Method for the preparation of gelatin hydrolysate having a low endotoxin content

Abstract

Described is a method for the preparation of a gelatin hydrolysate having a decreased endotoxin content, comprising the steps of incubating a solution of gelatin of gelatin hydrolysate at a temperature of 70-125° C. at a pH of 3.5 or less for a time period of at least 15 minutes, and recovering the gelatin hydrolysate. Further a gelatin hydrolysate thus obtained is described.

Claims

1. A method for the preparation of a gelatin hydrolysate having a decreased endotoxin content, comprising the steps of: a) incubating a solution of gelatin or gelatin hydrolysate at a temperature of 80-96° C. at a pH of 3.5 or less for a time period of at least 15 minutes, and b) recovering the gelatin hydrolysate having a decreased endotoxin content, wherein in the method, no surfactant is added to the gelatin or gelatin hydrolysate.

2. The method of claim 1, wherein the pH is 2.7 or less.

3. The method of claim 1, wherein the pH is 3.0 or less.

4. The method of claim 1, wherein the time period is 30 minutes or more.

5. The method of claim 4, wherein the time period is 1-5 hours.

6. The method of claim 1, being free of an enzymatic treatment step.

7. The method of claim 1, wherein in step a) the gelatin or gelatin hydrolysate has a molecular weight of more than 70 kDa.

8. The method of claim 1, wherein the gelatin hydrolysate of step b) has a molecular weight of 30 kDa or less.

9. The method of claim 1, wherein the gelatin hydrolysate of step b) has an endotoxin level of 20 EU/g gelatin hydrolysate or less.

Description

EXAMPLE 1

(1) Effect of Temperature and pH on Endotoxin Levels in Gelatine Hydrolysate

(2) Temperatures varied from 60, 70, 80, 90, and 95° C.,

(3) pH values were 2, 2.5, 3, ‘5’(unchanged),

(4) Hydrolysis was performed up to 3 hours.

(5) Gelatin used: #140P117162B1, pig skin gelatin type A, Rousselot, Ghent, Belgium (high bloom, a molecular weight (MW) of 150 kDa, having an endotoxin content of 14000 EU/g).

(6) For each hydrolysis temperature/pH value, 20 g gelatin was dissolved in 180 g water by heating at 55° C. for at least 30 min to prepare 200 g of a 10% gelatin solution. It is also possible to prepare a solution having a lower or higher gelatin concentration.

(7) Samples of 10 g were taken and these were put in to a water bath that has the correct hydrolysis temperature until the solution reached this temperature (±3° C.).

(8) For pH analysis, a 5M H.sub.2SO.sub.4 solution was used; the needed amounts of acid per pH were been determined with the aid of a titration curve.

(9) Take samples were incubated for 0.1-0.5-1-1.5-2-3 h.

(10) At the end of the envisaged incubation period, 10M NaOH was admixed to bring the sample pH to 4.9 with the aid of a titration curve, as outlined in table 1, and the samples were kept in the freezer at −20° C. until the endotoxin level was measured.

(11) TABLE-US-00001 TABLE 1 titration values for NaOH addition pH 10M NaOH [μL] 2 .fwdarw. ~4.9 19.3 2.5 .fwdarw. ~4.9   14.8 3 .fwdarw. ~4.9 11.8 4 .fwdarw. ~4.9 5.9

(12) For the measurements of endotoxin level, the samples were thawed in a 40° C. water bath.

(13) The molecular weight has been measured according to the method described by Zhang, supra, and is shown in tables 2 and 3.

(14) TABLE-US-00002 TABLE 2 molecular weight at 95° C. at different pH pH 2.0 and pH 2.5 and pH 2.7 and 95° C. 95° C. 95° C. Time (h) MW (Da) MW (Da) MW (Da) 0 155 155 155 1 9.5 11.8 12.0 2 7.0 8.8 8.5 3 6.4 7.4 7.5 4 5.4 6.7 6.7

(15) TABLE-US-00003 TABLE 3 molecular weight at pH 2.0 at different temperature pH 2.0 and pH 2.0 and pH 2.0 and 80° C. 90° C. 95° C. Time (h) MW (Da) MW (Da) MW (Da) 0 150 150 155 1 13.8 9.5 9.5 2 12.8 7.4 7.0 3 11.2 6.3 6.4 4 10.6 5.6 5.4

(16) The same experiments were performed at a pH of 3, 4, and 5 at a temperature of 121° C. in an autoclave for 20 minutes.

(17) The results are given in the following tables 3—and in FIGS. 1-4

(18) TABLE-US-00004 TABLE 3 comparative example at pH 5 Hydrolysis time [h] Temp [° C.] EU/g 0 95 12017 0 80 12017 0 60 10834 3 60 7723 1 80 5421 2 80 7671 3 80 7371 1 90 7082 2 90 8000 3 90 7812 1 95 6520 2 95 6562 3 95 6323 0.33 121 4240

(19) At a pH of 5, no significant endotoxin removal could be observed. See also FIG. 1.

(20) TABLE-US-00005 TABLE 4 endotoxin levels at pH of 2 Hydrolysis time [h] Temp [° C.] EU/g 0.1 60 10000 1 60 5853 3 60 5285 0.1 70 9990 1 70 5084 3 70 825 0.1 80 10446 0.5 80 7911 1 80 4214 2 80 1875 3 80 731 1 90 751 2 90 67 3 90 13 0.1 95 6586 0.5 95 220 1 95 10 2 95 4 3 95 3 0.33 121 1

(21) See also FIG. 2. It can be seen that at 90 and 95° C., as well as at 121° C., low endotoxin levels were obtained to attractive values

(22) TABLE-US-00006 TABLE 5 Endotoxin levels at pH of 3 Hydrolysis time [h] Temp [° C.] EU/g 0.1 70 8900 0.5 70 5148 1 70 5708 3 70 5222 0.1 80 7359 0.5 80 5693 1 80 5024 3 80 1858 0.1 95 5924 0.5 95 1032 1 95 92 3 95 7 0.33 121 3

(23) See also FIG. 3. Again, at a pH of 3 and a temperature of above 90° C., such as 95° C., envisaged endotoxin levels are observed.

(24) TABLE-US-00007 TABLE 6 Endotoxin levels at a pH of 2.5 and a temperature of 95° Hydrolysis time [h] EU/g MW[kDa] 0 13919 150 0.25 7960 1 14.5 4534 3 <4 7.4 4 <4 6.7 4.5 <4 6.5 5 <4 6.3 6 <4 6.0 7 <4 5.7

(25) From table 6, the data are also shown in FIG. 4, it can be observed that the gelatin becomes hydrolysed to a molecular weight of below 4000 Da, while the endotoxin level is below 10 EU/g after more than an hour incubation time.

(26) In addition to the above data at 121° C. at pH values of 2, 3 and 5 and an incubation period of 20 minutes, also a measurement at pH 4 was taken, resulting in a value of 164 EU/g. The data are summarized in FIG. 5, showing attractive result at a pH value of 3.

(27) Similar results were obtained for type B gelatin.

BRIEF DESCRIPTION OF THE DRAWINGS

(28) FIG. 1 shows hydrolysis time for gelatin hydrolysates at pH 5.

(29) FIG. 2 shows hydrolysis time for gelatin hydrolysates at pH 2.

(30) FIG. 3 shows hydrolysis time for gelatin hydrolysates at pH 3.

(31) FIG. 4 shows hydrolysis time vs. LPS level at pH 2.5

(32) FIG. 5 shows LPS levels at different pH values.