Low-phenylalanine gelatin polypeptide and application thereof

Abstract

The invention discloses low-phenylalanine gelatin polypeptide and application thereof, and relates to the technical field of food. The preparation process of the low-phenylalanine gelatin polypeptide comprises the following steps: dissolving gelatin, carrying out two-step enzymolysis by using alkaline protease and flavourzyme, centrifuging enzymatic hydrolysate, collecting supernate, and adsorbing by using activated carbon to remove polypeptide containing aromatic amino acid (including phenylalanine) so as to obtain a low-phenylalanine gelatin polypeptide solution; and freeze-drying, grinding and sieving to obtain the low-phenylalanine gelatin polypeptide. Compared with the unadsorbed polypeptide, the low-phenylalanine gelatin polypeptide has lower fat binding capacity, higher emulsifying capacity and lower Fe < 2 + > chelating capacity. The molecular weight of the obtained low-phenylalanine gelatin polypeptide is thousands of thousands, compared with amino acid and oligopeptide, the low-phenylalanine gelatin polypeptide has better physiological activity and nutritional value, the content of phenylalanine is controllable (0.19-1.13 g / 100g), and the low-phenylalanine gelatin polypeptide has the potential of treating phenylalanine hydroxylase deficiency.

Description

Technical Field

[0001] The invention relates to the field of food, in particular to a low-phenylalanine gelatin polypeptide and application thereof. Background Art

[0002] Phenylalanine hydroxylase (PAH) deficiency (e.g., phenylketonuria and hyperphenylalaninemia) is an autosomal recessive disorder of amino acid (AA) metabolism. The underlying mechanism involves deficiency of phenylalanine hydroxylase or its cofactor tetrahydrobiopterin, which are responsible for converting phenylalanine (Phe) to tyrosine (Tyr). Insufficient conversion capacity results in high Phe levels in the blood and brain, with adverse effects on brain development and function. Therefore, a strict lifelong low-phenylalanine diet is the mainstay of treatment for PAH deficiency.

[0003] Gelatin is one of the most valuable proteins in the food and biopharmaceutical industries. It is obtained by hydrolysis of collagen from tissues such as skin, scales and bones of many animals such as mammals and fish. Collagen has many important functions in structural support, cell behavior, cell integrity and tissue homeostasis. In addition, collagen accounts for one-third of the total protein mass in the human body. Therefore, gelatin is a promising natural protein to be developed for the treatment of PAH deficiency.

[0004] Chinese patent CN112080542A discloses a method for preparing low-phenylalanine egg white protein peptides, providing a method for preparing low-phenylalanine peptides by two-step protease hydrolysis combined with adsorption to remove phenylalanine from egg white protein. The low-phenylalanine peptides prepared by this method are oligopeptides, which contain fewer amino acid residues and therefore have lower nutritional value.

[0005] At present, the special medical foods for PAH deficiency on the market mainly include low-phenylalanine milk powder, etc., most of which use artificial synthetic amino acids as protein sources, and a very small number use low-phenylalanine peptides as protein sources. Products with synthetic amino acids as protein sources have disadvantages such as high osmotic pressure and poor taste. In order to meet their own nutritional needs, patients need to eat a large amount of such products, resulting in poor quality of life for patients, and thus poor compliance with dietary therapy. It may even be that a large amount of free amino acids causes excessive osmotic pressure in the patient's gastrointestinal system, leading to diarrhea. Therefore, we propose a low-phenylalanine gelatin polypeptide and its application. Summary of the invention

[0006] The purpose of the present invention is to provide a low-phenylalanine gelatin polypeptide and its application to solve the problems raised in the prior art. The method of the present invention removes more than 97% of phenylalanine, and the polypeptide is mainly composed of polypeptides with a molecular weight of several thousand, and has high nutritional value. The prepared low-phenylalanine gelatin polypeptide has great application value in the field of special medical foods for PAH deficiency. The preparation method of the present invention can also be used for other proteins.

[0007] To achieve the above object, the present invention provides the following technical solution: a preparation process of low-phenylalanine gelatin polypeptide, comprising the following steps:

[0008] Step (1): alkaline protease hydrolysis: preheat the gelatin solution, adjust the pH to 8.0-9.0, add alkaline protease, stir in a water bath, and inactivate the enzyme in a boiling water bath to obtain a hydrolyzate;

[0009] Step (2): flavor protease enzymolysis: cool the hydrolyzate and then preheat it, adjust the pH to 6.0-7.0, add flavor protease, stir in a water bath, inactivate the enzyme in a boiling water bath, cool and centrifuge to obtain a supernatant;

[0010] Step (3): Activated carbon adsorption: preheat the supernatant, adjust the pH to 3.0-7.0, add activated carbon for adsorption, filter, freeze-dry the filtrate, grind, and sieve to obtain low-phenylalanine gelatin polypeptide powder.

[0011] Furthermore, the molecular weight of the obtained low-phenylalanine gelatin polypeptide is 3747-4583 Da, and the phenylalanine content is 0.19-1.13 g / 100 g.

[0012] Furthermore, in step (1), alkaline protease can be replaced by trypsin;

[0013] In step (2), flavor protease can be replaced by papain.

[0014] Furthermore, the time for inactivating the enzyme in the boiling water bath in step (1) and step (2) is 6 to 10 minutes.

[0015] Furthermore, in the step (1), the gelatin solution is obtained by mixing gelatin and ultrapure water and stirring in a water bath;

[0016] The ratio of gelatin to ultrapure water is 10-50 g / L;

[0017] The process conditions of water bath stirring are: temperature 35-45°C, rotation speed 160-180rpm, and time 20-30min.

[0018] Furthermore, in step (1), the preheating temperature is 45 to 55°C;

[0019] In step (1), the ratio of alkaline protease to gelatin is 1000-5000 U / g;

[0020] In step (1), the process conditions for water bath stirring are: temperature 45-55° C., rotation speed 160-200 rpm, and time 30-180 min.

[0021] Furthermore, a 30-mesh screen was used for sieving.

[0022] Furthermore, alkaline protease: CAS number 9014-01-1, enzyme activity 200U / mg, from Shanghai Yuanye Biotechnology Co., Ltd.;

[0023] Gelatin: CAS number is 9000-70-8, purity is 99%, and it comes from Shandong Pingju Biotechnology Co., Ltd.

[0024] Furthermore, in step (2), the process conditions for water bath stirring are: temperature 45-55° C., rotation speed 160-200 rpm, and time 30-180 min;

[0025] In step (2), the preheating temperature is 45 to 55° C.;

[0026] In step (2), the ratio of flavor protease to gelatin is 1000-5000 U / g;

[0027] In step (2), the centrifugal process conditions are: centrifugal force 1597-1697 g, time 6-10 min.

