Method for determining oligopeptide content of puffed feather meal

Abstract

The invention relates to the field of polypeptide determination, and particularly discloses a method for determining the oligopeptide content of puffed feather meal. According to the method, an existing national standard method is improved, the puffed feather meal which is stuck together can be completely dispersed, and the problem that extraction is not complete due to denaturation and lumping after an extracting agent is added into the puffed feather meal is effectively solved. According to the method, the adding mode and the adding amount of the extracting agent are limited, so that the stability is maintained, meanwhile, the sample is better dispersed and homogenized, and the interference of polypeptide aggregation or further denaturation caused by too low or too high local concentration on subsequent analysis steps is reduced; according to the method, the grinding procedure is added, and incomplete extraction due to insufficient oscillation time is avoided by controlling the oscillation grinding time, so that the content of oligopeptide in the expanded feather meal is remarkably increased, the blank in the aspect of determining the content of oligopeptide in the expanded feather meal at present is filled, and the problem of low oligopeptide determination amount caused by a traditional method is solved.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide extraction, and particularly relates to a method for determining the oligopeptide content of puffed feather powder. Background Art

[0002] Extruded feather meal is made from feathers, a byproduct of poultry slaughter. After being collected and cleaned, it is then processed through high-temperature and high-pressure hydrolysis, puffing, or steam explosion techniques. This process disrupts the fibrous structure of the feathers, making them more digestible. Containing up to 70%-80% crude protein, extruded feather meal is an excellent protein supplement. Its nutrients are more readily absorbed by animals, significantly improving digestibility. It can be used as livestock and poultry feed, aquatic feed, and pet food, as well as as an organic fertilizer, biomaterial, and industrial raw material. This fully utilizes a byproduct of the poultry industry, reducing waste while providing an effective nutritional resource. It is a cost-effective raw material with broad industrial applications.

[0003] Puffed feather meal is rich in protein, which is the main source of its nutritional value. During the processing of puffed feather meal, some disulfide bonds and peptide bonds in feather keratin are broken down, generating soluble small-molecule peptide fragments, including oligopeptides. These oligopeptides can be directly absorbed by the intestine without further decomposition, making them particularly suitable for young animals. Currently, there is no specific method for measuring the oligopeptide content in puffed feather meal. Industry professionals generally use the GB / T 22492-2008 soybean peptide powder and GB / T 22729-2008 marine fish oligopeptide methods to test the peptide content in puffed feather meal. However, these methods have been found to be difficult to extract completely, resulting in low measured oligopeptide contents. Therefore, it is necessary to provide a more suitable method for measuring the oligopeptide content in puffed feather meal. Summary of the Invention

[0004] Aiming at the problems that the current method of using soybean peptide powder and marine fish oligopeptides to determine the oligopeptide content in puffed feather powder has incomplete extraction and low measured oligopeptide content, the present invention provides a method for determining the oligopeptide content in puffed feather powder.

[0005] The present invention provides a method for determining the oligopeptide content in puffed feather powder, comprising the following steps:

[0006] S1. Accurately weigh the puffed feather powder and transfer it into a plastic centrifuge tube; then, add zirconium dioxide grinding beads to the centrifuge tube. Then, add 15% trichloroacetic acid solution to the centrifuge tube in batches, let it stand for 3 minutes, and seal it. The ratio of the total volume of trichloroacetic acid added to the weight of the puffed feather powder is (20:1)-(40:1);

[0007] S2. Grind the sealed centrifuge tube by shaking for 10-20 minutes, mix thoroughly, and let it stand for 10-20 minutes;

[0008] S3, centrifuging after standing, and taking the supernatant after centrifugation;

[0009] S4. Determine the acid-soluble protein hydrolysate content X1 and the free amino acid content X2 in the supernatant respectively, and calculate the oligopeptide content X=X1-X2.

[0010] In the above-mentioned method for determining the oligopeptide content of puffed feather powder, the plastic centrifuge tube is a round-bottom plastic centrifuge tube.

[0011] In the above-mentioned method for determining the oligopeptide content of puffed feather powder, the amount of zirconium dioxide grinding beads added in step S1 is 1.5-3 times the mass of the puffed feather powder.

