阅读说明：本技术 一种牛骨胶原蛋白多肽的消化特性评价方法 (Digestion characteristic evaluation method of bovine bone collagen polypeptide ) 是由 朱瑶迪 李苗云 赵改名 赵静 陈夏男 李婷婷 于 2021-01-20 设计创作，主要内容包括：本发明公开了一种牛骨胶原蛋白多肽的消化特性评价方法,包括以下步骤：(a)配置消化液,包括配置模拟唾液SSF、模拟胃液SGF、模拟肠液SIF,利用GI20体外模拟消化系统进行模拟体外消化过程,包括ⅰ口腔、ⅱ胃和ⅲ小肠；(b)测定不同分子量胶原蛋白多肽的消化特性,包括检测步骤(a)中ⅰ口腔、ⅱ胃和ⅲ小肠中的消化前后消化物的消化率。本发明提供的牛骨胶原蛋白多肽的消化特性评价方法,测定不同酶解条件下不同分子量胶原蛋白多肽的消化特性,采用完全模拟的方式进行快速获得数据,为企业大规模生产不同分子量胶原蛋白多肽提供有效的手段。(The invention discloses a method for evaluating the digestion characteristics of bovine bone collagen polypeptide, which comprises the following steps: (a) preparing digestive juice, including preparing simulated saliva SSF, simulated gastric fluid SGF and simulated intestinal fluid SIF, and performing simulated in-vitro digestion process by using a GI20 in-vitro simulated digestion system, including i oral cavity, ii stomach and iii small intestine; (b) determining the digestion characteristics of collagen polypeptides of different molecular weights comprising determining the digestibility of the digest in step (a) before and after digestion in the mouth i, stomach ii and small intestine iii. The digestion characteristic evaluation method of the bovine bone collagen polypeptide provided by the invention is used for measuring the digestion characteristics of the collagen polypeptides with different molecular weights under different enzymolysis conditions, and rapidly acquiring data by adopting a complete simulation mode, thereby providing an effective means for large-scale production of the collagen polypeptides with different molecular weights for enterprises.)

1. A method for evaluating the digestion characteristics of bovine bone collagen polypeptide is characterized by comprising the following steps:

(a) preparing digestive juice, including preparing simulated saliva SSF, simulated gastric fluid SGF and simulated intestinal fluid SIF, and performing simulated in-vitro digestion process by using a GI20 in-vitro simulated digestion system, including i oral cavity, ii stomach and iii small intestine;

(b) determining the digestion characteristics of collagen polypeptides of different molecular weights comprising determining the digestibility of the digest in step (a) before and after digestion in the mouth i, stomach ii and small intestine iii.

2. The method for evaluating the digestion performance of bovine collagen polypeptide according to claim 1, wherein said simulating digestion of i oral cavity in vitro in step (a) comprises: adding collagen polypeptide into SSF solution, adding salivary amylase and lysozyme, simulating chewing with oral cavity simulator, mixing, and placing in GI-20 in vitro gastrointestinal simulation instrument.

3. The method of claim 1, wherein the step (a) of simulating digestion of the ii-stomach in vitro comprises: taking simulated oral digests, adding SGF solution and pepsin, incubating at constant temperature, and placing in boiling water bath for enzyme deactivation treatment.

4. The method for evaluating the digestion profile of bovine bone collagen polypeptide according to claim 1, wherein said method for simulating in vitro digestion of the small intestine in step (a) comprises: taking gastric digests, adding SIF solution, trypsin and bile salt, adjusting the pH value by using NaOH, incubating at constant temperature, and then putting in a boiling water bath for enzyme deactivation treatment.