[0028] Furthermore, flavor protease: CAS number is 9001-92-7, enzyme activity is 150000U / g, and it is sourced from Shanghai McLean Biochemical Technology Co., Ltd.

[0029] Furthermore, in step (3), the preheating temperature is 30 to 50°C;

[0030] In step (3), the ratio of activated carbon to supernatant is 10 to 60 g / L;

[0031] In step (3), the adsorption process conditions are: rotation speed 60-100 rpm, time 1.0-5.0 h;

[0032] In step (3), the freeze-drying process conditions are: temperature: -30 to -50°C, time 12 to 48 hours.

[0033] Furthermore, an aqueous mixed fiber filter membrane with a pore size of 0.22 μm was used for filtration, which was sourced from Haining Delu New Material Technology.

[0034] In the above technical scheme, the release of phenylalanine in natural protein is mainly through protease hydrolysis, so that the peptide bond composed of phenylalanine is broken under appropriate conditions, thereby releasing phenylalanine from the protein polypeptide chain. Alkaline protease belongs to endopeptidase, has more enzyme cleavage sites, has a good hydrolysis effect on aromatic amino acids, and has high hydrolysis efficiency. Flavor protease contains two activities of endopeptidase and exopeptidase, which can degrade the hydrophobic groups exposed to the outside to a large extent, and hydrolyze some flavor precursors at the same time, which helps the enzymatic hydrolysis products to form good flavor and improve the taste of the product. Alkaline protease and flavor protease can also be replaced by trypsin and papain. Trypsin is an efficient protein hydrolase that mainly acts on the peptide bonds in proteins, especially at the carboxyl end of aromatic amino acids and hydrophobic amino acids, and can quickly decompose proteins into large molecular polypeptides and amino acids. Papain belongs to thiol protease, which can hydrolyze most peptide bonds and is very sensitive to the peptide bonds formed by the carboxyl groups of arginine, lysine, and phenylalanine in proteins or polypeptides. The present invention adopts a step-by-step enzymatic hydrolysis method (endo-peptidase + exopeptidase) to hydrolyze the protein. Compared with other biodegradation methods or chemical degradation, enzymatic hydrolysis has the advantages of controllable process, few side reactions, high product quality and large-scale commercialization. Activated carbon has low cost, simple operation, and selective adsorption of amino acids. Its adsorption principle is mainly the π-π stacking force between the graphene ring structure and the benzene ring of the phenylalanine side chain, and the hydrogen bond force between the amino group of phenylalanine and the oxygen-containing group of activated carbon. The isoelectric point of phenylalanine is 5.48, so under pH 2 conditions, both activated carbon and phenylalanine solution are positively charged, and the electrostatic repulsion between the two will reduce the adsorption capacity of activated carbon. At this time, the adsorption mainly depends on the hydrophobic force; at pH 4-6, activated carbon is negatively charged, and amino acids are positively and negatively charged at the same time. Therefore, no matter which adsorption principle is used, the electrostatic attraction between the protonated amino group and the activated carbon surface will promote the adsorption.

[0035] The activated carbon used for adsorption is replaced by a macroporous adsorption resin, and the other process conditions and parameters remain unchanged. A low-phenylalanine gelatin polypeptide and its application include the following steps:

[0036] Step (1): alkaline protease hydrolysis: preheat the gelatin solution, adjust the pH to 8.0-9.v, add alkaline protease, stir in a water bath, inactivate the enzyme in a boiling water bath, and obtain a hydrolyzate;

[0037] Step (2): flavor protease enzymolysis: cool the hydrolyzate and then preheat it, adjust the pH to 6.0-7.0, add flavor protease, stir in a water bath, inactivate the enzyme in a boiling water bath, cool and centrifuge to obtain a supernatant;

[0038] Step (3): adsorption with macroporous adsorption resin: preheat the supernatant, adjust the pH to 3.0-7.0, add macroporous adsorption resin for adsorption, filter, freeze-dry the filtrate, grind, and sieve to obtain low-phenylalanine gelatin polypeptide powder.

[0039] Furthermore, in step (3), the macroporous adsorption resin is modified, and the specific process is as follows:

[0040] The chloromethylated macroporous adsorption resin and dimethylformamide are mixed, allowed to stand and swell for 1 to 2 hours, N-methylimidazole is added, reacted at 60 to 80°C for 36 to 48 hours, filtered, washed with anhydrous ethanol, the product is placed in anhydrous ethanol, stirred for reaction, and vacuum dried to obtain a modified macroporous adsorption resin.

[0041] Further, the mass ratio of chloromethylated macroporous adsorption resin, dimethylformamide, and N-methylimidazole is 1:(2-4):(0.3-0.5);

[0042] The mass ratio of product to anhydrous ethanol is 1:(2-6).

[0043] Furthermore, the process conditions of the stirring reaction are: temperature 55-65° C., time 12-24 h.

[0044] Furthermore, the process conditions of vacuum drying are: temperature 50-70° C., time 8-12 h.

[0045] Further, dimethylformamide: CAS number is 68-12-2, purity is 99%, and it is sourced from Jinan Xinjinshuo New Materials Co., Ltd.;

[0046] N-Methylimidazole: CAS number is 616_47-7, purity is 99%, from Wuhan Smik Biotechnology Co., Ltd.

[0047] Anhydrous ethanol: CAS number is 64-17-5, sourced from Shandong Hengshuo Chemical Co., Ltd.

[0048] Further, the chloromethylated macroporous adsorption resin is prepared by the following process:

[0049] The macroporous adsorption resin and 1,4-dichloromethoxybutane are mixed, and allowed to stand for swelling for 8 to 12 hours. Zinc chloride and sodium chloride are then added, and the mixture is ultrasonically stirred and washed to obtain the chloromethylated macroporous adsorption resin.

[0050] Furthermore, the mass ratio of the macroporous adsorption resin, 1,4-dichloromethoxybutane, zinc chloride and sodium chloride is 1:(2-4):(0.3-0.5):(2.5-4.5).

[0051] Furthermore, the process conditions of ultrasonic stirring are: power 65-75W, temperature 40-50°C, and time 16-24h.

[0052] Further, macroporous adsorption resin: model D101, particle size 0.315-1.25 mm, sourced from Bengbu Sanyi Technology Co., Ltd.;

[0053] 1,4-Dichloromethoxybutane: CAS number is 13483-19-7, derived from McLean reagent;

[0054] Zinc chloride: CAS number is 7646-85-7, purity is 99%, from Merck reagent;

[0055] Sodium chloride: CAS number is 7647-14-5, purity is 99%, and it comes from Shandong Haihe Chemical Co., Ltd.

