A preparation process and product of enzymatically hydrolyzed chicken collagen peptide juice

Abstract

This application relates to a preparation process and product of enzymatically hydrolyzed chicken collagen peptide juice, belonging to the field of biotechnology. The preparation process includes the following steps: Enzymatic hydrolysis: Chicken collagen raw material is mixed with water at a weight ratio of 1:3-8, the pH is adjusted to 6.0-8.0, a complex protease is added, and enzymatic hydrolysis is carried out at 45-55℃ for 2-6 hours. After enzyme inactivation, an enzymatic hydrolysate is obtained; Fermentation: A carbon source and a nitrogen source are added to the enzymatic hydrolysate, the pH is adjusted to 5.5-7.0, a complex probiotic is inoculated, and fermentation is carried out at 30-37℃ for 12-48 hours to obtain a fermentation broth; Post-treatment: The fermentation broth is centrifuged, filtered, and sterilized to obtain the peptide juice. This application combines enzymatic hydrolysis with probiotic fermentation, resulting in a chicken collagen peptide juice with a high peptide content, rich in lactic acid and characteristic flavor substances, small molecular weight, easy absorption, and excellent taste and flavor.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a preparation process and product of enzymatic hydrolysis of chicken collagen peptide juice. Background Technology

[0002] Collagen peptides are a mixture of small-molecule peptides obtained from collagen through enzymatic or hydrolytic hydrolysis. Due to their good biocompatibility, low antigenicity, and easy absorption, they are widely used in the food and cosmetic industries. Chicken collagen is mainly derived from byproducts such as chicken skin, bones, and carcasses, making it widely available and inexpensive. Collagen peptides prepared from chicken collagen have potential benefits such as promoting skin health and improving joint function.

[0003] In existing technologies, the most common method for preparing chicken collagen peptides is a single enzymatic hydrolysis method. For example, specific proteases (such as alkaline protease, papain, etc.) are used to hydrolyze chicken skin, carcass, or bones, followed by filtration and drying to obtain collagen peptide powder or solution. While this enzymatic hydrolysis process can break down large collagen molecules into smaller peptides, the products typically have a strong fishy or bitter taste, resulting in poor flavor and limiting their direct application in oral liquids, beverages, and other products. Furthermore, the composition of simple enzymatic hydrolysis products is relatively simple, mainly consisting of peptides and amino acids, lacking beneficial organic acids, vitamins, and other probiotic metabolites.

[0004] To address flavor issues, some existing technologies attempt to improve taste by adding masking agents such as flavorings and sweeteners, but this does not fundamentally alter the product's composition and nutritional characteristics. Other technologies employ microbial fermentation to process animal protein, but these typically involve long fermentation cycles and lack specificity for the raw materials, making it difficult to effectively enrich specific small-molecule peptides and flavor compounds while simultaneously degrading collagen. Therefore, developing a preparation process that can efficiently degrade chicken collagen while imparting a good flavor and rich nutrition to the product is a pressing technical problem in this field. Summary of the Invention

[0005] The purpose of this application is to overcome the above-mentioned technical problems and provide a preparation process and product of enzymatic hydrolysis of chicken collagen peptide juice.

[0006] This application provides a process for preparing enzymatically hydrolyzed chicken collagen peptide juice, comprising the following steps: (1) Enzymatic hydrolysis: Chicken collagen raw material is mixed with water at a weight ratio of 1:3~8, the pH is adjusted to 6.0~8.0, a compound protease is added, and the mixture is enzymatically hydrolyzed at 45~55℃ for 2~6 hours. After the enzyme is inactivated, the enzymatic hydrolysate is obtained. (2) Fermentation: Add carbon and nitrogen sources to the enzymatic hydrolysate obtained in step (1), adjust the pH to 5.5-7.0, inoculate with compound probiotics, and ferment at 30-37℃ for 12-48 hours to obtain fermentation broth; (3) Post-processing: The fermentation liquid obtained in step (2) is centrifuged, filtered and sterilized in sequence to obtain the enzymatically hydrolyzed chicken collagen peptide juice.

[0007] Preferably, the chicken collagen raw material in step (1) is one or more of chicken skin, chicken cartilage, and chicken carcass; the complex protease is a combination of two or three of alkaline protease, neutral protease, trypsin, and papain.

