Process for extracting collagen peptide based on enzyme fermentation method

Abstract

The invention belongs to the field of biological extraction, and particularly relates to a process for extracting collagen peptide based on an enzyme fermentation method. The collagen peptide which is easy to digest and absorb and has relatively good taste and flavor is obtained through a process of combining enzymolysis with probiotic fermentation, not only is relatively high extraction rate achieved, but also the obtained product is high in polypeptide content and small in molecular weight and has excellent functions of resisting oxidation and quenching free radicals.

Description

Technical Field

[0001] This invention belongs to the field of bio-extraction, specifically relating to a process for extracting collagen peptides based on enzymatic fermentation. Background Technology

[0002] During fish processing, a large amount of processing byproducts (fish bones, skin, viscera, and scales, etc.) are inevitably generated, accounting for approximately 40% to 55% of the raw materials. These aquatic products are often discarded as waste or processed into animal feed for reuse, resulting in a significant waste of these byproduct resources. The main components of fish bones are protein and ash. Taking cod bones as an example, ash and protein account for 52.64% and 35.78% respectively, with a fat content of only 1%. Cod bones are a high-quality protein source and processing raw material, and the extraction of collagen peptides from cod bones is of great significance.

[0003] Currently, enzymatic hydrolysis of animal and plant proteins is the main method for preparing short peptides. However, collagen peptides extracted from fish bones using conventional methods have a certain fishy and bitter taste, and research on complex extraction processes for collagen peptides is still relatively lacking. Summary of the Invention

[0004] To address the above problems, this invention provides a process for extracting collagen peptides based on enzymatic fermentation, comprising the following steps:

[0005] (1) Take fresh cod bones, remove the fish meat, add deionized water and lipase to carry out enzymatic hydrolysis;

[0006] (2) Wash the fish bones after the reaction with deionized water, drain them, and then crush them to less than 2 mm to obtain cod bone powder;

[0007] (3) Mix cod bone powder with deionized water, add compound enzyme for enzymatic hydrolysis, and obtain enzymatic hydrolysate after enzyme inactivation;

[0008] (4) After sterilizing the enzymatic hydrolysate, inoculate it with the compound fermentation culture and add sugar source to ferment it to obtain fermentation broth;

[0009] (5) Centrifuge the fermentation broth, collect the supernatant and freeze dry to obtain collagen peptides.

[0010] Further, in step (1), the material-to-liquid ratio is 1:6-10 g / ml, the amount of lipase added is 1-5% of the weight of the mixture, the enzymatic hydrolysis temperature is 35-55℃, the pH is 6.5-7.5, and the time is 20-30 min.

[0011] Further, in step (1), the material-to-liquid ratio is 1:6-10 g / ml, the compound enzyme is papain and neutral protease, and the amount of compound enzyme added is 0.5-2% of the weight of the mixture.

[0012] Furthermore, the enzymatic hydrolysis temperature is 45-65℃, the pH is 6.5-7.5, and the time is 3-6h. The enzyme inactivation conditions are incubation at 80-100℃ for 10-20min.

[0013] Furthermore, the sterilization is irradiation sterilization.

[0014] Further, the compound fermentation broth is prepared as follows: Lactobacillus rhamnosus, Bifidobacterium longum and Bacillus subtilis are activated and cultured to OD600 = 0.8-1.2 respectively, and then mixed in a volume ratio of 1:1:2 to obtain the compound fermentation broth.

[0015] Furthermore, the amount of the compound fermentation broth added is 1-5% of the weight of the mixture.

[0016] Furthermore, the sugar source is sucrose, and the amount added is 2-5% of the weight of the mixture.

[0017] Furthermore, the fermentation temperature is 35-38℃, the pH is 7.0-7.5, and the time is 12-48h.

[0018] Furthermore, the process further includes sterilizing the fermentation broth after fermentation is completed. Further, the sterilization includes irradiation sterilization.