[0056] In the above technical scheme, the macroporous adsorption resin has a porous structure, no dissociable groups, and a large specific surface area. The non-polar macroporous adsorption resin uses polystyrene as a matrix, and the benzene rings are cross-linked on the main chain to form a plane. The electron distribution is uniform, there is no positive or negative charge, and it has a strong adsorption capacity for aromatic compounds. By introducing chloromethyl groups on the macroporous adsorption resin, the surface properties of the resin are changed, the adsorption capacity for phenylalanine is enhanced, and the selectivity and adsorption efficiency of the macroporous adsorption resin are improved; chloromethylation also increases the functional diversity of the macroporous adsorption resin. Through the chloromethylation reaction, N-methylimidazole functional groups are introduced into the macroporous adsorption resin, and the hydrogen bonds, van der Waals forces and other interactions between the resin and phenylalanine are strengthened, so as to achieve highly selective adsorption of phenylalanine, thereby achieving better separation selectivity and separation efficiency.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] 1. Gelatin peptides containing aromatic amino acids have higher fat binding capacity, lower emulsification and higher Fe content than other peptides. 2+ Chelating ability, and the molecular weight of the polypeptide is larger than that of the amino acid, the osmotic pressure is greatly reduced, and diarrhea caused by excessive osmotic pressure is avoided. The present invention uses gelatin as a raw material and adopts a two-step enzymatic hydrolysis combined with adsorption method to prepare low-phenylalanine gelatin polypeptide. The prepared gelatin polypeptide has a low phenylalanine content. Compared with other biodegradation methods or chemical degradation, enzymatic hydrolysis has the advantages of controllable process, few side reactions, high product quality and large-scale commercialization. Flavor protease contains two activities, endopeptidase and exopeptidase, which can degrade the hydrophobic groups exposed to the outside to a large extent, and hydrolyze some flavor precursors, which helps the enzymatic hydrolysis products to form a good flavor and improve the taste of the product; activated carbon has selective adsorption of amino acids, and has the advantages of low cost and simple operation, and has good economic benefits.

[0059] 2. By introducing chloromethyl groups on the macroporous adsorption resin, the surface properties of the resin are changed, the adsorption capacity of phenylalanine is enhanced, and the selectivity and adsorption efficiency of the macroporous adsorption resin are improved. Chloromethylation also increases the functional diversity of the macroporous adsorption resin. Through the chloromethylation reaction, N-methylimidazole functional groups are introduced on the macroporous adsorption resin, which strengthens the hydrogen bonding, van der Waals force and other interactions between the resin and phenylalanine, and achieves highly selective adsorption of phenylalanine, thereby achieving better separation selectivity and separation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a picture of the low-phenylalanine gelatin polypeptide powder product obtained in Example 1 of the present invention.

[0061] Figures 2 to 7 This is a freeze-dried product of low phenylalanine gelatin polypeptide obtained when the ratio of activated carbon to supernatant in S3 of the present invention is 10 to 60 g / L.

[0062] Figure 8 This is a gel permeation chromatogram of the non-adsorbed gelatin polypeptide in Example 1 of the present invention.

[0063] Figures 9 to 14 The gel permeation chromatograms of the low phenylalanine gelatin polypeptides prepared in Examples 34 to 38 of the present invention and Example 1 when the ratio of activated carbon to supernatant is 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, and 60 g / L, respectively.

[0064] In the figure, the ordinate represents absorbance, and the abscissa represents retention time.

[0065] Fig.15 It is a function diagram of the effect of gelatin solution concentration on gelatin hydrolysis degree in Example 1 and Examples 4 to 7 of the present invention.

[0066] In the figure, the ordinate represents the degree of hydrolysis, and the abscissa represents the concentration of gelatin solution; the data were fitted using cubic polynomial, Log3P1 and linear function; different lowercase letters represent significant differences in each figure (p<0.05).

[0067] Fig.16 It is a function diagram of the effect of the amount of alkaline protease on the degree of hydrolysis in Examples 1 and 8 to 11 of the present invention.

[0068] In the figure, the ordinate represents the degree of hydrolysis, and the abscissa represents the amount of alkaline protease used; the data were fitted using cubic polynomial, Log3P1 and linear function; different lowercase letters represent significant differences in each figure (p<0.05).

[0069] Fig.17It is a function diagram of the effect of the water bath stirring time of S1 on the hydrolysis degree in Example 1 and Examples 12 to 16 of the present invention.

[0070] In the figure, the ordinate represents the degree of hydrolysis, and the abscissa represents the first hydrolysis time; the data were fitted using cubic polynomial, Log3P1 and linear function; different lowercase letters represent significant differences in each figure (p<0.05).

[0071] Fig.18 It is a function graph showing the effect of the amount of flavor protease on the degree of hydrolysis in Examples 1 and 17 to 20 of the present invention.

[0072] In the figure, the ordinate represents the degree of hydrolysis, and the abscissa represents the amount of flavor protease used; the data were fitted using cubic polynomial, Log3P1 and linear function; different lowercase letters represent significant differences in each figure (p<0.05).

[0073] Fig.19 It is a function diagram of the effect of the water bath stirring time of S2 on the hydrolysis degree in Examples 1 and 21 to 25 of the present invention.

[0074] In the figure, the ordinate represents the degree of hydrolysis, and the abscissa represents the second hydrolysis time; the data were fitted using cubic polynomial, Log3P1 and linear function; different lowercase letters represent significant differences in each figure (p<0.05).

[0075] Fig. 20 This is a function graph showing the effect of the preheating temperature of the supernatant on the phenylalanine removal rate in Examples 1 and 26 to 29 of the present invention.

[0076] In the figure, the ordinate represents the phenylalanine removal rate, and the abscissa represents the preheating temperature of the supernatant; the data were fitted with a cubic polynomial and a Log3P1 function; different lowercase letters represent significant differences in each figure (p<0.05).

[0077] Fig.21 This is a function diagram of the effect of the pH value of the supernatant of S3 on the phenylalanine removal rate in Example 1 and Examples 30 to 33 of the present invention.

[0078] In the figure, the ordinate represents the phenylalanine removal rate, and the abscissa represents the pH value of the supernatant; the data were fitted with a cubic polynomial and a Log3P1 function; different lowercase letters represent significant differences in each figure (p<0.05).

[0079] Fig. 22 It is a function graph showing the effect of activated carbon concentration on phenylalanine removal rate in Example 1 and Examples 34 to 39 of the present invention.

[0080] In the figure, the ordinate represents the phenylalanine removal rate, and the abscissa represents the activated carbon concentration; the data were fitted with a cubic polynomial and a Log3P1 function; different lowercase letters represent significant differences in each figure (p<0.05).

[0081] Fig.23 This is a function graph showing the effect of the adsorption time of S3 on the removal rate of phenylalanine in Example 1 and Examples 40 to 43 of the present invention.

[0082] In the figure, the ordinate represents the phenylalanine removal rate, and the abscissa represents the adsorption time; the data were fitted with a cubic polynomial and a Log3P1 function; different lowercase letters represent significant differences in each figure (p<0.05).