[0008] By adopting the above technical solutions, by selecting raw materials from different parts, byproducts can be fully utilized. Furthermore, by combining complex proteases and utilizing the cleavage sites of different enzymes, collagen can be hydrolyzed more thoroughly into small peptides and amino acids, thereby improving the bioavailability of the products.

[0009] Preferably, the amount of the compound protease added in step (1) is 0.1% to 1.5% of the total mass of the chicken collagen raw material, and the stirring rate is maintained at 50 to 200 rpm during the enzymatic hydrolysis process.

[0010] By adopting the above technical solution and controlling the amount of enzyme and the stirring rate, the enzymatic hydrolysis reaction can be carried out under mild and efficient conditions, avoiding excessive hydrolysis that produces too many free amino acids and affects the peptide content, while ensuring the homogeneity of the reaction system.

[0011] Preferably, the carbon source in step (2) is one or more of glucose, sucrose, and fructooligosaccharides, and the amount added is 0.5% to 3% of the mass of the enzymatic hydrolysate; the nitrogen source is one or more of yeast extract, soybean peptone, and corn steep liquor, and the amount added is 0.2% to 1.5% of the mass of the enzymatic hydrolysate.

[0012] By adopting the above technical solution, sufficient carbon and nitrogen sources are provided for subsequent probiotic fermentation, promoting the rapid proliferation and metabolism of probiotics, thereby producing abundant beneficial metabolites and flavor substances such as lactic acid and short-chain fatty acids.

[0013] Preferably, the compound probiotics in step (2) are at least two of Lactobacillus plantarum, Streptococcus thermophilus, and Saccharomyces cerevisiae, with a total inoculation amount of 1% to 5% of the volume of the enzymatic hydrolysate and a live bacteria concentration of 10^6 to 10^8 CFU / mL.

[0014] By adopting the above technical solution, a specific combination of compound probiotics can work synergistically in the enzymatic hydrolysate system. Lactic acid bacteria produce lactic acid to lower the pH, inhibit the growth of miscellaneous bacteria, and at the same time give the product a refreshing sour flavor. Yeast can produce aroma substances such as esters and alcohols, further improving the product flavor.

[0015] Preferably, the fermentation in step (2) includes a combination of static fermentation and intermittent aeration fermentation: first, static fermentation for 6 to 12 hours, and then intermittent aeration fermentation for 6 to 24 hours with an aeration rate of 0.1 to 0.5 vvm.

[0016] By adopting the above technical solution, the initial static fermentation is conducive to the growth and metabolic acid production of facultative anaerobic bacteria such as lactic acid bacteria; the later intermittent aeration fermentation can provide a micro-oxygen environment for aerobic or facultative anaerobic bacteria such as brewer's yeast, promoting their growth and the synthesis of flavor substances. Through dissolved oxygen control at different stages, the segmented synergistic fermentation of a variety of probiotics is realized.

[0017] Preferably, the centrifugation in step (3) is performed at 4000~8000 rpm for 10~30 minutes; the filtration uses a microfiltration membrane with a pore size of 0.22~1.0 μm; the sterilization is pasteurization or ultra-high temperature instantaneous sterilization, wherein the pasteurization temperature is 65~75℃ and lasts for 30 minutes; and the ultra-high temperature instantaneous sterilization temperature is 135~140℃ and lasts for 3~5 seconds.

[0018] By adopting the above technical solutions, high-speed centrifugation and microfiltration membrane filtration can effectively remove bacteria and insoluble impurities from the fermentation broth, resulting in a clear and transparent peptide juice. The sterilization method used can kill residual microorganisms and ensure product safety while maximizing the retention of peptides, amino acids and heat-sensitive flavor substances.

[0019] This application also provides an enzymatically hydrolyzed chicken collagen peptide juice, prepared by the above-mentioned preparation process, wherein the peptide content is ≥75%, the free amino acid content is ≥2.5 g / L, the lactic acid content is ≥3.0 g / L, and it has characteristic flavor substances produced by probiotic metabolism.

[0020] In summary, the present invention has at least the following beneficial effects: Because this invention employs a combined process of "enzymatic hydrolysis followed by fermentation," enzymatic hydrolysis breaks down large collagen molecules into smaller peptides, providing an easily usable nitrogen source for subsequent probiotic fermentation. The fermentation process not only further degrades some peptides, producing free amino acids, but also generates beneficial substances such as lactic acid and esters through probiotic metabolism. Compared to a single enzymatic hydrolysis process, the peptide juice obtained in this application has a higher peptide content (≥75%), smaller molecular weight, and is more easily absorbed by the human body. Simultaneously, the lactic acid content is significantly increased (≥3.0 g / L), resulting in better probiotic functions.

