Aquatic collagen peptide prebiotics based on Maillard reaction as well as preparation method and application of aquatic collagen peptide prebiotics

Abstract

The invention relates to the field of food science, in particular to aquatic collagen peptide prebiotics based on Maillard reaction as well as a preparation method and application of the aquatic collagen peptide prebiotics. According to the preparation method of the aquatic collagen peptide prebiotics based on the Maillard reaction, provided by the invention, the antioxidant activity of the aquatic collagen peptide can be remarkably enhanced; meanwhile, the prebiotic activity of the product has a remarkable and continuous growth promoting effect on lactobacillus casei, lactobacillus pentosus, bifidobacterium longum and bifidobacterium bifidum. The preparation process provided by the invention is simple, large-scale production is easy, the product can be used as prebiotics in the fields of food, functional food and the like, and the added value of the aquatic collagen peptide can be greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of food science, and particularly relates to a marine collagen peptide prebiotic based on the Maillard reaction, its preparation method and application. Background Art

[0002] The human gut microbiota is a complex and dynamic ecosystem, and the dysregulation of the microecology is associated with a variety of diseases, including diabetes, cardiovascular diseases, obesity, and inflammatory bowel disease (IBD). Prebiotics and probiotics play a crucial role in regulating the composition and metabolic functions of the gut microbiota. A prebiotic is an organic substance that is not digested by the host but can selectively promote the growth of probiotics in the gut of the host, thereby enhancing the health of the host organism. Amino acids are also crucial for the growth of probiotics. Most dietary proteins are absorbed in the small intestine, which may lead to a lack of nitrogen sources in the interstitial fluid between colon cells, thus hindering the growth of probiotics. Therefore, researchers have begun to focus on the potential prebiotic activity of nitrogen-containing compounds such as dietary proteins and amino acids. It has been found that they can affect host health by regulating immunity, enhancing gut barrier function, reducing oxidative stress, regulating the gut microbiota, and the production of their metabolites. It has been reported that collagen peptides also have prebiotic activity. However, most collagen peptides are absorbed in the intestine, so it is difficult for them to be fermented and utilized by gut microorganisms, and single collagen peptides do not meet the requirements for being a prebiotic.

[0003] The Maillard reaction is a non-enzymatic browning reaction that occurs between the reducing carbonyl group of carbohydrates and the free amino group in amino acids / peptides / proteins. During the Maillard reaction, a large number of complex Maillard reaction products are generated. Among them, some of the generated reductones and melanoidins have good antioxidant activity, and some Amadori products and melanoidins have potential prebiotic activity. And it has been found in previous studies that the Maillard reaction can reduce the digestibility of proteins. Therefore, the Maillard reaction can be used to conjugate proteins / peptides and carbohydrates while generating bioactive substances, thereby preparing protein / peptide-containing prebiotics with multiple biological activities. There are currently no invention patents or research reports on the prebiotic potential of glycosylated collagen peptides. In 2023, the total output of aquatic products in China was approximately 71 million tons, of which fish accounted for about 60%. During the processing of fish products, by-products such as fish skin, fish scales, fish bones, and internal organs, which account for 40%-55% of their total mass, are generated. Fish skin, fish scales, and fish bones contain a large amount of collagen. Utilizing these collagens can greatly increase the added value of fish processing, contribute to the in-depth exploration of the new track of the marine economy, and generate greater economic and social benefits.

[0004] Galactooligosaccharides (GOS) is a natural and highly effective prebiotic that can selectively stimulate the growth of probiotics in the human colon, such as Bifidobacterium and Lactobacillus, thereby producing a series of health effects, such as resisting intestinal virus infections, improving human mineral absorption, preventing allergies and intestinal inflammation, and inhibiting the growth of pathogenic intestinal microorganisms. Existing studies have reported that the Maillard reaction products of galactooligosaccharides and proteins / peptides have good digestive resistance, antioxidant activity, and prebiotic activity. Therefore, galactooligosaccharides are a good carbohydrate source for Maillard reaction conjugates. However, research and invention patents on the glycosylation modification of galactooligosaccharides on aquatic collagen peptides as functional foods have not been reported. Summary of the Invention

[0005] The present invention provides an aquatic collagen peptide-based prebiotic based on the Maillard reaction, which has antioxidant activity and prebiotic activity and can be used as a key component of functional foods.

