Application and method of collagen hydrolysate in preparing products for improving acid resistance and / or bile resistance and / or proliferation activity of probiotics

Abstract

The present invention relates to an application of collagen hydrolyzate in the preparation of a product for improving the acid resistance and / or bile salt resistance and / or proliferation activity of probiotics, a method for improving the acid resistance and / or bile salt resistance and / or proliferation activity of probiotics, and a probiotic product. On the basis of the collagen hydrolyzate providing growth factors for probiotics (such as Lactobacillus rhamnosus), the collagen hydrolyzate is added under acidic and bile salt culture medium conditions to significantly improve the growth activity of Lactobacillus rhamnosus, and can improve the activity of probiotics (such as Lactobacillus rhamnosus) in gastric juice and intestines when ingested.

Description

Technical Field

[0001] The present invention relates to the field of probiotics, and in particular to an application of collagen hydrolyzate in the preparation of a product for improving the acid resistance and / or bile resistance and / or proliferation activity of probiotics, a method for improving the acid resistance and / or bile resistance and / or proliferation activity of probiotics, and a probiotic product. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Probiotics are a type of microorganisms that are of great significance to human health. Common lactic acid bacteria include Bifidobacterium, Lactobacillus, Lactococcus, and Streptococcus thermophilus. Probiotics have the functions of promoting the body's absorption of nutrients, improving the body's immunity, improving the body's antioxidant level, and inhibiting intestinal inflammation. They play an important role in human health. How to efficiently promote the growth of probiotics has become a problem that needs to be solved. At present, many studies have reported that functional oligosaccharides can selectively promote the proliferation of probiotics. Common functional oligosaccharides include oligofructose, oligogalactose, oligomaltosan, etc. However, these rapidly fermented oligosaccharides have many disadvantages, including a relatively simple production method, high price, low yield, and can cause diarrhea and flatulence in humans. During the processing, transportation, storage, sales and consumption of probiotic products, changes in temperature, light, oxygen, water activity and other factors will affect the survival ability of probiotics, ultimately leading to a rapid decline in the number of live bacteria.

[0004] Therefore, increasing the number of live probiotics and improving the resistance of probiotics to adverse environmental factors such as oxygen, gastric acid, and bile has become the focus of current research.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] Purpose of the Invention

[0007] The object of the present invention is to provide an application of collagen hydrolyzate in the preparation of a product for improving the acid resistance and / or bile resistance and / or proliferation activity of probiotics, a method for improving the acid resistance and / or bile resistance and / or proliferation activity of probiotics, and a probiotic product.

[0008] On the basis of providing growth factors for probiotics (such as Lactobacillus rhamnosus), the collagen hydrolyzate of the present invention is added under acidic and bile salt culture medium conditions to significantly improve the growth activity of Lactobacillus rhamnosus and to improve the activity of probiotics (such as Lactobacillus rhamnosus) in gastric juice and intestines when ingested.

[0009] Solution

[0010] To achieve the purpose of the present invention, in a first aspect, the present invention provides a use of a collagen hydrolyzate in the preparation of a product for improving the acid resistance and / or bile resistance and / or proliferation activity of probiotics.

[0011] Furthermore, the collagen hydrolysate is fish-derived collagen hydrolysate, and optionally the collagen hydrolysate is fish-derived bone collagen hydrolysate; optionally the collagen hydrolysate is tuna bone collagen hydrolysate.

[0012] Furthermore, the collagen hydrolysate is a collagen hydrolysate hydrolyzed by alkaline protease; optionally, the collagen before enzymatic hydrolysis is fish-derived collagen that has been decalcified, deproteinized and / or delipidated; optionally, the collagen before enzymatic hydrolysis is collagen powder.

[0013] Furthermore, the collagen hydrolyzate contains a polypeptide as shown in SEQ ID NO: 1: GLpGPLGPpGPR (since p (hydroxylated proline) cannot be correctly displayed in the sequence list, that is, P at positions 3 and 9 in the sequence list are hydroxylated proline);

[0014] Furthermore, the content of the collagen hydrolyzate in the product is ≥2 mg / mL, optionally 0.25 mg / mL to 6 mg / mL, optionally 0.25 mg / mL to 4 mg / mL, optionally ≥4 mg / mL, optionally 0.4 mg / mL to 6 mg / mL.