5. The method for evaluating the digestion characteristics of bovine bone collagen polypeptide according to claim 1, wherein the collagen polypeptides with different molecular weights in step (b) are prepared by the following steps:

(1) crushing and cleaning fresh bovine bones, adding purified water, extracting ossein by high-efficiency hot-pressing extraction, standing, separating three phases, removing bone oil and a middle mixed layer, and concentrating by a tube centrifuge to obtain bovine ossein;

(2) adding an EDTA solution into the bovine ossein obtained in the step (1), and removing calcium ions in the bovine ossein to obtain a first crude collagen polypeptide product;

(3) adding a NaCl solution into the first crude collagen polypeptide product obtained in the step (2), further purifying by a salting-out method, and performing low-temperature centrifugal separation to obtain a second crude collagen polypeptide product;

(4) adding an acetic acid solution into the second crude collagen polypeptide product obtained in the step (3) for dissolving, then carrying out sectional dialysis, and carrying out dialysis by using the acetic acid solution and then distilled water to obtain a third crude collagen polypeptide product;

(5) and (4) carrying out enzymolysis on the third crude collagen polypeptide product in the step (4) by selecting multi-gradient enzymolysis to obtain a collagen polypeptide stock solution.

(6) And (5) repeatedly dialyzing and purifying the collagen polypeptide stock solution in the step (5) through semipermeable membranes with different molecular weights to obtain collagen polypeptides with different molecular weights.

6. The method for evaluating the digestion characteristics of bovine bone collagen polypeptide according to claim 5, wherein the multi-gradient enzymolysis in step (5) comprises: firstly, adding alkaline protease with the pH value of 9.0 and the enzymolysis temperature of 50 ℃, carrying out enzymolysis for 2 hours, then adjusting the pH value of an enzymolysis solution to 8.0, then adding trypsin and compound flavor protease with the enzymolysis temperature of 40 ℃, and carrying out enzymolysis for 2 hours; finally, enzyme deactivation treatment is carried out for 20min at 90 ℃.

7. The method for evaluating the digestion characteristics of bovine bone collagen polypeptide according to any one of claims 1 to 6, wherein the molecular weight distribution of said polypeptide is:

molecular weight range Ratio of molecular weight <1kDa 25％～44％ 1kDa-1.5kDa 15～40％ 1.5kDa-2.5kDa 6％～10％ 2.5kDa～3kDa 2％～5％ >3kDa 0.5％～1％

Technical Field

The invention belongs to the technical field of collagen polypeptides, and particularly relates to a digestion characteristic evaluation method of bovine bone collagen polypeptides.

Background

With the intensification of beef cattle breeding and the development of meat processing industry in China, beef cattle slaughtering processing byproducts are increased, particularly beef bones, the total meat yield in 2018 is about 8517 ten thousand tons according to the statistics of the national statistical bureau, the beef bones account for 1/4 of the total world yield, the fresh bones produced by the beef cattle slaughtering processing byproducts can reach 2500 ten thousand tons, and the meat with improper bones is the traditional consumption concept of human beings.

The livestock and poultry bone is rich in nutrient components such as lipid, protein ossein, chondroitin, mineral elements and the like, can be used as a high-quality protein resource and lipid source, has important practical significance for carrying out deep comprehensive utilization and high-value high-efficiency transformation on the protein resource and protecting the environment and recycling the resource, particularly contains rich collagen in the bovine bone, is a biological polymer, is a necessary component for maintaining normal tissue structure and performance, and has good biochemistry and biocompatibility. The bovine bone collagen is subjected to segmented enzymolysis to obtain small molecular protein polypeptides with different molecular weights, and compared with the collagen, the bovine bone collagen has higher utilization rate after being eaten, is easy to be absorbed by a human body, and has great difference in functions, for example, a component with the molecular weight of less than 10kD has strong antibacterial activity on escherichia coli, has higher free radical clearance rate of less than 3kDa, can be directly absorbed by the human body without digestion of less than 1kDa, and is greatly helpful for human metabolism. The method not only enables the livestock and poultry bones to be utilized with high added value, but also can bring good economic benefit, avoid environmental pollution and realize green sustainable development.