[0019] Beneficial effects of the invention

[0020] This invention utilizes a process combining enzymatic hydrolysis and probiotic fermentation to obtain collagen peptides that are easily digestible and absorbable, and have excellent taste and flavor. The resulting product not only has a high extraction rate but also boasts a high peptide content, small molecular weight, and excellent antioxidant and free radical quenching functions. Detailed Implementation

[0021] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0022] The lipase used in the examples was purchased from Beijing Bio-Lab Technology Co., Ltd., with an enzyme activity of 20,000 U / g; the papain was purchased from Hebei Baiwei Biotechnology Co., Ltd., with an enzyme activity of 60,000 U / g; and the neutral protease was purchased from Shandong Jiusheng Biotechnology Co., Ltd., with an enzyme activity of 200,000 U / g.

[0023] The MRS liquid culture medium used consisted of: 10.0 g / L peptone, 8.0 g / L beef meal, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1.0 g / L Tween 80. The pH was adjusted to 6.5 with 1 mol / L NaOH, and the medium was sterilized at 121°C for 20 min.

[0024] The Lactobacillus rhamnosus (ATCC7469), Bifidobacterium longum subsp. Infantis (ATCC15697), and Bacillus subtilis (ATCC6633) used were all commercially obtained.

[0025] Example 1:

[0026] A process for extracting collagen peptides based on enzymatic fermentation includes the following steps:

[0027] (1) Take fresh cod bones, remove the fish meat on them, add deionized water at a ratio of 1:8g / ml, add lipase at 3% of the weight of the mixture, and react at 40℃ and 7.0pH for 30min.

[0028] (2) Wash the fish bones after the reaction with deionized water, drain them, and then crush them to less than 2 mm to obtain cod bone powder;

[0029] (3) Mix cod bone powder with deionized water at a material-to-liquid ratio of 1:8 g / ml, add a compound enzyme consisting of papain and neutral protease mixed at a mass ratio of 1:1, adjust the pH to 7 with 1 mol / L NaOH aqueous solution or 1 mol / L H3PO4 aqueous solution, and enzymatically hydrolyze at 50℃ for 5 h. After the enzymatic hydrolysis is completed, incubate at 90℃ for 15 min to inactivate the enzyme and obtain the enzymatic hydrolysate.

[0030] (4) The freeze-dried powders of Lactobacillus rhamnosus and Bifidobacterium longum subsp. infantis were activated by anaerobic culture in MRS liquid medium at 37°C for 24 h. The activated bacterial solutions were then inoculated into MRS liquid medium and cultured at 37°C at 250 r / min until the bacterial concentration reached OD600 of 1.0 ± 0.1. The freeze-dried powder of Bacillus subtilis was activated by aerobic culture in MRS liquid medium at 37°C for 24 h. The activated bacterial solutions were then inoculated into MRS liquid medium and cultured at 37°C at 250 r / min until the bacterial concentration reached OD600 of 1.0 ± 0.1.

[0031] Then, the expanded cultures of Lactobacillus rhamnosus, Bifidobacterium longum subsp. infantis, and Bacillus subtilis were mixed in a volume ratio of 1:1:2 to obtain a compound fermentation culture.

[0032] The enzymatic hydrolysate obtained in step (3) was sterilized by irradiation. Then, the above-mentioned compound fermentation liquid was inoculated at 4% of the weight of the enzymatic hydrolysate and 5% of the weight of the mixed liquid was added with sucrose. The mixture was fermented at 36°C, 7.2 pH and 150 r / min for 36 h to obtain the fermentation liquid.

[0033] (5) Sterilize the fermentation broth by irradiation, centrifuge at 6000 r / min for 15 min, collect the supernatant and freeze dry to obtain collagen peptides.

[0034] Example 2:

[0035] A process for extracting collagen peptides based on enzymatic fermentation includes the following steps:

[0036] (1) Take fresh cod bones, remove the fish meat on them, add deionized water at a ratio of 1:8g / ml, add lipase at 3% of the weight of the mixture, and react at 40℃ and 7.0pH for 30min.

[0037] (2) Wash the fish bones after the reaction with deionized water, drain them, and then crush them to less than 2 mm to obtain cod bone powder;

[0038] (3) Mix cod bone powder with deionized water at a material-to-liquid ratio of 1:8 g / ml, add a compound enzyme consisting of papain and neutral protease mixed at a mass ratio of 3:1, adjust the pH to 7 with 1 mol / L NaOH aqueous solution or 1 mol / L H3PO4 aqueous solution, and enzymatically hydrolyze at 50℃ for 5 h. After the enzymatic hydrolysis is completed, incubate at 90℃ for 15 min to inactivate the enzyme and obtain the enzymatic hydrolysate.