[0015] In a second aspect, a method for improving the acid resistance and / or bile resistance and / or proliferation activity of probiotics is provided, comprising: adding collagen hydrolysate to a probiotic product, wherein the content of the collagen hydrolysate in the product is ≥2 mg / mL.

[0016] Furthermore, the content of the collagen hydrolyzate in the product is 0.25 mg / mL to 6 mg / mL, optionally 0.25 mg / mL to 4 mg / mL, optionally ≥ 4 mg / mL, optionally 0.4 mg / mL to 6 mg / mL.

[0017] Furthermore, the collagen hydrolysate contains the polypeptide shown in SEQ ID NO: 1: GLpGPLGPpGPR.

[0018] Furthermore, the preparation method of the collagen hydrolysate comprises:

[0019] Alkaline protease is added to the animal-derived collagen powder for hydrolysis to obtain a hydrolyzate. The hydrolysis conditions are optionally 50-60° C., pH 8.5-9.5, and the hydrolysis time is 1.5-3 hours.

[0020] Furthermore, the animal-derived collagen powder is fish-derived bone collagen powder. Optionally, the preparation method thereof includes: acid-decalcifying the fish-derived bone, washing it, removing fat by adding lipase, and dehydrating and crushing the fish-derived bone.

[0021] In a third aspect, a probiotic product is provided, comprising collagen hydrolysate, wherein the content of the collagen hydrolysate in the product is ≥2 mg / mL.

[0022] Furthermore, the content of the collagen hydrolyzate in the product is 0.25 mg / mL to 6 mg / mL, optionally 0.25 mg / mL to 4 mg / mL, optionally ≥ 4 mg / mL, optionally 0.4 mg / mL to 6 mg / mL.

[0023] Furthermore, the collagen hydrolysate contains the polypeptide shown in SEQ ID NO: 1: GLpGPLGPpGPR.

[0024] Beneficial Effects

[0025] (1) On the basis of providing growth factors for probiotics (such as Lactobacillus rhamnosus), the collagen hydrolyzate of the present invention is added under acidic and bile salt medium conditions to significantly improve the growth activity of Lactobacillus rhamnosus and improve the activity of probiotics (such as Lactobacillus rhamnosus) in gastric juice and intestines when ingested.

[0026] (2) The present invention has conducted amino acid sequence analysis and biological activity research on fish bone collagen hydrolysate, and found a collagen peptide with high biological activity, namely, a collagen peptide with an amino acid sequence of GLpGPLGPpGPR. Collagen hydrolysate containing this sequence plays a significant role in promoting the growth activity of Lactobacillus rhamnosus.

[0027] (3) The collagen hydrolyzate in the present invention is derived from natural materials, has mild preparation conditions, does not contain any additives, will not cause hemolytic toxicity to probiotics, and has high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0029] Figure 1 This is the preparation process of tuna collagen hydrolysate in Example 1 of the present invention.

[0030] Figure 2This is the growth curve of Lactobacillus rhamnosus cultured in normal MRS medium in Example 1-3 of the present invention, wherein CH represents tuna collagen hydrolyzate.

[0031] Figure 3 It is the growth curve of Lactobacillus rhamnosus cultured in acidic MRS medium or MRS medium containing bile salts in Examples 1-3 of the present invention, wherein CH represents tuna collagen hydrolysate; A is the growth curve of Lactobacillus rhamnosus in acidic MRS medium, and B is the growth curve of Lactobacillus rhamnosus in MRS medium containing bile salts.

[0032] Figure 4 This is the activity of the probiotics after gastric juice and intestinal juice simulation in Example 4 of the present invention, wherein CH represents tuna collagen hydrolysate. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "including" or its variations such as "comprising" or "including" will be understood to include the stated elements or components, without excluding other elements or other components.

[0034] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0035] In the following examples, all raw materials are commercially available products.

[0036] In the following examples, peptide sequence analysis was performed using liquid chromatography-mass spectrometry.

[0037] In the following examples, tuna bones were purchased from Rongcheng Lanrun Biotechnology Co., Ltd.