[0083] Fig.24 Schematic diagram of the foaming behavior of gelatin polypeptide (GP) and low phenylalanine gelatin polypeptide (LPP1-6) obtained in Example 1 and Examples 34 to 38 of the present invention at 0 min.

[0084] Fig.25 Schematic diagram of the foaming behavior of gelatin polypeptide (GP) and low phenylalanine gelatin polypeptide (LPP1-6) obtained in Example 1 and Examples 34 to 38 of the present invention at 30 minutes.

[0085] Fig.26 Schematic diagram of the water holding behavior of gelatin polypeptide (GP) and low phenylalanine gelatin polypeptides (LPP1-6) obtained in Examples 1 and 34 to 38 of the present invention.

[0086] Fig. 27 Schematic diagram of the fat binding behavior of gelatin polypeptide (GP) and low phenylalanine gelatin polypeptides (LPP1-6) obtained in Examples 1 and 34 to 38 of the present invention.

[0087] Fig.28 This is a comparison chart of the fat binding capacity of the gelatin polypeptide of the present invention and the low phenylalanine gelatin polypeptides (LPP1-6) obtained in Examples 1 and Examples 34 to 38.

[0088] In the figure, the ordinate represents the fat binding capacity, the abscissa GP represents the gelatin polypeptide, and LPP1-6 represents the low phenylalanine gelatin polypeptide prepared when the activated carbon concentration is 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, and 60 g / L, respectively.

[0089] Fig.29 This is a comparison chart of the emulsifying activity of the gelatin polypeptide of the present invention and the low phenylalanine gelatin polypeptides (LPP1-6) obtained in Examples 1 and 34-38.

[0090] In the figure, the ordinate represents the emulsification activity index, the abscissa GP represents the gelatin polypeptide, and LPP1-6 represents the low phenylalanine gelatin polypeptide prepared when the activated carbon concentration is 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, and 60 g / L, respectively.

[0091] Fig.30 This is a comparison chart of the emulsification stability of the gelatin polypeptide of the present invention and the low phenylalanine gelatin polypeptides (LPP1-6) obtained in Examples 1 and Examples 34 to 38.

[0092] In the figure, the ordinate represents the emulsification stability index, the abscissa GP represents the gelatin polypeptide, and LPP1-6 represents the low phenylalanine gelatin polypeptide prepared when the activated carbon concentration is 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, and 60 g / L, respectively.

[0093] Fig.31 This is a comparison chart of the DPPH scavenging ability of the gelatin polypeptide of the present invention and the low phenylalanine gelatin polypeptides (LPP1-6) obtained in Examples 1 and Examples 34 to 38.

[0094] In the figure, the ordinate represents the DPPH scavenging ability, the abscissa GP represents gelatin polypeptide, and LPP1-6 represents low phenylalanine gelatin polypeptides prepared when the activated carbon concentration is 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, and 60 g / L, respectively.

[0095] Fig.32 This is a comparison chart of the reducing abilities of the gelatin polypeptide of the present invention and the low phenylalanine gelatin polypeptides (LPP1-6) obtained in Examples 1 and 34 to 38.

[0096] In the figure, the ordinate represents reducing ability, the abscissa GP represents gelatin polypeptide, and LPP1-6 represents low phenylalanine gelatin polypeptide prepared when the activated carbon concentration is 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, and 60 g / L, respectively.

[0097] Fig.33 The Fe of the gelatin polypeptide of the present invention and the low phenylalanine gelatin polypeptide (LPP1-6) obtained in Examples 1 and Examples 34 to 38 2+ Comparison chart of chelating capacity.

[0098] In the figure, the vertical axis represents Fe 2+ Chelating ability, the horizontal axis GP represents gelatin polypeptide, and LPP1-6 represents low phenylalanine gelatin polypeptide prepared when the activated carbon concentration is 10g / L, 20g / L, 30g / L, 40g / L, 50g / L, and 60g / L, respectively. DETAILED DESCRIPTION

[0099] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only 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.

[0100] In the following specific implementations,

[0101] Alkaline protease: CAS number is 9014-01-1, enzyme activity is 200U / mg, from Shanghai Yuanye Biotechnology Co., Ltd.;

[0102] Gelatin: CAS number is 9000-70-8, purity is 99%, sourced from Shandong Pingju Biotechnology Co., Ltd.

[0103] Flavor protease: CAS number is 9001-92-7, enzyme activity is 150000U / g, from Shanghai MacLean Biochemical Technology Co., Ltd.;

[0104] Aqueous phase mixed fiber filter membrane, pore size 0.22[tm, from Haining Delv New Material Technology;

[0105] Dimethylformamide: CAS number is 68-12-2, purity is 99%, sourced from Jinan Xinjinshuo New Materials Co., Ltd.

[0106] N-Methylimidazole: CAS number is 616_47-7, purity is 99%, from Wuhan Smik Biotechnology Co., Ltd.

[0107] Anhydrous ethanol: CAS number is 64-17-5, from Shandong Hengshuo Chemical Co., Ltd.

[0108] Macroporous adsorption resin: Model D101, particle size 0.315-1.25 mm, from Bengbu Sanyi Technology Co., Ltd.

[0109] 1,4-Dichloromethoxybutane: CAS number is 13483-19-7, derived from McLean reagent;

[0110] Zinc chloride: CAS number is 7646-85-7, purity is 99%, from Merck reagent;

[0111] Sodium chloride: CAS number is 7647-14-5, purity is 99%, from Shandong Haihe Chemical Co., Ltd.;

[0112] Trypsin: CAS number is 9002-07-7, enzyme activity is 4000U / g, from Shaanxi Rankang Biotechnology Co., Ltd.

[0113] Papain: CAS number is 9001-73_4, enzyme activity is 10000U / g, from Guangzhou Huayu Biotechnology Co., Ltd.

[0114] Activated carbon: CAS number is 7440_44-0, sourced from Xi'an Qianyecao Biotechnology Co., Ltd.