[0039] (4) The freeze-dried powders of Lactobacillus rhamnosus and Bifidobacterium longum subsp. infantis were activated by anaerobic culture in MRS liquid medium at 37°C for 24 h. The activated bacterial solutions were then inoculated into MRS liquid medium and cultured at 37°C at 250 r / min until the bacterial concentration reached OD600 of 1.0 ± 0.1. The freeze-dried powder of Bacillus subtilis was activated by aerobic culture in MRS liquid medium at 37°C for 24 h. The activated bacterial solutions were then inoculated into MRS liquid medium and cultured at 37°C at 250 r / min until the bacterial concentration reached OD600 of 1.0 ± 0.1.

[0040] Then, the expanded cultures of Lactobacillus rhamnosus, Bifidobacterium longum subsp. infantis, and Bacillus subtilis were mixed in a volume ratio of 1:2:1 to obtain a compound fermentation culture.

[0041] The enzymatic hydrolysate obtained in step (3) was sterilized by irradiation. Then, the above-mentioned compound fermentation liquid was inoculated at 4% of the weight of the enzymatic hydrolysate and 5% of the weight of the mixed liquid was added with sucrose. The mixture was fermented at 36°C, 7.2 pH and 150 r / min for 36 h to obtain the fermentation liquid.

[0042] (5) Sterilize the fermentation broth by irradiation, centrifuge at 6000 r / min for 15 min, collect the supernatant and freeze dry to obtain collagen peptides.

[0043] Comparative Example 1:

[0044] A process for enzymatic extraction of collagen peptides, comprising the following steps:

[0045] (1) Take fresh cod bones, remove the fish meat on them, add deionized water at a ratio of 1:8g / ml, add lipase at 3% of the weight of the mixture, and react at 40℃ and 7.0pH for 30min.

[0046] (2) Wash the fish bones after the reaction with deionized water, drain them, and then crush them to less than 2 mm to obtain cod bone powder;

[0047] (3) Mix cod bone powder with deionized water at a material-to-liquid ratio of 1:8 g / ml, add a compound enzyme consisting of papain and neutral protease mixed at a mass ratio of 1:1, adjust the pH to 7 with 1 mol / L NaOH aqueous solution or 1 mol / L H3PO4 aqueous solution, and enzymatically hydrolyze at 50℃ for 5 h. After the enzymatic hydrolysis is completed, incubate at 90℃ for 15 min to inactivate the enzyme and obtain the enzymatic hydrolysate.

[0048] (4) Sterilize the enzymatic hydrolysate by irradiation, centrifuge at 6000 r / min for 15 min, collect the supernatant and freeze dry to obtain collagen peptides.

[0049] Comparative Example 2:

[0050] A process for extracting collagen peptides based on enzymatic fermentation includes the following steps:

[0051] (1) Take fresh cod bones, remove the fish meat on them, add deionized water at a ratio of 1:8g / ml, add lipase at 3% of the weight of the mixture, and react at 40℃ and 7.0pH for 30min.

[0052] (2) Wash the fish bones after the reaction with deionized water, drain them, and then crush them to less than 2 mm to obtain cod bone powder;

[0053] (3) Mix cod bone powder with deionized water at a material-to-liquid ratio of 1:8 g / ml, add a compound enzyme consisting of papain and neutral protease mixed at a mass ratio of 1:1, adjust the pH to 7 with 1 mol / L NaOH aqueous solution or 1 mol / L H3PO4 aqueous solution, and enzymatically hydrolyze at 50℃ for 5 h. After the enzymatic hydrolysis is completed, incubate at 90℃ for 15 min to inactivate the enzyme and obtain the enzymatic hydrolysate.

[0054] (4) Add dry yeast to deionized water at a ratio of 1:8 g / ml, add 5% sucrose by weight of the mixture, stir at 38°C for 20 minutes, cool to 35°C to obtain yeast solution, and adjust the bacterial count in the solution to 5 × 10⁻⁶. 8 cfu / mL;

[0055] The enzymatic hydrolysate obtained in step (3) was sterilized by irradiation. Then, the above yeast solution was inoculated at 4% of the weight of the enzymatic hydrolysate and 5% of the weight of the mixture was added with sucrose. The mixture was fermented at 35°C, 7.0 pH and 150 r / min for 36 h to obtain the fermentation broth.