[0006] On the one hand, the present invention provides a preparation method of an aquatic collagen peptide-based prebiotic based on the Maillard reaction, specifically including the following steps:

[0007] a. Prepare a glycopeptide aqueous solution with an aquatic collagen peptide concentration of 5% and a high-purity galactooligosaccharide concentration of 20%; subject the glycopeptide aqueous solution to ultrasonic treatment to fully mix and obtain a mixed solution.

[0008] b. Place the mixed solution in step a under water bath conditions for reaction, and after the reaction is completed, cool it to room temperature to obtain a Maillard reaction product solution.

[0009] c. Rotate evaporate and dry the Maillard reaction product solution in step b to obtain an aquatic collagen peptide-based prebiotic based on the Maillard reaction.

[0010] Further, the purity of the high-purity galactooligosaccharides described in step a is not less than 80%.

[0011] Further, the average molecular weight of the aquatic collagen peptide described in step a is less than 2500 Da.

[0012] Further, the aquatic collagen peptide described in step a can be derived from the fish skin, fish bones, and fish scales of fish such as tilapia, grass carp, giant salamander, sea bream, and cod.

[0013] Further, the ultrasonic temperature in step a is 30°C, and the ultrasonic time is 5 min.

[0014] Further, for the water bath reaction described in step b, the temperature is 70-95°C, and the time is 60-180 min.

[0015] Further, the drying in step c is spray drying or vacuum freeze drying.

[0016] On the other hand, the present invention provides a Maillard reaction product of aquatic collagen peptide obtained by the above preparation method.

[0017] On the other hand, the present invention provides the application of the above-mentioned aquatic collagen peptide prebiotic based on the Maillard reaction as an antioxidant.

[0018] On the other hand, the present invention provides the application of the above-mentioned aquatic collagen peptide prebiotic based on the Maillard reaction as a prebiotic active product.

[0019] The present invention has the following advantages and effects compared with the prior art:

[0020] 1) The aquatic collagen peptide prebiotic based on the Maillard reaction provided by the present invention makes full use of the collagen peptides extracted from various by-products such as fish scales, fish bones, and fish skins, and produces a peptide-containing prebiotic product, which further improves the added value of fish processing and expands the application scenarios of aquatic collagen peptides.

[0021] 2) The Maillard reaction product of aquatic collagen peptide provided by the present invention has good antioxidant activity and prebiotic activity, and can be widely applied to functional foods.

[0022] 3) The preparation method of the aquatic collagen peptide prebiotic based on the Maillard reaction provided by the present invention is simple and easy to be industrially produced. Description of the Drawings

[0023] Figure 1 . Influence of digestion products on the growth density of four probiotic strains, where A: Lactobacillus casei B: Lactobacillus pentosus C: Bifidobacterium longum D: Bifidobacterium bifidum.

[0024] Figure 2 . Influence of fermentation products on the growth density of four probiotic strains, where A: Lactobacillus casei B: Lactobacillus pentosus C: Bifidobacterium longum D: Bifidobacterium bifidum. Detailed Embodiments

[0025] The following further elaborates the present invention in detail with reference to specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention.

[0026] All experimental materials of the present invention are from commercial sources. Tilapia fish scale collagen peptide is purchased from Hainan Huayan Collagen Technology Co., Ltd., and galactooligosaccharide is purchased from Yunfu Xinjinshan Biotechnology Co., Ltd.