[0115] Example 1: A process for preparing a low-phenylalanine gelatin polypeptide, comprising the following steps:

[0116] S1: Alkaline protease hydrolysis: gelatin and ultrapure water were mixed, stirred in a water bath at 180 rpm for 30 min at 45°C to obtain a gelatin solution, preheated to 55°C, adjusted to pH 9.0, added alkaline protease at 3000 U / g gelatin, stirred in a water bath at 200 rpm for 150 min at 55°C, and inactivated in a boiling water bath for 10 min to obtain a hydrolyzate; the ratio of gelatin to ultrapure water was 30 g / L; S2: Flavor protease hydrolysis: the hydrolyzate was cooled and preheated to 55°C, adjusted to pH 7.0, added flavor protease at 3000 U / g gelatin, stirred in a water bath at 200 rpm for 120 min at 55°C, and inactivated in a boiling water bath. Enzyme for 10 minutes, cool and centrifuge to obtain supernatant; the process conditions of centrifugation are: centrifugal force 1697g, time 10min; S3: activated carbon adsorption: preheat the supernatant to 40℃, adjust the pH to 4.0, add activated carbon, adsorb, filter, freeze-dry the filtrate, grind through a 30-mesh sieve to obtain low phenylalanine gelatin polypeptide powder; the ratio of activated carbon to supernatant is 60g / L; the process conditions of adsorption are: rotation speed 100rpm, time 3h; the process conditions of freeze-drying are: temperature -50℃, time 48h; the molecular weight of the obtained low phenylalanine gelatin polypeptide is 3747Da, and the phenylalanine content is 0.20g / 100g.

[0117] Example 2: A process for preparing low-phenylalanine gelatin polypeptide, comprising the following steps:

[0118] S1: Alkaline protease hydrolysis: gelatin and ultrapure water were mixed, stirred in a water bath at 170 rpm for 25 min at 40°C to obtain a gelatin solution, preheated to 50°C, adjusted to pH 8.5, added alkaline protease at 2500 U / g gelatin, stirred in a water bath at 180 rpm for 125 min at 50°C, inactivated in a boiling water bath for 8 min to obtain a hydrolyzate; the ratio of gelatin to ultrapure water was 30 g / L; S2: Flavor protease hydrolysis: the hydrolyzate was cooled and preheated to 50°C, adjusted to pH 6.5, added flavor protease at 2500 U / g gelatin, stirred in a water bath at 180 rpm for 100 min at 50°C, inactivated in a boiling water bath Inactivate the enzyme for 8 minutes, cool and centrifuge to obtain a supernatant; the process conditions for centrifugation are: centrifugal force 1650g, time 8min; S3: activated carbon adsorption: preheat the supernatant to 38°C, adjust the pH to 3.5, add activated carbon, adsorb, filter, freeze-dry the filtrate, and grind through a 30-mesh sieve to obtain a low-phenylalanine gelatin polypeptide powder; the ratio of activated carbon to supernatant is 30g / L; the process conditions for adsorption are: rotation speed 80rpm, time 2h; the process conditions for freeze-drying are: temperature -40°C, time 24h; the molecular weight of the obtained low-phenylalanine gelatin polypeptide is 4165Da, and the phenylalanine content is 0.70g / 100g.

[0119] Example 3: A process for preparing low-phenylalanine gelatin polypeptide, comprising the following steps:

[0120] S1: Alkaline protease hydrolysis: gelatin and ultrapure water were mixed, stirred in a water bath at 160 rpm for 20 min at 35°C to obtain a gelatin solution, preheated to 45°C, adjusted to pH 8.0, added alkaline protease at 2000 U / g gelatin, stirred in a water bath at 160 rpm for 100 min at 45°C, and inactivated in a boiling water bath for 6 min to obtain a hydrolyzate; the ratio of gelatin to ultrapure water was 10 g / L; S2: Flavor protease hydrolysis: the hydrolyzate was cooled and preheated to 45°C, adjusted to pH 6.0, added flavor protease at 2000 U / g gelatin, stirred in a water bath at 160 rpm for 80 min at 45°C, and inactivated in a boiling water bath. Enzyme for 6 minutes, cool and centrifuge to obtain supernatant; the process conditions of centrifugation are: centrifugal force 1597g, time 6min; S3: activated carbon adsorption: preheat the supernatant to 36°C, adjust the pH to 3.0, add activated carbon, adsorb, filter, freeze-dry the filtrate, and grind through a 30-mesh sieve to obtain low phenylalanine gelatin polypeptide powder; the ratio of activated carbon to supernatant is 10g / L; the process conditions of adsorption are: speed 60rpm, time 1h; the process conditions of freeze-drying are: temperature -30°C, time 12h; the molecular weight of the obtained low phenylalanine gelatin polypeptide is 4583Da, and the phenylalanine content is 1.12g / 100g.

[0121] Examples 4 to 7: With Example 1 as a comparison, the concentrations of the gelatin solutions in Examples 4 to 7 are 10 g / L, 20 g / L, 40 g / L, and 50 g / L, respectively, and the other conditions remain unchanged. The hydrolysis degree of the gelatin is tested, and the specific data are as follows:

[0122] Example 4 Example 5 Example 6 Example 7 Degree of hydrolysis / % 8.03 9.83 10.13 9.47

[0123] Table 1 Effect of different gelatin solution concentrations on hydrolysis degree

[0124] Examples 8 to 11: In comparison with Example 1, alkaline protease was added to gelatin at 1000 U / g, 2000 U / g, 4000 U / g, and 5000 U / g in Examples 8 to 11, and the other conditions remained unchanged. The hydrolysis degree of gelatin was tested. The specific data are shown in the following table:

[0125] Example 8 Example 9 Example 10 Embodiment 11 Degree of hydrolysis / % 7.14 8.51 9.79 10.27

[0126] Table 2 Effect of different alkaline protease dosage on hydrolysis degree

[0127] Examples 12 to 16: Compared with Example 1, the water bath stirring time of S1 in Examples 12 to 16 is 30 min, 60 min, 90 min, 120 min, and 180 min, respectively, and the other conditions remain unchanged. The hydrolysis degree of gelatin is tested, and the specific data are as follows:

[0128] Example 12 Example 13 Embodiment 14 Embodiment 15 Example 16 Degree of hydrolysis / % 8.03 8.59 8.91 9.47 10.76

[0129] Table 3 Effect of different water bath stirring time on hydrolysis degree of S1

[0130] Examples 17-20: Compared with Example 1, in Examples 17-20, flavor protease was added to gelatin at 1000 U / g, 2000 U / g, 4000 U / g, and 5000 U / g, respectively, and the other conditions remained unchanged. The hydrolysis degree of gelatin was tested, and the specific data are as follows:

[0131] Embodiment 17 Embodiment 18 Embodiment 19 Embodiment 20 Degree of hydrolysis / % 17.60 20.80 24.62 24.80

[0132] Table 4 Effect of different flavor protease dosage on hydrolysis degree

[0133] Examples 21-25: Compared with Example 1, the water bath stirring time of S2 in Examples 21-25 is 30min, 60min, 90min, 150min, and 180min, respectively, and the other conditions remain unchanged. The hydrolysis degree of gelatin is tested, and the specific data are as follows:

[0134] Embodiment 21 Embodiment 22 Embodiment 23 Embodiment 24 Embodiment 25 Degree of hydrolysis / % 17.64 19.59 21.04 24.54 25.42

[0135] Table 5 Effect of different water bath stirring time on hydrolysis degree of S2