[0021] This invention preferably employs a specific combination of complex proteases and complex probiotics, and defines the process parameters for staged fermentation (first static setting followed by intermittent aeration). Due to precise control over the growth characteristics and metabolic needs of different bacterial strains, the enzymatic hydrolysis and fermentation processes are highly efficient and synergistic. The resulting peptide juice not only has a pure flavor and no fishy or bitter taste, but is also rich in characteristic flavor substances produced by probiotic metabolism, resulting in a significantly superior taste compared to existing products. It can be consumed directly as a functional beverage or used as a base material. Attached Figure Description

[0022] Figure 1 This is a process flow diagram covering the entire process from raw material mixing, pH adjustment, enzymatic hydrolysis to enzyme inactivation and defatting to obtain the enzymatic hydrolysate.

[0023] Figure 2 This is a flowchart of the subsequent fermentation process.

[0024] Figure 3 This is a process flow diagram for the post-processing stage. Detailed Implementation

[0025] The present application will be further described below with reference to the accompanying drawings: (Since this solution is a method and product invention and does not involve specific structural drawings, the following detailed implementation will be described in detail in conjunction with the process steps.)

[0026] Example 1 This embodiment discloses a process for preparing enzymatically hydrolyzed chicken collagen peptide juice, including the following steps: (1) Raw material preparation, enzymatic hydrolysis and defatting: Weigh chicken skin and chicken carcass as chicken collagen raw materials and mix them at a weight ratio of 1:1. Add the mixed raw materials and water at a weight ratio of 1:5 into the enzymatic hydrolysis tank. Adjust the pH of the mixture to 7.0 using food-grade sodium hydroxide or hydrochloric acid. Add a complex protease to the tank. In this embodiment, the complex protease consists of alkaline protease and neutral protease at an enzyme activity ratio of 1:1, and the total amount added is 0.8% of the total mass of the chicken collagen raw materials. Turn on the stirring device and set the stirring speed to 120 rpm. At the same time, heat and control the temperature inside the tank at 50°C. Enzymatic hydrolysis is carried out for 4 hours under these conditions. After enzymatic hydrolysis, heat to 85°C and maintain for 15 minutes to inactivate the enzyme, and obtain the enzymatic hydrolysate. Cool the obtained enzymatic hydrolysate to 55-65°C and defatt it by physical centrifugation. Transfer the enzymatic hydrolysate to a disc centrifuge and centrifuge at 6000-8000 rpm and 55-65°C for 10-15 minutes. After centrifugation, the upper oil phase (animal fat) and the lower aqueous phase (defatting enzymatic hydrolysate) are collected separately. The lower aqueous phase is then introduced into step (2) of the fermentation process.

[0027] (2) Fermentation: After the enzymatic hydrolysate obtained in step (1) has cooled naturally to 35°C, a carbon source and a nitrogen source are added to it. Glucose is selected as the carbon source, and the amount added is 1.5% of the mass of the enzymatic hydrolysate; yeast extract is selected as the nitrogen source, and the amount added is 0.8% of the mass of the enzymatic hydrolysate. The pH is adjusted to 6.2 again. Then, compound probiotics are inoculated. In this embodiment, the compound probiotics are composed of Lactobacillus plantarum and Saccharomyces cerevisiae, with a live bacteria ratio of 2:1. The total inoculation volume is 3% of the volume of the enzymatic hydrolysate, and the initial live bacteria concentration is adjusted to about 10^7 CFU / mL. After inoculation, staged fermentation is carried out: First, the fermentation is allowed to stand at 35°C for 8 hours; then, aeration and stirring are turned on, and intermittent aeration fermentation is carried out at an aeration rate of 0.3 vvm (for example, aeration for 10 minutes, stopping for 20 minutes, and repeating). The total fermentation time reaches 24 hours (i.e., the intermittent aeration stage is 16 hours). After the fermentation is completed, the fermentation broth is obtained.