[0027] Example 1

[0028] a. Prepare a glycopeptide aqueous solution with a concentration of 5% tilapia scale collagen peptide and 20% galactooligosaccharide at 80%. Ultrasonically treat the glycopeptide aqueous solution for sufficient mixing. The ultrasonic temperature is 30°C and the ultrasonic time is 5 min to obtain a mixed solution.

[0029] b. Place the mixed solution from step a under water bath conditions for reaction. The water bath temperature is 90°C and the reaction time is 60 min. After the reaction, cool it to room temperature to obtain a Maillard reaction product solution.

[0030] c. Rotate evaporate and vacuum freeze-dry the Maillard reaction product solution from step b at 45°C to obtain a kind of aquatic collagen peptide prebiotic based on the Maillard reaction. Store the product in a polyethylene food-grade self-sealing bag.

[0031] Example 2

[0032] a. Prepare a glycopeptide aqueous solution with a concentration of 5% tilapia scale collagen peptide and 20% galactooligosaccharide at 80%. Ultrasonically treat the glycopeptide aqueous solution for sufficient mixing. The ultrasonic temperature is 30°C and the ultrasonic time is 5 min to obtain a mixed solution.

[0033] b. Place the mixed solution from step a under water bath conditions for reaction. The water bath temperature is 90°C and the reaction time is 150 min. After the reaction, cool it to room temperature to obtain a Maillard reaction product solution.

[0034] c. Rotate evaporate and vacuum freeze-dry the Maillard reaction product solution from step b at 45°C to obtain a kind of aquatic collagen peptide prebiotic based on the Maillard reaction. Store the product in a polyethylene food-grade self-sealing bag.

[0035] Comparative Example 1

[0036] a. Prepare a glycopeptide aqueous solution with a concentration of 5% tilapia scale collagen peptide and 20% galactooligosaccharide at 80%. Ultrasonically treat the glycopeptide aqueous solution for sufficient mixing. The ultrasonic temperature is 30°C and the ultrasonic time is 5 min to obtain a mixed solution.

[0037] b. Rotate evaporate and vacuum freeze-dry the mixed solution from step a at 45°C to obtain a physical mixture of tilapia scale collagen peptide - galactooligosaccharide. Store the product in a polyethylene food-grade self-sealing bag.

[0038] Experimental Example 1, Determination of Antioxidant Activity

[0039] Determine the antioxidant activity of Example 1, Example 2 and Comparative Example 1.

[0040] The steps for determining the antioxidant activity are as follows:

[0041] (1) Determination of ABTS radical scavenging activity

[0042] React 7 mmol / L ABTS with 2.45 mmol / L potassium persulfate, and place it in the dark at room temperature for 12 h to obtain ABTS solution. Keep the ABTS solution stable in the dark for 2 days. Dilute the ABTS solution with 5 mM phosphate buffer solution (pH 7.4) to an absorbance of 0.70 ± 0.02 at 730 nm to obtain the ABTS working solution. Reconstitute the sample solutions of Example 1, Example 2 and Comparative Example 1 with water to a concentration of 15.625 mg / mL. Take 0.1 mL of the sample solution, add 2 mL of the ABTS working solution, mix well and react in the dark at room temperature for 20 min, then measure the absorbance at 730 nm. The ABTS radical scavenging ability of the sample solution is expressed as μmol water-soluble vitamin E equivalent (trolox equivalent, TE) / g peptide.

[0043] (2) Determination of reducing power

[0044] Mix 10 mmol / L TPTZ solution, 20 mmol / L ferric chloride solution and 300 mmol / L sodium acetate buffer solution (pH 3.6) in a volume ratio of 1:1:10 to obtain the FRAP working solution. Reconstitute the sample solutions of Example 1, Example 2 and Comparative Example 1 with water to a concentration of 62.5 mg / mL. Take 0.1 mL of the sample solution, add 4.9 mL of the FRAP working solution, mix well and react in the dark at 37°C for 30 min, then measure the absorbance at 595 nm. The reducing power of the sample solution is expressed as μmol water-soluble vitamin E equivalent (trolox equivalent, TE) / g peptide.