The biological activity in the collagen peptide is closely related to the diversity of polypeptide compositions, for example, Asp-Gly-Glu-Ala can promote the transformation from bone marrow cells to osteoblasts, and the collagen peptide can play an important role in the physiological metabolism and regulation of cells from simple dipeptide to macromolecular polypeptide with a ring structure through modification such as glycosylation, phosphorylation, acylation and the like, and different polypeptide compositions also have obvious influence on the digestion and absorption of human bodies. The composition, function and the like of different polypeptides are obviously different. Therefore, a rapid and nondestructive evaluation method for the digestion characteristics of the collagen polypeptide is needed to be developed to explore the change of the digestion characteristics of the bovine bone collagen polypeptide in different digestion stages, so that the additional value of bovine bone can be improved, and a technical means is provided for the development of different active peptides.

Disclosure of Invention

The invention aims to provide a simple, convenient, rapid and efficient method for evaluating the digestion characteristics of bovine bone collagen polypeptide.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for evaluating the digestion characteristics of bovine bone collagen polypeptide comprises the following steps:

(a) preparing digestive juice, including preparing simulated saliva SSF, simulated gastric fluid SGF and simulated intestinal fluid SIF, and performing simulated in-vitro digestion process by using a GI20 in-vitro simulated digestion system, including i oral cavity, ii stomach and iii small intestine;

(b) determining the digestion characteristics of collagen polypeptides of different molecular weights comprising determining the digestibility of the digest in step (a) before and after digestion in the mouth i, stomach ii and small intestine iii.

Further, the method for simulating digestion of the i oral cavity in vitro in the step (a) comprises the following steps: adding collagen polypeptide into SSF solution, adding salivary amylase and lysozyme, simulating chewing with oral cavity simulator, mixing, and placing in GI-20 in vitro gastrointestinal simulation instrument.

Further, the method for simulating in vitro digestion of the ii stomach in the step (a) comprises the following steps: taking simulated oral digests, adding SGF solution and pepsin, incubating at constant temperature, and placing in boiling water bath for enzyme deactivation treatment.

Further, the method for simulating in vitro digestion of iii small intestine in step (a) comprises: taking gastric digests, adding SIF solution, trypsin and bile salt, adjusting the pH value by using NaOH, incubating at constant temperature, and then putting in a boiling water bath for enzyme deactivation treatment.

Further, the collagen polypeptides with different molecular weights in the step (b) are prepared by the following method:

(1) crushing and cleaning fresh bovine bones, adding purified water, extracting ossein by high-efficiency hot-pressing extraction, standing, separating three phases, removing bone oil and a middle mixed layer, and concentrating by a tube centrifuge to obtain bovine ossein;

(2) adding an EDTA solution into the bovine ossein obtained in the step (1), and removing calcium ions in the bovine ossein to obtain a first crude collagen polypeptide product;

(3) adding a NaCl solution into the first crude collagen polypeptide product obtained in the step (2), further purifying by a salting-out method, and performing low-temperature centrifugal separation to obtain a second crude collagen polypeptide product;

(4) adding an acetic acid solution into the second crude collagen polypeptide product obtained in the step (3) for dissolving, then carrying out sectional dialysis, and carrying out dialysis by using the acetic acid solution and then distilled water to obtain a third crude collagen polypeptide product;

(5) and (4) carrying out enzymolysis on the third crude collagen polypeptide product in the step (4) by selecting multi-gradient enzymolysis to obtain a collagen polypeptide stock solution.

(6) And (5) repeatedly dialyzing and purifying the collagen polypeptide stock solution in the step (5) through semipermeable membranes with different molecular weights to obtain collagen polypeptides with different molecular weights.

Further, the method for multi-gradient enzymolysis in the step (5) comprises the following steps: firstly, adding alkaline protease with the pH value of 9.0 and the enzymolysis temperature of 50 ℃, carrying out enzymolysis for 2 hours, then adjusting the pH value of an enzymolysis solution to 8.0, then adding trypsin and compound flavor protease with the enzymolysis temperature of 40 ℃, and carrying out enzymolysis for 2 hours; finally, enzyme deactivation treatment is carried out for 20min at 90 ℃.