[0136] Examples 26-29: Compared with Example 1, the supernatant of S3 in Examples 26-29 was preheated to 30°C, 35°C, 45°C, and 50°C in sequence, and the other conditions remained unchanged. The removal rate of phenylalanine in the low-phenylalanine gelatin polypeptide was detected and calculated. The specific data are as follows:

[0137] Embodiment 26 Embodiment 27 Embodiment 28 Embodiment 29 Removal rate / % 93.91 94.68 95.01 90.52

[0138] Table 6 Effect of different supernatant preheating temperatures on removal rate

[0139] Examples 30-33: With Example 1 as a comparison, the pH of the supernatant of S3 in Examples 30-33 was adjusted to 3.0, 5.0, 6.0, and 7.0 respectively, and the other conditions remained unchanged. The removal rate of phenylalanine in the low-phenylalanine gelatin polypeptide was detected and calculated. The specific data are as follows:

[0140] Embodiment 30 Embodiment 31 Embodiment 32 Embodiment 33 Removal rate / % 94.30 94.63 90.43 70.62

[0141] Table 7 Effect of different supernatant pH on removal rate

[0142] Examples 34-39: Compared with Example 1, the ratios of activated carbon and supernatant in S3 in Examples 34-39 are 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, and 70 g / L, respectively, and the other conditions remain unchanged. The removal rate of phenylalanine in low phenylalanine gelatin polypeptide is detected and calculated. The specific data are as follows:

[0143]

[0144] Table 8 Effect of different activated carbon dosage on removal rate

[0145] Examples 40-43: Compared with Example 1, the adsorption time of S3 in Examples 40-43 is 1.0h, 2.0h, 4.0h, and 5.0h, respectively, and the other conditions remain unchanged. The removal rate of phenylalanine in the low-phenylalanine gelatin polypeptide is detected and calculated. The specific data are as follows:

[0146] Embodiment 40 Embodiment 41 Embodiment 42 Embodiment 43 Removal rate / % 92.28 93.77 95.08 95.45

[0147] Table 9 Effect of different adsorption time on removal rate

[0148] Example 44: Taking Example 1 as a comparison, the activated carbon is replaced by a modified macroporous adsorption resin, the ratio of the modified macroporous adsorption resin to the supernatant is 10 g / L, and the remaining steps are the same as in Example 1.

[0149] Example 45: Compared with Example 1, the ratio of modified macroporous adsorption resin to supernatant is 35 g / L, and the remaining steps are the same as Example 1.

[0150] Example 46: Compared with Example 1, the ratio of modified macroporous adsorption resin to supernatant is 60 g / L, and the remaining steps are the same as Example 1.

[0151] Comparative Example 1: Using Example 1 as a comparison, alkaline protease was replaced by trypsin, and the remaining steps were the same as those in Example 1.

[0152] Comparative Example 2: Using Example 1 as a comparison, the flavor protease was replaced by papain, and the remaining steps were the same as those in Example 1.

[0153] Comparative Example 3: Taking Example 44 as a comparison, the macroporous adsorption resin was not modified, and the remaining steps were the same as those in Example 1.

[0154] experiment:

[0155] Molecular weight distribution test: Gel permeation chromatography was used to determine the molecular weight distribution of gelatin polypeptide and low phenylalanine gelatin polypeptide prepared in Example 1 and Examples 34-38. The test method is as follows: the polypeptide powder is dissolved in ultrapure water at a concentration of 1.5 g / L and centrifuged at 106×g for 10 min. The supernatant is then injected into a high performance liquid chromatography system using a UV detector (220 nm) and a BEH 200 column (4.6 mm×150 mm, 3.5 μm), a column temperature of 30°C, a mobile phase of 50 mmol / L phosphate buffer (pH 6.7), and a flow rate of 0.3 mL / min. The protein standards are 1350, 17000, 44000, 158000 and 670000 Da, respectively;

[0156] Figure 8 is the gel permeation chromatogram of gelatin polypeptide. Figure 9-14 It is a gel permeation chromatogram of low phenylalanine gelatin polypeptide prepared when the activated carbon concentration is 10-60g / L; the abscissa (Retention time) is the retention time, and the ordinate (AU) represents the absorbance.

[0157] From the above molecular weight distribution results, it can be seen that the obtained low phenylalanine gelatin polypeptides are all single peaks in the chromatogram, with a molecular weight of 3747-4583Da, no oligopeptides with a molecular weight of 100-300Da, no gelatin with a molecular weight of 50-100kDa, and no proteins and polypeptides with other molecular weights.

[0158] Peptide yield determination: determined by Kjeldahl nitrogen determination method; based on GB5009.5-2016, the protein content in low phenylalanine gelatin polypeptide and gelatin was determined, and the calculation formula is as follows:

[0159] Peptide yield = (protein content in low phenylalanine gelatin polypeptide / protein content in gelatin) × 100%;

[0160] Phenylalanine removal rate: amino acid analyzer is used to determine the phenylalanine content in raw materials and products. The calculation formula is as follows:

[0161] Phenylalanine removal rate = (phenylalanine content in raw material - phenylalanine content in product) × 100% / phenylalanine content in raw material;

[0162] Hydrolysis degree test: The hydrolysis degree of low phenylalanine gelatin polypeptide was determined by OPA method, and the calculation formula is as follows:

[0163] Degree of hydrolysis = number of peptide bonds cleaved per gram of protein after hydrolysis × 100% / millimoles of peptide bonds per gram of raw protein;

[0164] Peptide yield (%) Degree of hydrolysis (%) Phenylalanine removal rate (%) Example 1 22.62 100 97.79 Example 2 21.95 99 97.72 Example 3 20.73 99 97.65 Embodiment 44 22.35 99 97.85 Embodiment 45 22.57 99 97.98 Embodiment 46 22.88 100 98.82 Comparative Example 1 20.11 98 97.58 Comparative Example 2 21.89 98 97.64 Comparative Example 3 22.15 100 96.39

[0165] Table 10 Peptide yield and phenylalanine removal rate of low phenylalanine gelatin polypeptide

[0166] According to the data in the above table, we can clearly draw the following conclusions:

[0167] The low phenylalanine gelatin polypeptides obtained in Examples 1 to 3 and Examples 44 to 46 were compared with the low phenylalanine gelatin polypeptides obtained in Comparative Examples 1 to 3. The test results show that compared with Example 1, in Comparative Example 1, the alkaline protease was replaced by trypsin, and the peptide yield and hydrolysis degree decreased. This is because the action site of trypsin is the basic amino acid residues on the peptide chain, that is, the carboxyl side of lysine and arginine, while the action site of alkaline protease is usually the carboxyl side of aromatic amino acid residues or hydrophobic amino acid residues on the peptide chain, such as tyrosine, tryptophan and phenylalanine. Therefore, alkaline protease is preferentially selected as the main endonuclease; compared with Example 1, Comparative Example 2 replaces flavor protease with papain, and the peptide yield and hydrolysis degree decrease because flavor protease mainly acts on the carboxyl side of hydrophobic amino acids, such as leucine, isoleucine, phenylalanine and tryptophan, while papain mainly acts on the carboxyl side of arginine and lysine. Therefore, flavor protease is preferentially selected as the exonuclease; compared with Example 1, Comparative Example 3 does not modify the macroporous adsorption resin, and the peptide yield and hydrolysis degree do not change significantly, and the phenylalanine removal rate decreases. Examples 44 to 46 replace activated carbon with modified macroporous adsorption resin, and the peptide yield and phenylalanine removal rate increase overall. From the above conclusions, it can be seen that the setting of the process and the materials used in this application can promote the improvement of the peptide yield of the prepared gelatin polypeptide, and at the same time improve the removal rate of phenylalanine in the gelatin polypeptide.

