Jean Amiot

High Performance Size Exclusion Liquid Chromatography of Small Molecular Weight Peptides from Protein Hydrolysates Using Methanol as a Mobile Phase Additive

Abstract The separation of low molecular weight peptides according to their molecular weight has been a challenge. The earlier works using chaotropic additives in the mobile phase such as SDS or guanidium chloride failed to give a linear response for the semilogarithmic graph of molecular weight versus retention time. Here we report a mobile phase composition suited to the size exclusion separation of the peptides of molecular weight between 6000 and 250 on a TSK-SW 2000

Application of reversed-phase high-performance liquid chromatography to the separation of peptides from phosphorylated and dephosphorylated casein hydrolysates

Abstract Peptides from phosphorylated and dephosphorylated casein hydrolysates were fractionated on a TSK G2000SW size-exclusion column. The fractionated peptides were separated by reversed-phase high-performance liquid chromatography on a C 18 column using aqueous trifluoroacetic acid as the mobile phase and acetonitrile as the mobile phase modifier in the linear gradient elution system. The separation of the dephosphorylated and phosphorylated hydrolysates gave 213 and 187 peptides, respectively, of which 116 and 99, respectively, were reported. A study of their composition and retention times verified that the peptide separation mechanism includes ionic interactions, hydrogen bonding and peptide characteristics, in addition to overall peptide hydrophobicity.

Cationic balance in skim milk during bipolar membrane electroacidification

Abstract Bipolar membrane electroacidification (BMEA) technology has been applied to skim milk protein, in order to produce high purity casein isolates. BMEA uses a property of bipolar membrane to split water and the action of monopolar membranes for demineralization. When the H + are generated at the bipolar membrane, one cationic charge inherent to the solution being acidified must cross the cation exchange membrane (CEM) to keep the solution electrically neutral. The purpose of this study was to monitor the migration of charge through the CEM to counterbalance H + generated at the bipolar membranes. K + ions were identified as being the predominant ions that electromigrate from the skim milk solution until pH 5.0 is reached, at which point its concentration becomes insufficient to counterbalance H + . The other cations partially replaced K + to assure the electroneutrality of the solution. It appears that K + is a necessary ionic species to ensure the best efficiency of the BMEA and to limit the non-desired migration of H + across the CEM. Therefore, the electrical efficiency of BMEA is decreased by a loss of electrogenerated H + due to a lack of sufficiently mobile ions such as potassium. Consequently, the enrichment of the skim milk with some potassium would be required in order to obtain a better electrical efficiency.

Protease production by immobilized growing cells of Serratia marcescens and Myxococcus xanthus in calcium alginate gel beads

SummarySerratia marcescens and Myxococcus xanthus cells were immobilized in calcium alginate gel beads. Immobilization under various conditions had no effect on the extracellular proteolytic activity of S. marcescens cells. Protease production seemed rather to depend on the free cells in the medium. However, the stability over time of enzyme production was enhanced, as immobilization increased protease production half-life from 5 to 12 days. On the other hand, Myxococcus xanthus produced proteases inside the gel beads which could diffuse into the medium. The proteolytic activity increased as a function of the initial cell content of the beads and of the bead inoculum. Compared to free cells, immobilized cells of Myxococcus xanthus could produce 8 times more proteolytic activity, with a very low free-cell concentration in the medium.

Effect of membrane permselectivity on the fouling of cationic membranes during skim milk electroacidification

Abstract A previous study on skim milk demonstrated that bipolar-membrane electroacidification (BMEA) is a technology that can be used to produce isoelectric precipitation of casein from milk. However, a deposit, suspected to be calcium hydroxide, was observed on the cation-exchange membrane (CEM) side in contact with the base. Also, the performance of the process decreased as well as the lifetime of the membrane. The aim of the present study was therefore to evaluate two types of CEM with different permselectivities in terms of electrodialysis efficiency and membrane parameters. BMEA allowed the separation of high purity bovine milk casein (about 97% protein), and the permselectivity of both membranes tested does not influence the purity of the isolates produced. However, the migration of cations through the cationic membrane was found to be influenced by the permselectivity leading to isolates with different ash contents. The CSV™ membrane slowed down the migration of cations in comparison with CMX™ membranes. The membrane fouling of cation-exchange membrane was identified as precipitating calcium and magnesium hydroxides. Moreover, the fouling formed at the outer layer surface of the CEM was demonstrated to be reversible for both membranes while the fouling of the CSV™ membrane inner layer was irreversible.