[0045] Table 1 Results of antioxidant activity test

[0046] Sample Name ABTS Radical Scavenging Capacity (μmol TE / g peptide) Reducing Power (μmol TE / g peptide) Example 1 <![CDATA[190.79±0.85 a > <![CDATA[15.52±0.97 a <!-- 3 -->]]> Example 2 <![CDATA[198.52±0.23 b > <![CDATA[21.45±0.44 b > Comparative Example 1 <![CDATA[175.33±0.85 c > <![CDATA[12.65±0.12 c >

[0047] Note: Different letters indicate significant differences in the ABTS radical scavenging ability or reducing power of different samples (P < 0.05).

[0048] Experimental Example 2, Determination of antioxidant stability under digestion conditions

[0049] Determine the antioxidant stability of Example 1, Example 2 and Comparative Example 1 under digestion conditions.

[0050] The steps for determining the antioxidant stability under digestion conditions are as follows:

[0051] (1) In vitro simulated gastrointestinal digestion

[0052] A. The preparation methods of simulated gastric juice and simulated intestinal juice are shown in Table 2.

[0053] Table 2 Components for preparing simulated gastric juice and simulated intestinal juice

[0054] Component KCl <![CDATA[KH2PO4]]> <![CDATA[NaHCO3]]> NaCl <![CDATA[MgCl2(H2O)6]]> <![CDATA[(NH4)2CO3]]> <![CDATA[H2O]]> Stock Solution g / L 37.3 68 84 117 30.5 48 SGF(1.25x) mL 3.45 0.45 6.25 5.9 0.2 0.25 183.5 SIF(1.25x) mL 5.1 0.6 31.875 7.2 0.825 - 254.4

[0055] B. Redissolve the samples of Example 1, Example 2 and Comparative Example 1 with water to a sample solution with a concentration of 250 mg / mL. Conduct simulated gastric digestion: Mix 12 mL of the sample solution, 8.4 mL of SGF, 1.2 mL of pepsin at 40000 U / mL, and 6 μL of 0.3 mol / L CaCl2, make up the volume to 24 mL with distilled water, and adjust the pH to 3.0 with 1 mol / L HCl. Oscillate in a shaker at 37 °C for 2 h.

[0056] C. Conduct simulated intestinal digestion: Mix 12 mL of the gastric digest, 7.2 mL of SIF, 2.4 mL of trypsin at 1000 U / mL, 24 μL of 0.3 mol / L CaCl2, make up the volume to 24 mL with distilled water, and adjust the pH to 7.0 with 1 mol / L NaOH. Oscillate in a shaker at 37 °C for 4 h. Bile salts are not added in this intestinal digestion process because bile salts mainly act on lipid components and most bile salts will be reabsorbed by the human intestine, and bile salts have no important role in this simulated intestinal digestion process.

[0057] (2) Determination of ABTS radical scavenging activity and reducing power

[0058] The steps for determining the antioxidant stability under digestion conditions are the same as those in the above antioxidant activity determination steps.

[0059] Table 3 Results of antioxidant stability test under digestion conditions

[0060] Sample Name ABTS Radical Scavenging Capacity (μmol TE / g peptide) Reducing Power (μmol TE / g peptide) Example 1 <![CDATA[189.98±0.23 a > <![CDATA[15.66±0.73 a > Example 2 <![CDATA[206.80±0.23 b > <![CDATA[22.64±0.44 b > Comparative Example 1 <![CDATA[179.94±0.62 c > <![CDATA[11.68±0.55 c >

[0061] Note: Different letters indicate significant differences in the ABTS radical scavenging ability or reducing power of different samples (P < 0.05).

[0062] Experimental Example 3, Determination of prebiotic activity

[0063] Vacuum freeze-dry the digestive juices of Example 1, Example 2 and Comparative Example 1 obtained in Experimental Example 2 to obtain digestive products, and determine the prebiotic activity of the digestive products.