[0168] Amino acid content and removal rate test: The gelatin polypeptides prepared in Examples 1 and 34-38 were tested using a fully automatic amino acid analyzer in accordance with the Chinese national standard GB5009.124-2016. 0.020 g of dried sample, 10 mL of HCl solution (6 mol / L) and 3 drops of phenol were mixed in a hydrolysis tube. After being placed in a 4 ° C refrigerator for 5 min, the hydrolysis tube was vacuum sealed by a vacuum pump for 10 min, and then incubated at 110 ° C for 22 h in a constant temperature drying oven (DHG-9023A, Shanghai Jinghong Experimental Equipment Co., Ltd.). After the sample was cooled to room temperature, it was filtered with filter paper and rinsed with 6 mol / L hydrochloric acid into a 50 mL volumetric flask, and the volume was set to 50 mL. 1 mL of sample was placed in a vacuum oven (VC50, Salvis, Switzerland) and dried at 55 ° C. 1 mL of ultrapure water was then added and dried again in a vacuum oven. Sodium citrate buffer (2.00 mL, pH 2.2) was then added and the sample was vortexed to dissolve. Finally, the solution was filtered through a 0.22 μm mixed cellulose filter membrane, and the amino acid content was determined using an amino acid automatic analyzer (LA-8080, Hitachi, Japan) and commercial amino acid standards (H type, Wako Pure Chemical Industries, Ltd., Japan). The content of each amino acid in the dried sample was calculated according to the following formula:

[0169]

[0170] Wherein, V1 is the volume of sodium citrate buffer (2.00 mL), V2 is the sample volume (50.0 mL), M is the mass of amino acids detected in the injection volume (mg), V3 is the injection volume (0.02 mL), V4 is the dilution volume (1.0 mL), and M is the mass of the sample used (0.020 g);

[0171] The removal rate of each amino acid in the peptide sample was calculated according to the following formula:

[0172]

[0173] Where PY HS is the yield of the peptide sample, AAC HS AAC is the amino acid content in the peptide sample. G It is the amino acid content in gelatin.

[0174]

[0175] Table 11 Amino acid (AA) content in peptides (g / 100g dry sample) and removal rate results Note: LPP1 and LPP6 represent low phenylalanine peptides prepared at activated carbon concentrations of 10 and 60 g / L, respectively; bold fonts indicate that the amino acid content (AAs) is significantly lower than that of other amino acids (AAs); different letters indicate significant differences in each row.

[0176] According to the data in the above table, compared with gelatin, gelatin polypeptides (GPs) have almost no methionine (Met) after the first and second hydrolysis, which is due to the destruction of methionine during the alkaline hydrolysis process; in the third step of activated carbon adsorption, aromatic amino acids phenylalanine (Phe) and tyrosine (Tyr) are removed, and the phenylalanine removal rate increases with the increase of activated carbon dosage. Therefore, the amino acid removal rate of low phenylalanine polypeptides is significantly higher than that of gelatin polypeptides; high activated carbon dosage (LPP6) has a higher amino acid removal rate than low activated carbon dosage (LPP1); in addition, the removal rates of aromatic amino acids phenylalanine (Phe) and tyrosine (Tyr) are significantly higher than those of other amino acids, among which the contents of low activated carbon dosage (LPP1) and high activated carbon dosage (LPP6) are 1.12±0.01g / 100g and 0.20±0.01g / 100g, respectively. Therefore, the prepared low phenylalanine polypeptide powder may be used as a low phenylalanine polypeptide for the treatment of PAH deficiency.

[0177] Fat binding capacity test: 0.25 g of gelatin polypeptide powder and low-phenylalanine gelatin polypeptide powder obtained in Example 1 and Examples 34-38 were added to a centrifuge tube, and 5 mL of fish oil was added; the sample was allowed to stand at room temperature for 30 min, vortexed for 5 s every 10 min, and photographed with a digital camera; the sample was centrifuged at 424×g for 20 min, and the centrifuge tube was tilted at 45° and placed on filter paper for 30 min to discharge the fish oil;

[0178] Fig. 27 The vertical axis (FBC) is the fat binding capacity, the horizontal axis GP is the gelatin polypeptide, LPP1-6 is the low phenylalanine gelatin polypeptide obtained when the activated carbon concentration is 10-60g / L, and different letters indicate that each column has a significant difference (p<0.05);

[0179] Fat binding capacity (g / g) = (mass of fat-bound sample - mass of peptide sample used) / mass of peptide sample;

[0180] Emulsification activity and emulsification stability test: The emulsification activity index (EAI) and stability index (ESI) of gelatin polypeptide and low phenylalanine gelatin polypeptide obtained in Example 1 and Examples 34-38 were tested by ultraviolet spectrophotometry. Fish oil and polypeptide solution (0.005 g / mL) were mixed in a volume ratio of 1:3 and homogenized at 18000 rpm for 60 seconds using a homogenizer. At 0 min and 10 min, 50 μL of emulsion near the bottom of the glass bottle was diluted with 1 g / L sodium dodecyl sulfate solution (dilution factor: 100). After vortexing for 10 seconds, the absorbance of the sample was measured at 500 nm using a NanoDrop 2000C spectrophotometer. Calculation of EAI and ESI:

[0181]

[0182]

[0183] In the formula, A0 is the absorbance of the sample at 0 min, N is the dilution factor, is the volume fraction of oil (0.25), C is the polypeptide concentration (g / mL), A 10 is the absorbance of the sample at 10 min, and Δt is the time interval (10 min).

[0184] Fig.28 The vertical axis (EAI) is the emulsification activity index. Fig.29 The ordinate (ESI) is the emulsion stability index. In the abscissa of the two figures, GP is gelatin polypeptide, and LPP1-6 is low phenylalanine gelatin polypeptide obtained when the activated carbon concentration is 10-60 g / L. Different letters indicate that each column has significant differences (p<0.05).