Nutritive value of protein fractions extracted from soybean, rapeseed and wheat flours in the rat

The nutritive value of various protein fractions was studied. Fractions 2S and 12S from rapeseed, 2S, 7S and 11S from soybean were obtained by dissolution in ammonium sulfate solutions. Albumin-globulin, gluten, glutenin and gliadin fractions from wheat were obtained by dissolution in salted water (albumin-globulin), acetic acid (glutenin) and alcohol (gliadin). Liveweight gains, protein efficiency ratio (PER) and apparent digestibility coefficient (ADC) were used as measures of the nutritive value. The protein fractions had a lower nutritive value than the unfractionated proteins except for the albumin-globulin fraction of wheat which had a nutritive value higher than that of the unfractionated wheat protein. PER obtained with the rapeseed 2S and 12S fractions were 2.49 and 2.21, respectively, as compared to 2.64 for unfractionated rapeseed. With soybean fractions, PER were 0.92 for 2S, — 0.007 for 7S and 1.47 for 11S, as compared to 2.19 for the original protein. The wheat albumin-globulin fraction gave a PER of 2.78, as compared to 1.45 for the unfractionated wheat protein. Gluten, glutenin and gliadin fractions had a lower PER than that of unfractionated wheat protein. ADC of all fractions were higher than those of the original proteins. The difference in liveweight gains and PER observed between protein fractions can be partially explained on the basis of the essential amino acid content.

Neutralization of hydroxide generated during skim milk electroacidification and its effect on bipolar and cationic membrane integrity

Abstract The aim of the present study was to evaluate the effect of neutralizing the OH− electrogenerated during bipolar membrane electroacidification (BMEA) on the integrity of the cationic and bipolar membranes used. The OH− were neutralized by acidification of the anionic side compartment of the bipolar membrane during BMEA of skim milk. The integrity of the membranes was characterized by membrane parameters and microscopic membrane surface elemental analysis and mapping. It appeared from these results that the resistance of cationic membranes increased, due to an exchange of monovalent counter-cations of the membrane by divalent cations less mobile and to a slight protein fouling. However, integrity and physical characteristics of bipolar membranes were preserved during BMEA.

Enhancement of glucose uptake in muscular cell by soybean charged peptides isolated by electrodialysis with ultrafiltration membranes (EDUF): Activation of the AMPK pathway

Soy peptides consumption has been associated with beneficial effects in type 2 diabetes patients. However, the peptide fractions responsible for these effects, and their mechanisms of action, have not been identified yet. In this study, we have isolated soybean peptides by electrodialysis with an ultrafiltration membrane (EDUF) at 50 V/100 kDa, and tested them for their capacity to improve glucose uptake in L6 muscle cells. We observed that these fractions were able to significantly enhance glucose uptake in the presence of insulin. The reported bioactivity would be due to the low molecular weight peptides (300-500 Da) recovered. Moreover, we observed that an enhancement of glucose uptake was correlated to the activation of the AMPK enzyme, well known for its capacity to increase glucose uptake in muscle cells. To our knowledge, this is the first time that bioactive peptides with glucose uptake activity have been isolated from a complex soy matrix, and that the implication of AMPK in it is demonstrated.

High-performance liquid chromatography of casein hydrolysates phosphorylated and dephosphorylated. I. Peptide mapping.

A mixture of small peptides of molecular weight averaging 1000 daltons, obtained by controlled hydrolysis of casein with proteases, chymotrypsin and trypsin, was separated by size-exclusion and reversed-phase high-performance liquid chromatography. Peptides were identified and located in the known casein structures from their amino acid content and their N- and C-terminal amino acid analyses. The primary structure of peptides identified from casein hydrolysate phosphorylated and casein hydrolysate dephosphorylated is presented.

Biological availability of calcium and magnesium from dairy products

Abstract The bioavailability of calcium (Ca) and magnesium (Mg) from dairy products was studied in rats. Apparent intestinal absorption, bone deposition of minerals and bone resistance were measured. The results indicate that apparent absorption of calcium from cheese was higher than from skim milk, evaporated milk, yogurt and skim milk powder in the young animal, while in adults apparent absorption of calcium was lower from cheese than from other milk products. Nevertheless, the deposition of calcium in tibia and femur bones was the same for all diets. For magnesium, apparent absorption results indicated lower values for cheese than for other dairy products in the young and adult animal. This lower absorption was associated with a lower bone deposition from cheese. Bone resistance was the same for all dairy products.