[0028] (3) Post-processing: The fermentation broth obtained in step (2) was transferred to a tubular centrifuge and centrifuged at 6000 rpm for 20 minutes to remove the cells and precipitate. The supernatant was collected and filtered using a hollow fiber microfiltration membrane with a pore size of 0.45 μm to obtain a clear filtrate. Finally, the filtrate was sterilized. In this embodiment, pasteurization was used, and the solution was kept at 70°C for 30 minutes. After cooling, the solution was aseptically filled to obtain the enzymatically hydrolyzed chicken collagen peptide juice.

[0029] The implementation principle of Example 1 is as follows: This process first utilizes a complex of proteases (alkaline and neutral proteases) to perform targeted hydrolysis of collagen in chicken skin and feet under mild conditions, breaking down its macromolecular structure into smaller peptide fragments, forming an enzymatic hydrolysate rich in peptides and amino acids. The resulting hydrolysate is cooled to 55-65°C and defatted using physical centrifugation. The hydrolysate is then transferred to a disc centrifuge and centrifuged at 6000-8000 rpm and 55-65°C for 10-15 minutes. After centrifugation, the upper oil phase (animal fat) and the lower aqueous phase (defatted enzymatic hydrolysate) are collected separately. The lower aqueous phase enters the fermentation process. Subsequently, carbon and nitrogen sources are added to this hydrolysate, and a complex probiotic culture is inoculated. In the early stages of fermentation, static culture provides an anaerobic environment for *Lactobacillus plantarum*, allowing it to rapidly proliferate and metabolize, producing large amounts of lactic acid, lowering the pH of the system, inhibiting other bacteria, and imparting a tangy flavor to the product. In the later stages of fermentation, a microaerobic environment is created through intermittent aeration to activate the metabolic activity of the brewing yeast. This allows the yeast to synthesize aromatic compounds such as esters and alcohols from the sugars and peptides in the enzymatic hydrolysate, further enhancing the product's flavor. Finally, centrifugation and microfiltration remove the microbial cells and insoluble matter, followed by gentle pasteurization. This process ensures microbial safety while fully preserving small-molecule peptides, free amino acids, and heat-sensitive flavor components, resulting in a collagen peptide juice with high peptide content, excellent flavor, and rich nutrients.

[0030] Example 2

[0031] The difference in this embodiment lies in the substitution of raw materials, enzymes, and strains, as well as the adjustment of process parameters: Differences in the enzymatic hydrolysis steps: In this example, only chicken cartilage was selected as the raw material for chicken collagen. The weight ratio of raw material to water was 1:8. The pH was adjusted to 6.0. A mixture of trypsin and papain was selected as the complex protease, and the amount added was 1.5% of the total mass of the chicken collagen raw material. The enzymatic hydrolysis temperature was 55℃, the time was 2 hours, and the stirring speed was 200 rpm.

[0032] Differences in fermentation steps: Sucrose is used as the carbon source, added at 0.5% of the enzymatic hydrolysate mass; soybean peptone is used as the nitrogen source, added at 0.2%. The pH is adjusted to 5.5. The compound probiotics used are *Streptococcus thermophilus* and *Saccharomyces cerevisiae*, with a total inoculum volume of 5% of the enzymatic hydrolysate volume and a viable cell concentration of 10^6 CFU / mL. The fermentation process is as follows: first, static fermentation for 12 hours, followed by intermittent aeration at 0.1 vvm for 6 hours.

[0033] Differences in post-processing steps: Centrifugation speed is 4000 rpm for 30 minutes. Filter membrane pore size is 1.0 μm. Sterilization method is ultra-high temperature instantaneous sterilization at 135℃ for 5 seconds.

[0034] The implementation principle of Example 2 is basically the same as that of Example 1, except that chicken cartilage is used as the raw material. Its collagen structure differs slightly from that of skin and claws. Combined with specific cleavage sites of trypsin and papain, products with different peptide compositions can be obtained. The application of ultra-high temperature instantaneous sterilization can kill microorganisms in a very short time, providing better protection for the flavor and heat-sensitive components of the product, making it especially suitable for producing refreshing beverages with extremely high taste requirements.

[0035] Example 3

[0036] The difference in this embodiment is that some technical features have been removed, specifically: the "intermittent aeration fermentation" step in Embodiment 1 has been omitted, and static fermentation is used throughout the process.