[0038] In the following examples, alkaline protease was purchased from Shanghai Yuanye Biotechnology, BR, 200 U / mg.

[0039] In the following examples, Lactobacillus rhamnosus was purchased from Beina Biotechnology with strain number BNCC186192.

[0040] In the following examples, the acidic MRS medium is a medium in which the pH of the MRS medium is adjusted to 2 using 1 M HCl.

[0041] In the following examples, bile salt MRS medium is an MRS medium containing 0.1 wt % bile salt.

[0042] In the screening of collagen polypeptides in the following examples, the collagen peptides need to follow the structure of repeated Gly-XY units, Gly is glycine, X and Y represent any amino acid except glycine, X is more commonly proline, and Y is more commonly hydroxyproline. The amino acid sequence measured by liquid chromatography-mass spectrometry is compared to screen out peptides containing Gly-XY repeating units.

[0043] Example 1

[0044] Example 1-1 Preparation of tuna bone collagen hydrolysate

[0045] (1) Pretreatment: Tuna bones are first treated with 0.5M HCl to decalcify for 7.5 hours, and then the acid-soaked tuna bones are repeatedly washed with distilled water until neutral. After washing, the bones are soaked in 4% NaCl solution for 12 hours to remove the foreign proteins on the surface. The pH value of the solution is then adjusted to 8.0 (1M NaOH is used to adjust the pH), and 0.4% (w / v) lipase and 0.2% (w / v) yeast are added to the solution (to remove the fishy smell of the fish bones). The temperature is adjusted to 40°C and the treatment is carried out for 2 hours. The treated fish bones are dehydrated and placed in a grinder for pulverization to obtain tuna bone powder for use.

[0046] (2) Enzymatic hydrolysis: Take the tuna bone powder of step (1), add alkaline protease (the mass ratio of enzyme to substrate (tuna bone powder) is 1:25), and perform enzymatic hydrolysis for 2 hours at an enzymatic hydrolysis temperature of 55° C. and a pH value of 9. After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is treated in a 100° C. boiling water bath for 10 minutes to inactivate the enzyme and sterilize, and the stock solution is filtered using a filter membrane (pore size 0.22 μm), and finally the filtered liquid is freeze-dried to obtain tuna bone collagen hydrolyzate.

[0047] Example 1-2 Amino Acid Sequence Analysis of Tuna Collagen Hydrolysate

[0048] (1) Prepare 10 mg of the freeze-dried sample of Example 1-1, dissolve the sample in a mobile phase and filter it with a 0.22 μm filter membrane, and use liquid chromatography-mass spectrometry to test the sequence of tuna collagen hydrolyzate, wherein the mobile phase is acetonitrile: water: trifluoroacetic acid, with a volume ratio of 40:60:0.05.

[0049] (2) Screening and bioactivity analysis of collagen peptides that conform to the Gly-XY sequence, and ranking the peptides according to the predicted bioactivity scores (http: / / distilldeep.ucd.ie / PeptideRanker / ).

[0050] It was detected that the tuna collagen hydrolyzate in Example 1-1 contained collagen peptides of GLpGPLGPpGPR.

[0051] Example 1-3: Bioactivity analysis of tuna collagen hydrolysate

[0052] Effect of tuna collagen hydrolyzate of Example 1-1 on the growth activity of Lactobacillus rhamnosus

[0053] Turbidimetric method: The activated probiotic strain (Lactobacillus rhamnosus) was inoculated into the corresponding liquid culture medium (MRS culture medium, acidic MRS culture medium, bile salt MRS culture medium) at 1%, and cultured at 37°C for 24 hours under anaerobic conditions. A normal growth group and a sample addition group were set up (wherein, the normal growth group did not add tuna collagen hydrolysate, and the sample addition group added the tuna collagen hydrolysate of Example 1-1), and the culture was shaken at 37°C on a shaker. Samples were taken at different time points within 10 hours, and 200 μL of the sample was taken each time in a 96-well plate. The OD value (OD 600nm ), 3 replicates were set at each time point, and the average result was used as the OD 600nm The curves showing the changes of the values ​​over time are the growth curves of Lactobacillus rhamnosus.