Further, the molecular weight distribution of the polypeptide of the collagen polypeptide is:

the invention has the advantages that:

(1) the digestion characteristic evaluation method of the bovine bone collagen polypeptide provided by the invention is used for measuring the digestion characteristics of the collagen polypeptides with different molecular weights under different enzymolysis conditions, and rapidly acquiring data by adopting a complete simulation mode, thereby providing an effective means for large-scale production of the collagen polypeptides with different molecular weights for enterprises.

(2) According to the preparation method of the collagen polypeptides with different molecular weights, the bovine bone is treated by using a high-efficiency hot-pressing extraction and physical concentration mode, so that the pollution is safe; in the enzymolysis process, the type and the addition amount of suboptimal enzymes are excessive, particularly multi-gradient enzymolysis, so that each enzyme reacts under the optimal condition, and the next enzyme reaction is carried out on the basis of substrates after different enzyme reactions, thereby not only improving the enzymolysis efficiency, but also obtaining more abundant small peptide products.

Drawings

FIG. 1 is a schematic diagram of in vitro digestibility of bovine bone collagen polypeptide after enzymolysis.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the specific examples described herein are only for explaining the present invention and are not intended to limit the present invention.

The invention discloses a preparation method of bovine bone collagen polypeptide, which comprises the following steps: crushing fresh bovine bones, cleaning, hot-pressing extraction, degreasing, decalcification, acid dissolution extraction, separation and purification, enzymolysis, ultrafiltration separation and bovine bone collagen polypeptide.

(1) Taking a certain amount of fresh ox bone, crushing, cleaning in a tank to remove blood water, adding purified water according to a ratio of 1:1, extracting ox bone extract by a high-pressure extraction technology of 135 ℃ and 0.3Mpa, standing, separating three phases of ox bone oil, a mixture, lower layer bone soup and the like, removing bone oil and a middle mixed layer, and concentrating by a tube centrifuge to obtain the ox bone extract.

(2) Taking the bovine ossein extracted by hot pressing in the step (1) as a substrate, adding 0.5mol/L EDTA as a decalcifying agent, and removing calcium ions in the ossein to obtain a relatively pure first crude product of the collagen polypeptide;

(3) adding 0.9mol/L NaCl solution into the first crude collagen polypeptide product obtained in the step (2), further purifying by a salting-out method, and centrifuging at 4 ℃ and 10000rmp for 30min to obtain a second crude collagen polypeptide product;

(4) adding 0.5mol/L acetic acid solution into the second crude collagen polypeptide product obtained in the step (3) for dissolving, then carrying out sectional dialysis, firstly dialyzing with 0.2mol/L acetic acid solution, and then repeatedly dialyzing with distilled water for 2d to obtain a third crude collagen polypeptide product;

(5) carrying out enzymolysis on the third crude product of the collagen polypeptide in the step (4) by selecting multi-gradient enzymolysis, namely firstly adding 3% of alkaline protease, carrying out enzymolysis for 2 hours at the pH value of 9.0 and the enzymolysis temperature of 50 ℃, then adjusting the pH value of an enzymolysis solution to 8.0, then adding 2% of trypsin and 3% of compound flavourzyme, carrying out enzymolysis at the enzymolysis temperature of 40 ℃ for 2 hours; finally, inactivating enzyme at 90 ℃ for 20min to obtain collagen polypeptide stock solution.

TABLE 1 polypeptide molecular weight distribution of bovine bone collagen polypeptides

Molecular weight rangeEnclose	Ratio of molecular weight
		<1kDa	25％～44％
1kDa-1.5kDa	15～40％
		1.5kDa-2.5kDa	6％～10％
2.5kDa～3kDa	2％～5％
		>3kDa	0.5％～1％

Repeatedly dialyzing and purifying the collagen polypeptide stock solution by semipermeable membranes with different molecular weights of <1kDa, 1-1.5 kDa, 2-3 kDa and >3kDa to obtain collagen polypeptides with different molecular weights.