[0056] (5) Sterilize the fermentation broth by irradiation, centrifuge at 6000 r / min for 15 min, collect the supernatant and freeze dry to obtain collagen peptides.

[0057] Comparative Example 3:

[0058] A process for extracting collagen peptides based on enzymatic fermentation is disclosed. The difference between this process and Example 1 is that bromelain is used instead of papain, while the other operation steps are the same as in Example 1.

[0059] Comparative Example 4:

[0060] A process for extracting collagen peptides based on enzymatic fermentation is disclosed. The difference between this process and Example 1 is that Lactobacillus plantarum is used instead of Lactobacillus rhamnosus. All other operation steps are the same as in Example 1.

[0061] Comparative Example 5:

[0062] A process for extracting collagen peptides based on enzymatic fermentation is disclosed. The difference between this process and Example 1 is that Bifidobacterium animalis is used instead of Bifidobacterium longum subsp. infantis. All other operating steps are the same as in Example 1.

[0063] Test Example 1:

[0064] The peptide content and the content of short peptides with a relative molecular weight of less than 600 Da of the collagen peptides prepared in the examples and comparative examples were determined by the detection methods in Appendix A and Appendix B of GB / T 22492-2008. The results are shown in Table 1.

[0065] Table 1

[0066] peptide content Peptide content less than 600 Da Example 1 81.3％ 95.7％ Example 2 82.9％ 96.4％ Comparative Example 1 81.2％ 53.7％ Comparative Example 2 81.5％ 79.3％ Comparative Example 3 80.4％ 84.9％ Comparative Example 4 81.7％ 82.5％ Comparative Example 5 80.8％ 84.2％

[0067] The results in Table 1 show that the collagen peptides prepared by the process of this invention contain a large number of short peptides with low molecular weight, while the collagen peptides prepared in Comparative Examples 1 and 2 have lower contents of short peptides. The results of Comparative Example 3 show that when bromelain is used instead of papain, the content of short peptides with low molecular weight also decreases, indicating that the enzyme used in the enzymatic hydrolysis step also affects the subsequent microbial fermentation. Comparative Examples 4 and 5 show that when different probiotics are used for fermentation, the yield of short peptides of this invention cannot be achieved.

[0068] Test Example 2:

[0069] Sensory evaluation of fishy and bitter taste:

[0070] A sensory evaluation panel of 10 trained personnel (5 men and 5 women, aged 20-35) was selected to conduct sensory evaluations of the collagen peptides prepared in Examples 1-2 and Comparative Examples 1-2. In short, 1g of collagen peptide was added to 50mL of deionized water to prepare a collagen peptide solution. The sensory evaluation personnel fasted for two hours before the evaluation. At the start of the evaluation, they rinsed their mouths with water, then held 2-3mL of the collagen peptide solution sample in their mouths for 10 seconds, spitting it out and rinsing their mouths with water again. The time interval for evaluating bitterness was 5-10 minutes between different samples. The degree of fishy smell and bitterness was scored on a 10-point scale, divided into 5 levels (8 < no or slight fishy smell (or bitterness) ≤ 10; 6 < mild fishy smell (or bitterness) ≤ 8; 4 < moderate fishy smell (or bitterness) ≤ 6; 2 < strong fishy smell (or bitterness) ≤ 4; 0 < extremely strong fishy smell (or bitterness) ≤ 2). The higher the total score, the better the overall flavor. Each scoring was conducted individually by an evaluator, and the results are shown in Table 2.

[0071] Table 2:

[0072] Example 1 Example 2 Comparative Example 1 Comparative Example 2 Fishy smell rating 88 90 42 78 Bitterness rating 91 92 39 76

[0073] As shown in Table 2, based on sensory evaluation, the collagen peptides prepared in the embodiments of the present invention have relatively low fishy and bitter tastes, and basically no obvious fishy or bitter tastes.