[0185] DPPH scavenging ability test: The DPPH scavenging ability of gelatin polypeptide and low phenylalanine gelatin polypeptide samples obtained in Example 1 and Examples 34-38 was tested, with a mixture of 1.0 mL of polypeptide solution (1.0 g / L) and 1.0 mL of DPPH (0.1 mmol / L) ethanol solution as the sample, a mixture of 1.0 mL of polypeptide solution and 1.0 mL of ethanol as the control, and a mixture of 1.0 mL of ethanol and 1.0 mL of DPPH (0.1 mmol / L) ethanol solution as the blank. The mixture was incubated in the dark for 30 min. The absorbance of the mixture was measured at 517 nm using a NanoDrop 2000C spectrophotometer. Calculation of DPPH scavenging ability:

[0186]

[0187] In the formula, A 517.0 is the absorbance of the blank, A 517.1 is the absorbance of the control, A 517.2 is the absorbance of the sample;

[0188] Fig.30 The vertical axis is the DPPH scavenging ability, the horizontal axis is GP for gelatin polypeptide, LPP1-6 for low phenylalanine gelatin polypeptide obtained when the activated carbon concentration is 10-60 g / L, and different letters indicate that each column has significant differences (p<0.05);

[0189] Reducing ability test: The reducing ability of gelatin polypeptide and the low phenylalanine gelatin polypeptide samples obtained in Example 1 and Examples 34-38 was tested. 1.0 mL of polypeptide solution (1.0 g / L) was mixed with 2.5 mL of potassium ferricyanide solution (10 mg / mL). Incubate at 50°C for 20 min, add 2.5 mL of trichloroacetic acid solution (100 g / L), centrifuge at 1697×g for 10 min, then mix 2.5 mL of supernatant, 2.5 mL of ultrapure water and 0.5 mL of ferric chloride (10 g / L), and incubate at room temperature for 10 min. Finally, the absorbance (reducing ability) of the sample to ultrapure water was measured at 700 nm using a NanoDrop 2000C spectrophotometer;

[0190] Fig.31 The ordinate is the reducing ability, the abscissa is GP for gelatin polypeptide, LPP1-6 for low phenylalanine gelatin polypeptide obtained when the activated carbon concentration is 10-60 g / L, and different letters indicate significant differences in each column (p<0.05);

[0191] Fe 2+ Chelating ability: Gelatin polypeptide and low phenylalanine gelatin polypeptide samples obtained in Examples 1 and 34-38 Fe 2+ The chelating ability was tested, with a mixture of 0.8 mL of polypeptide solution (1.0 g / L), 10 μL of FeCl2 solution (2 mmol / L), and 20 μL of ferrozine solution (5 mmol / L) as the sample, a mixture of 0.8 mL of polypeptide solution (1.0 g / L), 10 μL of ultrapure water, and 20 μL of ferrozine solution (5 mmol / L) as the control, and a mixture of 0.8 mL of ultrapure water, 10 μL of FeCl2 solution (2 mmol / L), and 20 μL of ferrozine solution (5 mmol / L) as the blank. The reaction was allowed to proceed at room temperature for 10 min, and the absorbance was measured at 562 nm using a NanoDrop 2000C spectrophotometer. Fe 2+ Chelating ability:

[0192]

[0193] Among them, A 562.0 is the absorbance of the blank, A 562.1 is the absorbance of the control, A 562.2 is the absorbance of the sample;

[0194] Fig.32 The vertical axis is Fe 2+ Chelating ability, GP in the horizontal axis is gelatin polypeptide, LPP1-6 is low phenylalanine gelatin polypeptide obtained when the activated carbon concentration is 10-60g / L, different letters indicate significant differences in each column (p<0.05);

[0195] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A process for preparing low-phenylalanine gelatin polypeptide, characterized in that: The following steps are involved: Step (1): alkaline protease hydrolysis: preheat the gelatin solution, adjust the pH to 8.0-9.0, add alkaline protease, stir in a water bath, inactivate the enzyme in a boiling water bath, and obtain a hydrolyzate; Step (2): flavor protease enzymolysis: cool the hydrolyzate and then preheat it, adjust the pH to 6.0-7.0, add flavor protease, stir in a water bath, inactivate the enzyme in a boiling water bath, cool and centrifuge to obtain a supernatant; Step (3): Activated carbon adsorption: preheat the supernatant, adjust the pH to 3.0-7.0, add activated carbon for adsorption, filter, freeze-dry the filtrate, grind, and sieve to obtain low-phenylalanine gelatin polypeptide powder.

2. The preparation process of a low-phenylalanine gelatin polypeptide according to claim 1, characterized in that: In step (1), the ratio of alkaline protease to gelatin is 1000-5000 U / g.

3. The preparation process of a low-phenylalanine gelatin polypeptide according to claim 1, characterized in that: In step (2), the ratio of flavor protease to gelatin is 1000-5000 U / g.

4. The preparation process of a low-phenylalanine gelatin polypeptide according to claim 1, characterized in that: In step (1), the process conditions for water bath stirring are: temperature 45-55° C., rotation speed 160-200 rpm, and time 30-180 min.

5. The preparation process of a low-phenylalanine gelatin polypeptide according to claim 1, characterized in that: In step (2), the process conditions for water bath stirring are: temperature 45-55° C., rotation speed 160-200 rpm, and time 30-180 min.

6. The preparation process of a low-phenylalanine gelatin polypeptide according to claim 1, characterized in that: In step (3), the ratio of activated carbon to supernatant is 10 to 60 g / L.

7. The process for preparing a low-phenylalanine gelatin polypeptide according to claim 6, characterized in that: The activated carbon can be replaced by a macroporous adsorption resin, which is modified by the following specific process: The chloromethylated macroporous adsorption resin and dimethylformamide are mixed, allowed to stand and swell for 1 to 2 hours, N-methylimidazole is added, reacted at 60 to 80°C for 36 to 48 hours, filtered, washed with anhydrous ethanol, the product is placed in anhydrous ethanol, stirred for reaction, and vacuum dried to obtain a modified macroporous adsorption resin.

8. The process for preparing a low-phenylalanine gelatin polypeptide according to claim 7, characterized in that: The chloromethylated macroporous adsorption resin is prepared by the following process: The macroporous adsorption resin and 1,4-dichloromethoxybutane are mixed, and allowed to stand for swelling for 8 to 12 hours. Zinc chloride and sodium chloride are then added, and the mixture is ultrasonically stirred and washed to obtain the chloromethylated macroporous adsorption resin.

9. A low-phenylalanine gelatin polypeptide, characterized in that: The invention is prepared by the preparation process described in any one of claims 1 to 8.

10. The use of a low-phenylalanine gelatin polypeptide according to claim 9, characterized in that: Low phenylalanine gelatin polypeptide is used to prepare low phenylalanine milk powder.

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