[0012] In the above-mentioned method for determining the oligopeptide content of puffed feather powder, the particle size of the zirconium dioxide grinding beads is 2-4 mm.

[0013] In the above-mentioned method for determining the oligopeptide content of puffed feather powder, in step S1, the ratio of the total volume of trichloroacetic acid added to the mass of the puffed feather powder is (30:1)-(35:1).

[0014] In the above-mentioned method for determining the oligopeptide content of puffed feather powder, the oscillation frequency in step S2 is 2000-4000 r / min.

[0015] In the above-mentioned method for determining the oligopeptide content of puffed feather powder, the oscillation frequency in step S2 is 2500 r / min. In the above-mentioned method for determining the oligopeptide content of puffed feather powder, the oscillation grinding in step S2 is 20 minutes.

[0016] In the above-mentioned method for determining oligopeptide content in puffed feather powder, in step S3, the mixture is allowed to stand and then centrifuged at 8000 rpm for 10 minutes.

[0017] Compared with the prior art, the present invention has the following beneficial effects: the determination method of the present invention can completely disperse the puffed feather powder that is stuck together, effectively solving the problem of incomplete extraction caused by denaturation and clumping of puffed feather powder after adding the extractant. By limiting the method and amount of adding the extractant, the puffed feather powder stuck to the tube wall can be completely covered, while maintaining stability, so that the sample is better dispersed and homogenized, reducing the aggregation or further denaturation of polypeptides caused by local excessive concentration, and avoiding the problem of insufficient grinding due to low sample concentration, thus avoiding interference with subsequent analysis steps. The present invention adopts an oscillating grinding method and controls the oscillating grinding time to avoid incomplete extraction due to insufficient oscillation time, which can significantly increase the content of oligopeptides in puffed feather powder. This method fills the current gap in the determination of oligopeptide content in puffed feather powder and solves the problem of incomplete extraction and low oligopeptide measurement in traditional methods for determining the content of oligopeptides in puffed feather powder. DETAILED DESCRIPTION

[0018] To address the current lack of a standardized method for determining oligopeptide content in puffed feather meal, the present invention provides a method for determining oligopeptide content in puffed feather meal. This method can significantly increase the amount of oligopeptides extracted from puffed feather meal. The present invention is further described and illustrated below in conjunction with specific embodiments.

[0019] Example 1: This example provides a method for determining the oligopeptide content of puffed feather powder, which specifically comprises the following steps:

[0020] S1. Accurately weigh 1.000g of puffed feather powder and transfer it into a 50ml round-bottom plastic centrifuge tube. At the same time, add zirconium dioxide grinding beads to the centrifuge tube. The mass of the zirconium dioxide grinding beads is controlled to be 1.5-3 times that of the puffed feather powder according to the particle size of zirconium dioxide to ensure sufficient grinding without affecting subsequent centrifugation. Then, add a total of 35ml of 15% trichloroacetic acid solution to the centrifuge tube in three portions. Pay attention to the dropwise addition speed during the addition process to avoid excessive concentration of the extract in some locations, which will aggravate the clumping and stickiness of the sample. Seal tightly.

[0021] S2. Grind the sealed centrifuge tube at 2500 rpm for 20 min, mix thoroughly, and let stand for 20 min.

[0022] S3, after standing, centrifuge at 8000 rpm for 10 min, and collect 10 ml of supernatant;

[0023] S4. Determine the acid-soluble protein hydrolysate content X1 and the free amino acid content X2 in the supernatant respectively, and calculate the oligopeptide content X=X1-X2.

[0024] The above method was used to perform two measurements simultaneously, and the results are shown in Table 1 below.

[0025] Table 1 Oligopeptide content in puffed feather powder measured by the method of the present invention

[0026]

[0027] Example 2: This example provides a method for determining the oligopeptide content of puffed feather powder. The oligopeptide content is determined using the same batch of puffed feather powder as in Example 1 as a sample. The method mainly includes the following steps:

[0028] S1. Accurately weigh 1.000g of puffed feather powder and transfer it into a 50ml round-bottom plastic centrifuge tube. At the same time, add 3mm zirconium dioxide grinding beads to the centrifuge tube. The mass of zirconium dioxide grinding beads should be 1.5-3 times that of puffed feather powder. Specifically, 15-30 beads can be selected according to the amount of sample. Then, add 35ml of 15% trichloroacetic acid solution to the centrifuge tube in batches. Pay attention to the dropwise addition speed during the addition process to avoid excessive concentration of the extract in some parts, which will aggravate the clumping and stickiness of the sample.