[0037] Difference from Example 1: In step (2) of the fermentation process, only static fermentation is performed. That is, after inoculating with compound probiotics (Lactobacillus plantarum and Saccharomyces cerevisiae), the fermentation is carried out continuously at 35°C for 24 hours without intermittent aeration. The remaining steps, including the ratio of raw materials for enzymatic hydrolysis, the type and amount of enzymes, and the post-processing parameters, are exactly the same as in Example 1.

[0038] Implementation Principle and Effects: The implementation principle of this embodiment is basically the same as that of Embodiment 1. However, since the intermittent aeration step is omitted, the fermentation process is always in an anaerobic or static state. This allows the facultative anaerobic *Lactobacillus plantarum* to dominate throughout the fermentation cycle, while the growth and metabolism (especially aroma production ability) of *Saccharomyces cerevisiae* are significantly inhibited due to the lack of oxygen. Therefore, the lactic acid content in the peptide juice obtained in this embodiment may be higher or the acid production rate may be faster, but the types and contents of characteristic flavor substances (especially esters and higher alcohols produced by yeast metabolism) will be significantly lower than in Embodiment 1. This scheme is suitable for applications that prioritize a tangy taste and high lactic acid content, but do not require a high level of complex aroma.

[0039] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A process for preparing enzymatically hydrolyzed chicken collagen peptide juice, characterized in that, Includes the following steps: (1) Enzymatic hydrolysis: Chicken collagen raw material is mixed with water at a weight ratio of 1:(3~8), the pH is adjusted to 6.0~8.0, a compound protease is added, and enzymatic hydrolysis is carried out at 45~55℃ for 2~6 hours. After enzyme inactivation, the enzymatic hydrolysate is obtained. (2) Fermentation: Add carbon and nitrogen sources to the enzymatic hydrolysate obtained in step (1), adjust the pH to 5.5-7.0, inoculate with compound probiotics, and ferment at 30-37℃ for 12-48 hours to obtain fermentation liquid; (3) Post-processing: The fermentation broth obtained in step (2) is centrifuged, filtered and sterilized in sequence to obtain the enzymatically hydrolyzed chicken collagen peptide juice.

2. The preparation process according to claim 1, characterized in that, The chicken collagen raw material mentioned in step (1) is one or more of chicken skin, chicken cartilage, and chicken carcass; the complex protease is a combination of two or three of alkaline protease, neutral protease, trypsin, and papain.

3. The preparation process according to claim 1, characterized in that, The amount of compound protease added in step (1) is 0.1% to 1.5% of the total mass of chicken collagen raw material, and the stirring rate is maintained at 50 to 200 rpm during enzymatic hydrolysis.

4. The preparation process according to claim 1, characterized in that, The carbon source in step (2) is one or more of glucose, sucrose, and fructooligosaccharides, and the amount added is 0.5% to 3% of the mass of the enzymatic hydrolysate; the nitrogen source is one or more of yeast extract, soybean peptone, and corn steep liquor, and the amount added is 0.2% to 1.5% of the mass of the enzymatic hydrolysate.

5. The preparation process according to claim 1, characterized in that, The compound probiotics mentioned in step (2) are at least two of Lactobacillus plantarum, Streptococcus thermophilus, and Saccharomyces cerevisiae, with a total inoculation amount of 1% to 5% of the volume of the enzymatic hydrolysate and a live bacteria concentration of 10^6 to 10^8 CFU / mL.

6. The preparation process according to claim 1, characterized in that, The fermentation process in step (2) includes a combination of static fermentation and intermittent aeration fermentation: first, static fermentation for 6 to 12 hours, and then intermittent aeration fermentation for 6 to 24 hours with an aeration rate of 0.1 to 0.5 vvm.

7. The preparation process according to claim 1, characterized in that, The centrifugation in step (3) is 4000~8000 rpm for 10~30 minutes; the filtration uses a microfiltration membrane with a pore size of 0.22~1.0 μm; the sterilization is pasteurization at a temperature of 65~75℃ for 30 minutes or ultra-high temperature instantaneous sterilization at a temperature of 135~140℃ for 3~5 seconds.

8. An enzymatically hydrolyzed chicken collagen peptide juice, characterized in that, Prepared by any one of claims 1 to 7, wherein the peptide content is ≥75%, the free amino acid content is ≥2.5 g / L, the lactic acid content is ≥3.0 g / L, and it has characteristic flavor substances produced by probiotic metabolism.

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