[0064] The steps for determining the prebiotic activity are as follows:

[0065] (1) Preparation of modified MRS medium

[0066] Prepare MRS medium without glucose and peptone, add 0.5 g / L of L-cysteine, and sterilize. Add 2.5% (w / v) of the digestion product to the sterilized MRS medium as the sole carbon source and the main nitrogen source to obtain the modified MRS medium.

[0067] (2) Strain activation

[0068] Activate the purchased Lactobacillus casei, Lactobacillus pentosus, Bifidobacterium longum, and Bifidobacterium bifidum according to the instructions. Expand the bacterial liquid of each strain to the second generation respectively, and wait until its growth reaches OD 600 is 0.8 ± 0.01 to obtain the standby bacterial liquid.

[0069] (3) Determination of the growth curve of probiotics

[0070] Inoculate the standby bacterial liquid in step (2) into the modified MRS medium in step (1) at an inoculation amount of 2% (v / v), and perform anaerobic incubation at a constant temperature of 37 °C for 48 h. During this period, measure the OD of the culture solution every 2 h 600 .

[0071] Experimental Example 4, Determination of prebiotic persistence

[0072] Perform vacuum freeze-drying on the digestion fluids of Example 1, Example 2, and Comparative Example 1 obtained in Experimental Example 2 to obtain the digestion product, and perform the determination of prebiotic persistence on the digestion product.

[0073] The steps for determining prebiotic persistence are as follows:

[0074] (1) In vitro simulated colonic fermentation

[0075] A. Prepare the fermentation broth: Add 3.75 g of tryptone to about 250 mL of distilled water, adjust the pH value to 7, and make up the volume to 250 mL to obtain the fermentation medium; dissolve 78 mg of cysteine and 78 mg of sodium sulfide in distilled water, then add 0.5 ml of 1 mol / L NaOH, and make up the volume to 12.5 mL to obtain the reducing solution; prepare a 0.1% resazurin solution. Finally, mix 250 mL of the fermentation medium, 12.5 mL of the reducing solution, and 0.328 mL of the 0.1% resazurin solution evenly and sterilize to obtain the fermentation broth.

[0076] B. Prepare the inoculum: Collect fresh feces from four volunteers, and prepare 32% fecal suspensions with 0.1M phosphate buffer solution (pH 7) respectively. Centrifuge the fecal suspensions at 8000 g and 4 °C for 10 min, and take equal volumes of the supernatant of the centrifuged fecal suspensions and mix them evenly to obtain the inoculum.

[0077] C. Fermentation: Mix 3 g of the digestion product, 45 mL of fermentation broth, and 12 mL of the inoculum evenly, and perform anaerobic incubation at a constant temperature of 37 °C for 6 h. After the incubation, sterilize in a water bath at 100 °C for 6 min, centrifuge at 8000 g and 4 °C for 10 min, collect the supernatant, filter the supernatant through a 0.22 μm filter membrane, and lyophilize the filtrate to obtain the fermentation product.

[0078] (2) Preparation of the modified MRS medium

[0079] Prepare MRS medium without glucose and peptone, and add 0.5 g / L of L-cysteine, then sterilize. Add 2.5% (w / v) of the fermentation product to the sterilized MRS medium as the sole carbon source and the main nitrogen source to obtain the modified MRS medium.

[0080] (3) The determination of the growth curve of the probiotic is the same as that in the above-mentioned determination steps of the prebiotic activity.