[0029] S2. Grind the sealed centrifuge tube at 2500 rpm for 10 min, mix thoroughly, and let stand for 20 min.

[0030] S3, after standing, centrifuge at 8000 rpm for 10 min, and collect 10 ml of supernatant;

[0031] S4. Determine the acid-soluble protein hydrolysate content X1 and the free amino acid content X2 in the supernatant respectively, and calculate the oligopeptide content X=X1-X2.

[0032] The above method was used to perform two measurements simultaneously, and the results are shown in Table 2 below.

[0033] Table 2 Oligopeptide content in puffed feather powder measured by the method of the present invention

[0034]

[0035]

[0036] Example 3: This example provides a method for determining the oligopeptide content of puffed feather powder. The oligopeptide content is determined using the same batch of puffed feather powder as in Example 1 as a sample. The method mainly includes the following steps:

[0037] S1. Accurately weigh 1.000g of puffed feather powder and transfer it to a 50ml round-bottom plastic centrifuge tube. Add zirconium dioxide grinding beads to the centrifuge tube at a mass of 1.5-3 times that of the puffed feather powder. Then, add 30ml of 15% trichloroacetic acid solution to the centrifuge tube in batches. Pay attention to the dripping speed during the addition process to avoid excessive concentration of the extract in some parts, which will aggravate the sample clumping and sticking. Seal the tube.

[0038] S2. Grind the sealed centrifuge tube by shaking for 30 minutes, mix thoroughly, and let it stand for 20 minutes;

[0039] S3, after standing, centrifuge at 8000 rpm for 10 min, and collect the supernatant;

[0040] S4. Determine the acid-soluble protein hydrolyzate content X1 and the free amino acid content X2 in the supernatant, and calculate the oligopeptide content X=X1-X2. The oligopeptide content is 20.88 g / 100 g.

[0041] Comparative Example 1: This comparative example uses the same batch of puffed feather powder as Example 1 as a sample, adopts the method for determining the content of polypeptides in soybean peptide powder in GB / T22492-2008 and makes some adjustments to determine the oligopeptide content of the puffed feather powder. The main contents are as follows:

[0042] (1) Accurately weigh 1.000 g (accurate to 0.001 g) of puffed feather powder, add 20 ml of 15% trichloroacetic acid solution, and ultrasonically treat for 10 min at a power of 300 W and a frequency of 25 kHz. Then dissolve and dilute to 50 ml. Mix well and let stand for 5 min. Filter and remove the initial filtrate. The filtrate is used as a reserve solution.

[0043] (2) Take 10-20 ml of equal amounts of filtrate, respectively, and measure the acid-soluble protein content X1 (g / 100 g) and free amino acid content X2 (g / 100 g) of the filtrate, and calculate the oligopeptide content X=X1-X2.

[0044] Comparative Example 2: This comparative example uses the same batch of puffed feather powder as Example 1 as a sample, and adopts the method for determining the oligopeptide content in marine fish oligopeptide powder in GB / T22729-2008 to determine the oligopeptide content of the puffed feather powder. The main contents are as follows:

[0045] (1) Accurately weigh 2.000 g (accurate to 0.001 g) of puffed feather powder into a 50 ml centrifuge tube, add 10 ml of 15% trichloroacetic acid solution, mix well, and let stand for 10 min;

[0046] (2) After standing, centrifuge at 4000 rpm for 10 min and collect all the supernatant;

[0047] (3) Take equal amounts of the centrifuged supernatant and measure the acid-soluble protein content X1 (g / 100g) and free amino acid content X2 (g / 100g) of the filtrate, respectively, and calculate the oligopeptide content X=X1-X2.