[0054] Among them, the growth curve of Lactobacillus rhamnosus cultured in MRS medium is as follows Figure 2 As shown, the results showed that Lactobacillus rhamnosus proliferated according to a certain rule, and the addition of tuna collagen hydrolysate (abbreviated as CH) was beneficial to improving the proliferation efficiency of Lactobacillus rhamnosus, and the higher the concentration, the more obvious the effect.

[0055] The growth curve of Lactobacillus rhamnosus cultured in acidic MRS medium is as follows Figure 3 As shown in A, the results show that in the acidic MRS medium, the rhamnosus lactobacillus hardly reproduces, but after adding tuna collagen hydrolysate (abbreviated as CH), especially when the tuna collagen hydrolysate is added in an amount of 2 mg / mL, the rhamnosus lactobacillus can reproduce and improve its tolerance to acid.

[0056] The growth curve of Lactobacillus rhamnosus cultured in bile salt MRS medium is as follows Figure 3As shown in Figure B, the results show that in the bile salt MRS medium (not containing tuna collagen hydrolysate), the rhamnosus lactobacillus hardly reproduces, while after adding tuna collagen hydrolysate (abbreviated as CH), especially when the tuna collagen hydrolysate is added in an amount of 4 mg / mL, the rhamnosus lactobacillus can reproduce and improve its tolerance to bile salts, and the effect is not obvious at low concentrations.

[0057] Example 2

[0058] Example 2-1 Preparation of tuna collagen hydrolysate

[0059] (1) Pretreatment: Tuna bones are first treated with 1M HCl to decalcify for 8 hours, and then the acid-soaked bones are repeatedly washed with distilled water until neutral. After washing, the bones are soaked in 5% NaCl solution for 12 hours to remove the foreign proteins on the surface. The pH value of the solution is then adjusted to 8.0 (1M NaOH is used to adjust the pH), and 0.5% (w / v) lipase and 0.3% (w / v) yeast are added to the solution (to remove the fishy smell of the fish bones). The temperature is adjusted to 45°C and the treatment is carried out for 2 hours. The treated fish bones are dehydrated and placed in a grinder for pulverization to obtain tuna bone powder for use.

[0060] (2) Enzymatic hydrolysis: Take the tuna bone powder of step (1), add alkaline protease (enzyme to substrate mass ratio of 1:15), and perform enzymatic hydrolysis for 2 hours at an enzymatic hydrolysis temperature of 55° C. and a pH value of 9. After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is treated in a 100° C. boiling water bath for 10 minutes to inactivate the enzyme and sterilize, and the stock solution is filtered using a filter membrane (pore size 0.22 μm), and finally the filtered liquid is freeze-dried to obtain tuna bone collagen hydrolyzate.

[0061] Example 2-2 Amino Acid Sequence Analysis of Tuna Collagen Hydrolysate

[0062] (1) Prepare 10 mg of sample, dissolve the sample in mobile phase and filter with a 0.22 μm filter membrane, and use liquid chromatography-mass spectrometry to test the sequence of tuna collagen hydrolysate, wherein the mobile phase is acetonitrile: water: trifluoroacetic acid, with a volume ratio of 40:60:0.05.

[0063] (2) Screening and bioactivity analysis of collagen peptides that conform to the Gly-XY sequence, and ranking the peptides according to the predicted bioactivity scores (http: / / distilldeep.ucd.ie / PeptideRanker / ).

[0064] The tuna collagen hydrolyzate in Example 2-1 was detected to contain collagen peptides of GLpGPLGPpGPR.

[0065] Example 2-3: Bioactivity Analysis of Tuna Collagen Hydrolysate

[0066] The method for determining the growth activity of the tuna collagen hydrolyzate of Example 2-1 on Lactobacillus rhamnosus is as follows:

[0067] Turbidimetric method: The activated probiotic strain (Lactobacillus rhamnosus) was inoculated into the corresponding liquid culture medium (MRS culture medium, acid culture medium, bile salt culture medium) at 1%, and cultured at 37°C for 24 hours under anaerobic conditions. A normal growth group and a sample addition group were set up (wherein, the normal growth group did not add tuna collagen hydrolysate, and the sample addition group added the tuna collagen hydrolysate of Example 2-1), and the culture was shaken at 37°C on a shaker. Samples were taken at different time points within 10 hours, and 200 μL of the sample was taken each time in a 96-well plate. The OD value (OD 600nm ), 3 replicates were set at each time point, and the average result was used as the OD 600nm The curves showing the changes of the values ​​over time are the growth curves of Lactobacillus rhamnosus.