[0081] The ABTS radical scavenging ability and reducing ability can directly reflect the antioxidant ability of the sample. As can be seen from the results in Table 2, compared with Comparative Example 1, the antioxidant abilities of Example 1 and Example 2 are stronger, indicating that the Maillard reaction significantly enhances the antioxidant ability of aquatic collagen peptides, and the higher the degree of the Maillard reaction, the stronger the antioxidant improvement ability; as can be seen from the results in Table 3, compared with Comparative Example 1, the antioxidant abilities of Example 1 and Example 2 are stronger, and Example 2 still maintains the highest antioxidant activity, indicating that the aquatic collagen peptide prebiotic in the present invention can still maintain stable antioxidant ability after simulated gastrointestinal digestion, and the antioxidant activity of the Maillard reaction product will not decrease due to digestive conditions such as low pH and digestive enzymes. OD 600 can reflect the growth of the probiotic. The more the number of strains in the medium, the higher its OD 600 value. As Figure 1 can be seen from the results, compared with Comparative Example 1, the growth promotion effects of Example 1 and Example 2 on the four probiotic strains are both improved, and Example 2 shows the strongest growth promotion effect comprehensively, indicating that the Maillard reaction product of aquatic collagen peptides has a significant promoting effect on the growth of probiotics, and the growth promotion effect of Example 2 is stronger; as Figure 2 can be seen from the results, Comparative Example 1, Example 1 and Example 2 can still significantly promote the growth of the four probiotic strains, and compared with Comparative Example 1, the maximum OD 600 values of the four probiotic strains growing on Example 1 and Example 2 are generally higher, indicating that the aquatic collagen peptide prebiotic in the present invention has good fermentation persistence. In summary, the aquatic collagen peptide prebiotic developed in the present invention not only has strong antioxidant activity but also can significantly promote the growth of the tested probiotics. Therefore, the aquatic collagen peptide prebiotic developed in the present invention has great application potential in improving the application value and scenarios of aquatic collagen peptides and developing new multi-active peptide-containing prebiotics.

[0082] Finally, it should be noted that the above embodiments are preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A preparation method of aquatic collagen peptide prebiotic based on Maillard reaction, characterized in that, It includes the following steps: a. Prepare a glycopeptide aqueous solution with a concentration of 5% of aquatic collagen peptide and a concentration of 20% of high-purity galactooligosaccharide; subject the glycopeptide aqueous solution to ultrasonic treatment to fully mix and obtain a mixed solution. b. Place the mixed solution in step a under a water bath condition for reaction, and cool it to room temperature after the reaction ends to obtain a Maillard reaction product solution. c. Subject the Maillard reaction product solution in step b to rotary evaporation and drying to obtain an aquatic collagen peptide-based prebiotic based on the Maillard reaction.

2. The preparation method of a kind of aquatic collagen peptide prebiotic based on Maillard reaction according to claim 1, characterized in that, The purity of the high-purity galactooligosaccharide described in step S1 is not less than 80%.

3. The preparation method of a kind of aquatic collagen peptide prebiotic based on Maillard reaction according to claim 1, characterized in that, The average molecular weight of the aquatic collagen peptide described in step S1 is less than 2500 Da.

4. The preparation method of a kind of aquatic collagen peptide prebiotic based on Maillard reaction according to claim 1, characterized in that, The aquatic collagen peptide described in step S1 can be derived from the fish skin, fish bones and fish scales of fish such as tilapia, grass carp, giant salamander, sea bream, and cod.

5. The preparation method of a kind of aquatic collagen peptide prebiotic based on Maillard reaction according to claim 1, characterized in that, The ultrasonic temperature described in step S1 is 30 °C and the ultrasonic time is 5 min.

6. The preparation method of a kind of aquatic collagen peptide prebiotic based on Maillard reaction according to claim 1, wherein, For the water bath reaction described in step S2, the temperature is 70-95 °C and the time is 60-180 min.

7. A preparation method of a marine collagen peptide prebiotic based on the Maillard reaction according to claim 1, characterized in that, The drying described in step S3 is spray drying or vacuum freeze drying.

8. An aquatic collagen peptide-based prebiotic based on the Maillard reaction obtained by the preparation method according to any one of claims 1-7.

9. Use of the Maillard reaction product of the aquatic collagen peptide-based prebiotic described in claim 8 as an antioxidant.

10. Use of the aquatic collagen peptide-based prebiotic described in claim 8 as a prebiotic active product.

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