[0048] Comparative Example 3: This comparative example uses the same batch of puffed feather powder as in Example 1 to determine the oligopeptide content in the puffed feather powder. The main results are:

[0049] S1. Accurately weigh 1.000g of puffed feather powder and transfer it to a 50ml centrifuge tube. Add zirconium dioxide grinding beads to the centrifuge tube at the same time. The mass of zirconium dioxide grinding beads should be 1.5-3 times that of puffed feather powder, about 15-30 beads. Then, add 15% trichloroacetic acid solution to the centrifuge tube in batches, with a total amount of 35ml. Pay attention to the dripping speed during the addition process to avoid excessive concentration of the extract in some parts, which will aggravate the clumping and stickiness of the sample. Seal tightly.

[0050] S2. Grind the sealed centrifuge tube at 2500 rpm for 20 min, mix thoroughly, and let stand for 20 min.

[0051] S3. After standing, centrifuge at 4000 rpm for 10 min and collect the supernatant. It is found that some sticky residues on the wall of the centrifuge tube cannot be centrifuged.

[0052] S4. Determine the acid-soluble protein hydrolysate content X1 and the free amino acid content X2 in the supernatant respectively, and calculate the oligopeptide content X=X1-X2.

[0053] The oligopeptide contents measured in Comparative Examples 1-3 are shown in Table 3 below.

[0054] Table 3 Oligopeptide content of puffed feather powder measured by comparative method

[0055] Expanded feather powder Peptide content (g / 100g) Comparative Example 1 20.40 Comparative Example 2 18.06 Comparative Example 3 20.61

[0056] The above comparison shows that the oligopeptide content of the puffed feather meal measured using the treatment method of Example 1 of the present invention is significantly higher, increasing by 10.25% and 24.53% compared to the contents measured in Comparative Examples 1 and 2, respectively. This indicates that the method of the present invention can completely disperse the denatured and clumping puffed feather meal samples that are stuck together, resulting in more complete extraction. The peptide content measured in Example 2 is slightly lower than that in Example 1, possibly due to the slightly shorter grinding time and incomplete extraction of the extract. The slightly lower peptide content measured using the method of Comparative Example 3 may be due to the low centrifugation speed, which prevented the complete centrifugation of all residual sample on the walls of the centrifuge tube. The treatment method of the present invention can completely centrifuge the residual sample on the tube walls, further increasing the extraction yield of oligopeptides from the puffed feather meal.

[0057] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for determining oligopeptide content in puffed feather powder, characterized by: The following steps are involved: S1. Accurately weigh the puffed feather powder and transfer it into a plastic centrifuge tube. Then, add zirconium dioxide grinding beads to the centrifuge tube. Then, add 15% trichloroacetic acid solution to the centrifuge tube in batches, let it stand for 3 minutes, and seal it. The ratio of the total volume of trichloroacetic acid added to the weight of the puffed feather powder is (20:1)-(40:1). S2. Grind the sealed centrifuge tube by shaking for 10-20 minutes, mix thoroughly, and let it stand for 10-20 minutes; S3, centrifuging after standing, and taking the supernatant after centrifugation; S4. Determine the acid-soluble protein hydrolysate content X1 and the free amino acid content X2 in the supernatant, and calculate the oligopeptide content X = X1- X2.

2. The method for determining oligopeptide content in puffed feather powder according to claim 1, wherein: The plastic centrifuge tube is a round-bottom plastic centrifuge tube.

3. The method for determining oligopeptide content in puffed feather powder according to claim 1, wherein: In step S1, the amount of zirconium dioxide grinding beads added is 1.5-3 times the mass of the puffed feather powder.

4. The method for determining oligopeptide content in puffed feather powder according to claim 3, wherein: The particle size of the zirconium dioxide grinding beads is 2-4 mm.

5. The method for determining oligopeptide content in puffed feather powder according to claim 1, wherein: In step S1, the ratio of the total volume of trichloroacetic acid added to the mass of the puffed feather powder is (30:1)-(35:1).

6. The method for determining oligopeptide content in puffed feather powder according to claim 1, wherein: The oscillation frequency in step S2 is 2000-4000 r / min.

7. The method for determining oligopeptide content in puffed feather powder according to claim 6, wherein: The oscillation frequency in step S2 is 2500 r / min.

8. The method for determining oligopeptide content in puffed feather powder according to claim 6, wherein: In step S2, the mixture was shaken and ground for 20 min.

9. The method for determining oligopeptide content in puffed feather powder according to claim 1, wherein: After standing in step S3, the mixture was centrifuged at 8000 r / min for 10 min.

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