomegalovirus (HCMV) reference strain AD 169 were treated with different concentrations of methylated α-lactalbumin (Met-ALA) or methylated β-lactoglobulin (Met-BLG), as well as with their peptic hydrolysates, and with the highly basic polypeptides such as are L-polylysines (4–15 kDa). The antiviral activity was calculated by comparing the number of infected cells in the presence and absence of the tested substances. Both Met-ALA and Met-BLG, as well as their peptic hydrolysates, decreased the infectious activity of cytomegalovirus in fibroblast cells. As expected, L-polylysines showed the highest antiviral activity. However, the tested basic proteins and polypeptides despite their lower antiviral activities might be potentially quite useful in fight of arising drug resistance activities and the persistence capacities of this virus.

Susceptibility to trypsinolysis of esterified milk proteins

Methyl-, ethyl- and propyl-esters of beta-lactoglobulin, alpha-lactalbumin and beta-casein were prepared and then hydrolyzed with trypsin in various conditions. Resulting hydrolysates were analysed by SDS electrophoresis and RP-HPLC. The degree of hydrolysis of esterified samples was generally lower than those of the non-modified proteins. The highest degrees of hydrolysis were obtained at pH 7--8 with native and esterified protein samples. beta-Lactoglobulin propyl ester and beta-casein methyl ester yielded the lowest degrees of hydrolysis. Ethyl- and propyl-esters of beta-casein showed high resistance towards tryptic attack, even after 20 h of hydrolysis. SDS electrophoretic patterns of tryptic hydrolysates of native proteins showed bands corresponding to low molecular weights. Tryptic hydrolysates of esterified proteins showed bands with higher sizes. RP-HPLC profiles of tryptic hydrolysates of esterified samples showed peaks with longer elution times than those obtained with native proteins, indicating the presence of more hydrophobic peptide populations. A peptic pre-treatment improved tryptic action on esterified proteins. It resulted in a better resolution of RP-HPLC profiles and in a complete disappearance of the protein after 20 h tryptic hydrolysis.

Influenza virus A subtype H1N1 is inhibited by methylated β-lactoglobulin

Addition of methylated β-lactoglobulin (Met-BLG) in the medium of MDCK cell lines infected with influenza virus subtype H1N1 reduced hemagglutination activity (HA) in a concentration dependent manner. Antiviral activity of Met-BLG depended on its concentration, viral load, and duration of infection. Using 17 μg/ml of Met-BLG inhibited 50% of HA of H1N1 grown in MDCK cells at 1 MOI after 24 h incubation at 37°C and in 5% CO₂. Extension of incubation time enhanced antiviral action since the same concentration of Met-BLG inhibited about 61% of viral activity after 48 h. This viral inhibition was accompanied by a protection of MDCK cells as observed by using neutral red or by direct microscope examination. Reduction of viral RNA replication upon the addition of Met-BLG (50 μg/ml) was observed by real time-PCR showing a reduction of viral log value of about 0·9. When viral stock solution was mixed with 25 μg/ml Met-BLG in absence of cell lines, the morphology and viability of virus particles were significantly affected as observed by electron microscopy, and the number of intact virus particles was reduced by roughly 65%.

Amino acid grafting of β-lactoglobulin mediated by phosphorus oxychloride

β-Lactoglobulin was phosphorylated with 80 mol of POCI3/mol protein in the presence of triethylamine and amino acids or their esters added at a total molar excess of 6 mol base/mol POCI3. The extent of phosphorylation was reduced when the amino acids replaced triethylamine as the base. Arginine and lysine were grafted to protein molecules in amounts proportional to the β-lactoglobulin phosphorylation, while histidine grafting was very weak. The electrophoretic patterns of the modified proteins showed increased negative charges, reduced isoionic points and slight dimerization. The emulsifying properties of the modified proteins were improved.

Esterified milk proteins inhibit DNA replication in vitro

DNA replication was studied in vitro in the presence of native and esterified milk proteins [alpha-lactalbumin (ALA), beta-lactoglobulin (BLG) and beta-casein (BCN)]. Addition of unmodified proteins to the PCR medium did not change the result of the reaction seen by electrophoresis, even at excessive ratios of basic amino acids in proteins:phosphate groups in DNA as high as 100:1. Addition of esterified proteins greatly reduced the intensity of the bands corresponding to the newly synthesized DNA, at ratios as low as 1:1 and 5:1 in case of methylated-BLG and methylated-ALA, respectively. The inhibitory effect of esterified proteins was directly proportional to their extent of esterification and strongly related to their DNA-binding capacity. Generally, inhibition of PCR with esterified proteins was similar to what can be observed with histones. However, stronger inhibition was observed with highly esterified proteins when using a higher ratio of basic:acid residues (1:1) when compared with 0.5:1 ratio in case of histones. Highly esterified BCN did not exert any inhibitory effect because of its relatively lower pI when compared with that of other esterified milk proteins and due to its lower positive net charge at the pH used for PCR. During a second PCR run, only the addition of new DNA template was able to reinitiate the reaction, giving rise to new synthesized DNA. Addition of Taq DNA polymerase did not enhance DNA synthesis, showing that inhibition was performed only by binding of DNA template and not by the inhibition of the polymerase.

Mild method of simultaneous methionine grafting and phosphorylation of soybean globulins improves their functional properties

Soybean 7S and 11S globulins were phosphorylated to different extent as a function of the molar ratios of POCl 3 used (75, 150 and 300 mol POCl 3 /mol protein) in the presence of equivalent amounts of triethylamine. Simultaneous addition of L-methionine during the phosphorylation yielded methionylated globulins. In some experiments, the basic amino acid arginine was used as the sole base of the reaction, instead of triethylamine, at a ratio of 6 mol arginine/mol POCl 3 . Adding L-methionine in the course of the phosphorylation reaction reduced slightly the extent of protein phosphorylation, with increasing L-methionine content in the phosphorylated globulin up to about 280% of its original level. The use of L-arginine as the reaction base led to lower phosphorylation yields but could raise simultaneously both L-methionine and L-arginine to about 143% and 155% of their original levels, respectively. Isofocusing electrophoresis showed lower isoelectric points of the phosphorylated samples, compared to the starting aliquots. SDS-PAGE did not show major changes in the subunit distribution of the modified soybean globulins. Phosphorylation and amino acylation changed the solubility of the modified samples and also other related functional properties e.g. foaming and emulsifying which were remarkably improved, especially for the samples with grafted L-methionine.

Phosphorylation of β-lactoglobulin using amino acids as the sole base and nucleophile of the reaction

Abstractβ-Lactoglobulin was phosphorylated with 20, 40, and 80 mol of POCl3/mol protein in the presence of 4, 5, and 6 molar excess of basic amino acid per mol POCl3. Maximal phosphorylation yields of 5 and 3 mol P/mol protein were achieved when the highest stoichiometries of POCl3/arginine and lysine were used. Proportional high amounts of basic amino acids were also grafted to the protein molecule during its phosphorylation through the phosphoamide bond. Modified proteins displayed increased negative charges and reduced isoelectric points and were monomeric. The phosphorylated and phosphoamidatedβ-lactoglobulin showed improved functional properties.