[0068] Example 3 Effect of tuna collagen hydrolysate on the activity of Lactobacillus rhamnosus in a simulated digestive system

[0069] Simulated gastric fluid: Pepsin was sterilized by filtration, dissolved in a sterilized 0.5% NaCl solution, the final concentration of pepsin was 0.3%, and the pH was adjusted to 2 with 1 M hydrochloric acid.

[0070] Simulated intestinal fluid: Trypsin (0.1%) and porcine bile salt (0.1%) were dissolved in sterile 0.5% NaCl solution and the pH was adjusted to 6.8-7.4.

[0071] Establish an in vitro simulated gastrointestinal digestive system to evaluate the activity of Lactobacillus rhamnosus before and after digestion. The specific steps are as follows

[0072] The strain (Lactobacillus rhamnosus) was inoculated into MRS liquid culture medium at 2% (v / v), cultured at 37°C for 24 hours, the fermentation broth of the strain was collected and centrifuged (5000r / min, 10min), the bacterial precipitate was collected, the bacterial body was washed 3 times with sterile saline and then resuspended in sterile saline to obtain a bacterial suspension.

[0073] The concentration of bacterial suspension was adjusted to (10 6 -10 7CFU / mL) was placed in 5.0mL of the above-mentioned simulated gastric juice (pepsin 0.3%, pH 2.0). In this experiment, a normal group and a sample-added group were set up (wherein, the normal growth group did not add tuna collagen hydrolysate, and the sample-added group added the tuna collagen hydrolysate of Example 1-1), and incubated at 37°C for 2 hours with shaking at 100rpm. Then the pH of the system was adjusted to 6.8-7.4, an equal volume of simulated intestinal fluid was added, and the incubation continued at 37°C for 2h to simulate intestinal digestion. After the digestion was completed, 100μL of the solution of the appropriate dilution gradient was taken and coated in MRS solid culture medium, and the coated plate was placed under anaerobic conditions at 37°C for 24h, and the plate was taken out and the activity of the probiotics was observed.

[0074] The results of the 100-fold dilution plate culture are as follows Figure 4 As shown, the results show that after adding tuna collagen hydrolysate (abbreviated as CH), especially when the addition amount of tuna collagen hydrolysate is 2 mg / mL, the reproduction of Lactobacillus rhamnosus can be significantly improved, indicating that tuna collagen hydrolysate can improve the tolerance of Lactobacillus rhamnosus in gastric juice and intestines, which is beneficial to improve the survival ability of Lactobacillus rhamnosus when ingested into the body, especially the improvement effect of 4 mg / mL tuna collagen hydrolysate is very obvious.

[0075] Different concentrations of fish bone collagen hydrolysate were added to normal MRS medium, acidic MRS medium, and bile salt-containing MRS medium for culture. The results showed that the collagen hydrolysate not only promoted the growth activity of Lactobacillus rhamnosus under normal medium conditions, but also improved the activity of Lactobacillus rhamnosus in acidic and bile salt medium. In the simulated digestive system, it was confirmed that collagen hydrolysate improved the resistance of Lactobacillus rhamnosus to acid and bile salt environments, thereby improving the survival ability of Lactobacillus rhamnosus.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a collagen hydrolyzate in the preparation of a product for improving the acid resistance and / or bile resistance and / or proliferation activity of probiotics; the collagen hydrolyzate is a fish-derived collagen hydrolyzate; the collagen hydrolyzate is a collagen hydrolyzate hydrolyzed by alkaline protease; the collagen hydrolyzate contains a polypeptide as shown in SEQ ID NO: 1: GLpGPLGPpGPR, wherein p is hydroxylated proline; the probiotic is Lactobacillus rhamnosus.