[0074] Test Example 3:

[0075] Antioxidant test:

[0076] The ability of the collagen peptides prepared in the examples and comparative examples to scavenge DPPH free radicals was tested. A collagen peptide sample solution with a concentration of 0.5 mg / mL was prepared using anhydrous ethanol as the solvent; 2.0 mL of the sample solution and 2.0 mL of a 1.0 × 10⁻⁶ molar concentration were added to a 1 cm quartz cuvette. 4A mol / L DPPH solution was mixed thoroughly and placed in the dark for 30 minutes. Using a 95% ethanol aqueous solution as a reference solution, the absorbance (A) at 517 nm was measured. Similarly, the absorbance (A) at 517 nm was measured for a mixture of 2.0 mL of the sample solution and 2.0 mL of a 95% ethanol aqueous solution. o The molar concentration of 2.0 mL was then determined to be 1.0 × 10⁻⁶. 4 The inhibition rate is calculated using the following formula: The absorbance (A1) at 517 nm of a mixture of mol / L DPPH solution and 2.0 mL of 95% (v / v) ethanol aqueous solution is used to determine the inhibition rate.

[0077] Clearance rate (%) = [1 - (AA)] o ) / A1]×100.

[0078] The results are shown in Table 3.

[0079] Table 3

[0080] Group DPPH free radical scavenging rate Example 1 69.5％ Example 2 69.8％ Comparative Example 1 36.2％ Comparative Example 2 51.6％ Comparative Example 3 60.3％ Comparative Example 4 57.1％ Comparative Example 5 59.7％

[0081] As can be seen from the results in Table 3, the collagen peptides containing a large number of short peptides prepared in the embodiments of the present invention have good antioxidant properties, while the antioxidant properties of comparative examples 1-5, which contain fewer short peptides, are significantly worse.

[0082] It should be noted that while the preferred embodiments of the present invention are provided in this specification, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A process for extracting collagen peptides based on enzymatic fermentation, characterized in that, The process includes the following steps: (1) Take fresh cod bones, remove the fish meat, add deionized water and lipase to carry out enzymatic hydrolysis; (2) Wash the fish bones after the reaction with deionized water, drain them, and then crush them to less than 2 mm to obtain cod bone powder; (3) Mix cod bone powder with deionized water, add compound enzyme for enzymatic hydrolysis, and obtain enzymatic hydrolysate after enzyme inactivation; (4) After sterilizing the enzymatic hydrolysate, inoculate it with the compound fermentation culture and add sugar source to ferment it to obtain fermentation broth; (5) Centrifuge the fermentation broth, collect the supernatant and freeze dry to obtain collagen peptides.

2. The process according to claim 1, characterized in that, In step (1), the material-to-liquid ratio is 1:6-10 g / ml, the amount of lipase added is 1-5% of the weight of the mixture, the enzymatic hydrolysis temperature is 35-55℃, the pH is 6.5-7.5, and the time is 20-30 min.

3. The process according to claim 1, characterized in that, In step (1), the material-to-liquid ratio is 1:6-10 g / ml, the compound enzyme is papain and neutral protease, and the amount of compound enzyme added is 0.5-2% of the weight of the mixture.

4. The process according to claim 1, characterized in that, The enzymatic hydrolysis is performed at a temperature of 45-65℃, a pH of 6.5-7.5, and a time of 3-6 hours. The enzyme inactivation is performed by incubating at 80-100℃ for 10-20 minutes.

5. The process according to claim 1, characterized in that, The sterilization method is irradiation sterilization.

6. The process according to claim 1, characterized in that, The compound fermentation broth is prepared as follows: Lactobacillus rhamnosus, Bifidobacterium longum subsp. infantis and Bacillus subtilis are activated and cultured to OD600 = 0.8-1.2 respectively, and then mixed in a volume ratio of 1:1-2:1-2 to obtain the compound fermentation broth.

7. The process according to claim 1, characterized in that, The amount of the compound fermentation broth added is 1-5% of the weight of the mixture.

8. The process according to claim 1, characterized in that, The sugar source is sucrose, and the amount added is 2-5% of the weight of the mixture.

9. The process according to claim 1, characterized in that, The fermentation temperature is 35-38℃, the pH is 7.0-7.5, and the time is 12-48h.

10. The process according to claim 1, characterized in that, It also includes sterilizing the fermentation broth after fermentation is complete.