Composition Comprising Carbohydrates and Peptides which Comprise Tryptophan
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example 1
[0085]Hydrolysing Lysozyme Using Protex and Identity of the Peptides Formed.
[0086]A solution containing 10% (w / w) pure lysozyme was adjusted to pH 8.2 using NaOH and heated to 52 degrees C. Hydrolysis was started by adding 25 microliter of Protex / g of protein present. Under continuous stirring and maintaining the pH at 8.2, the hydrolysis was continued for 5.5 hours to yield an almost clear solution without a visible precipitate. After a heating step to inactivate the Protex activity, a sample was taken for DH analysis. The DH of the solution turned out to be almost 30%. The heat treated solution was ultrafiltered over a 10 kDa filter to yield a completely clear liquid. This clear liquid was used for LC / MS analysis, for molecular weight distribution of peptides and proteins present as well as for ion exchange chromatography.
[0087]To get an impression of the molecular weight distribution of peptides and proteins present, the clear liquid was subjected to a molecular size analysis as ...
example 2
[0090]Increasing the Tryptophan Content of the Hydrolysate.
[0091]Lysozyme incorporates a surprising high amount of the basic arginine and lysine residues. Furthermore the lysozyme molecule incorporates a significant number of the acid glutamate and aspartate residues. This data has been used to devise an innovative and elegant purification route towards hydrolysates featuring high tryptophan / LNAA ratios. Essential requirement for this purification route is, however, that only very few of the tryptophan residues show up in peptides also containing either an arginine or lysine residue or a glutamate or aspartate residue. As shown in Example 1, the specific hydrolysis route used here yields only few trytophan containing peptides containing an arginine residue and no peptides containing a lysine, glutamate or aspartate residue.
[0092]Theory predicts that a maximal charge difference between peptides with and without a glutamate or aspartate residue can be achieved around pH 3. A maximal c...
example 3
[0094]Large Scale Lysozyme Hydrolysis.
[0095]In larger scale lysozyme hydrolysis procedures, essentially the procedure as described in Example 1 was followed with some minor modifications. A solution containing 7.3% (w / w) pure lysozyme was heated to 65 degrees C. after which the pH was adjusted to pH 8.2 using NaOH. Hydrolysis was started by adding 25 microliter of Protex 6 L / g dry matter. Under continuous stirring and maintaining the pH at 8.2 and the temperature at 53 degrees C., the hydrolysis was continued for 2 hours. Then the pH value was increased to 9.0 and incubation was pursued for another 3.5 hours to yield a solution with some precipitate. Then the pH of the solution was lowered to 4.5 and the solution was cooled to below 4 degrees C. To obtain a completely clear product, the liquid was filtered over a Z 2000 filter (Pall) and subsequently excess water and salt was removed via nanofiltration. The resulting concentrate was then subjected to an UHT treatment of 7 seconds at...
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