2. The use according to claim 1, characterized in that: The collagen hydrolysate is tuna bone collagen hydrolysate.

3. The use according to claim 1, characterized in that: The collagen before enzymatic hydrolysis is fish-derived collagen that has been decalcified, deproteinized and / or de-fatified.

4. The use according to claim 1, characterized in that: The collagen is collagen powder before enzymatic hydrolysis.

5. The use according to claim 1, characterized in that: The collagen hydrolyzate is sterilized collagen hydrolyzate.

6. The use according to claim 1, characterized in that: The collagen hydrolyzate is a product obtained by filtering through a filter membrane and drying.

7. The use according to claim 6, characterized in that: The pore size of the filter membrane is 0.2~0.3μm.

8. The use according to claim 6, characterized in that: The pore size of the filter membrane is 0.22 μm.

9. The use according to any one of claims 1 to 8, characterized in that: The content of the collagen hydrolyzate in the product is ≥2 mg / mL.

10. The use according to any one of claims 1 to 8, characterized in that: The content of the collagen hydrolyzate in the product is 0.25 mg / mL to 6 mg / mL.

11. The use according to any one of claims 1 to 8, characterized in that: The content of the collagen hydrolyzate in the product is 0.25 mg / mL to 4 mg / mL.

12. The use according to any one of claims 1 to 8, characterized in that: The content of the collagen hydrolyzate in the product is ≥4 mg / mL.

13. The use according to any one of claims 1 to 8, characterized in that: The content of the collagen hydrolyzate in the product is 0.4 mg / mL to 6 mg / mL.

14. A method for improving the acid resistance and / or bile resistance and / or proliferation activity of probiotics, characterized in that: include: Adding collagen hydrolysate to the probiotic product, wherein the content of the collagen hydrolysate in the product is ≥2 mg / mL; The preparation method of the collagen hydrolysate comprises: adding alkaline protease to fish-derived collagen powder for hydrolysis to obtain a hydrolysate; The collagen hydrolyzate contains a polypeptide as shown in SEQ ID NO: 1: GLpGPLGPpGPR, wherein p is hydroxylated proline; The probiotic is Lactobacillus rhamnosus.

15. The method according to claim 14, characterized in that The content of the collagen hydrolyzate in the product is 0.25 mg / mL to 6 mg / mL.

16. The method according to claim 14, characterized in that The content of the collagen hydrolyzate in the product is 0.25 mg / mL to 4 mg / mL.

17. The method according to claim 14, characterized in that The content of the collagen hydrolyzate in the product is ≥4 mg / mL.

18. The method according to claim 14, characterized in that The content of the collagen hydrolyzate in the product is 0.4 mg / mL to 6 mg / mL.

19. The method according to any one of claims 14 to 18, characterized in that: The preparation method of the collagen hydrolysate comprises: enzymatic hydrolysis conditions of alkaline protease are 50-60°C, pH 8.5-9.5, and enzymatic hydrolysis for 1.5-3h.

20. The method according to any one of claims 14 to 18, characterized in that: The method for preparing the fish-derived collagen powder comprises: decalcifying the fish-derived bone with acid, washing it, removing fat with lipase, and dehydrating and crushing the fish-derived bone.

21. A probiotic product, characterized in that: Containing collagen hydrolysate, the content of the collagen hydrolysate in the product is ≥2 mg / mL; The collagen hydrolysate is fish-derived collagen hydrolysate; the collagen hydrolysate is collagen hydrolysate hydrolyzed by alkaline protease; the collagen hydrolysate contains a polypeptide as shown in SEQ ID NO: 1: GLpGPLGPpGPR, wherein p is hydroxylated proline; and the probiotic is Lactobacillus rhamnosus.

22. The probiotic preparation according to claim 21, characterized in that The content of the collagen hydrolyzate in the product is 0.25 mg / mL to 6 mg / mL.

23. The probiotic preparation according to claim 21, characterized in that The content of the collagen hydrolyzate in the product is 0.25 mg / mL to 4 mg / mL.

24. The probiotic preparation according to claim 21, characterized in that The content of the collagen hydrolyzate in the product is ≥4 mg / mL.

25. The probiotic preparation according to claim 21, characterized in that The content of the collagen hydrolyzate in the product is 0.4 mg / mL to 6 mg / mL.

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