The method for researching the digestion characteristic change of bovine bone collagen polypeptide at different digestion stages by utilizing a GI20 in-vitro gastrointestinal simulation system comprises the following steps:

(a) before the system is used, digestive juice is required to be prepared, wherein the digestive juice comprises simulated saliva SSF, simulated gastric fluid SGF and simulated intestinal fluid SIF, and a GI20 in-vitro simulated digestive system is used for simulating an in-vitro digestive process comprising i oral cavity, ii stomach and iii small intestine;

preparing a digestive juice: simulated Saliva (SSF) configuration: mainly comprises 15.1mL of KCl with the concentration of 37.3g/L and 68g/L KH₂PO₄3.7mL，84g/L NaHCO₃6.8 mL，30.5g/L MgCl₂(H₂O)60.5mL，48g/L(NH₄)2CO₃0.06 mL，44.1g/L CaCl₂(H₂O₂)0.025 mL. Simulated Gastric Fluid (SGF): 37.3g/L KCl 6.9mL, 68g/L KH₂PO₄0.9 mL，84g/L NaHCO₃12.5 mL，117g/L NaCl，11.8mL，30.5g/L MgCl₂(H₂O)60.4mL，48g/L(NH₄)2CO30.5 mL，6M HCl 1.3mL，44.1g/L CaCl₂(H₂O₂)0.005mL, pH 2.0 adjusted using HCl. ③ Simulated Intestinal Fluid (SIF): 37.3g/L KCl 6.8mL, 68g/L KH₂PO₄0.8 mL，84g/L NaHCO₃42.5 mL，117g/L NaCl 9.6mL，30.5g/L MgCl₂(H₂O)61.1mL，6M HCl 0.7mL，44.1g/L CaCl₂(H₂O₂)0.04mL, pH adjusted to 7.0 using NaOH.

Simulating an in vitro digestion process: oral cavity: taking 5mL of collagen polypeptide and 10mL of SSF, adding saliva amylase and lysozyme with certain enzyme activity, simulating chewing for 2min by using an oral simulator, fully mixing, and placing in a GI-20 in vitro gastrointestinal simulation instrument. ② ii stomach: to 10mL of the oral digest mimicking the oral cavity, 8mL of SGF and 2000U/mL of pepsin, 0.448mL of H₂And O, 200r/mim, incubating at the constant temperature of 37 ℃ for 2h, and after the reaction is finished, putting the sample into a boiling water bath for enzyme deactivation treatment. Iii small intestine: taking 20mL of gastric digest, adding 8mL of SIF, adding 100U/mL of trypsin and 3mL of 10mM bile salt, adjusting the pH value by using 5M NaOH, incubating at constant temperature of 200r/mim for 2h at 37 ℃, and placing the sample in a boiling water bath for enzyme deactivation treatment after the reaction is finished.

(b) Determining the digestion characteristics of collagen peptides of different molecular weights comprising measuring the digestibility of the digest in step (a) before and after digestion in the mouth i, stomach ii and small intestine iii. The method is characterized in that the digestion characteristics of the collagen polypeptides with different molecular weights are measured, mainly the digestion rates of digestates before and after digestion in small intestines of ii stomach and iii are measured, in particular, the change condition of total nitrogen of different digestates is measured by using a Kjeldahl method, and meanwhile, the change condition of the collagen polypeptides with different molecular weights after digestion is rapidly measured by using laser confocal Raman spectroscopy, and the absorption rates of the collagen polypeptides with different molecular weights before and after digestion are measured.

The in vitro digestibility is calculated as follows:

wherein: DT denotes the digestibility of the protein polypeptide after digestion with different enzyme solutions, W_O、W_DRepresents the polypeptide content before and after digestion, respectively.

In the embodiment of the invention, the digestibility of the collagen polypeptides with different molecular weights is obviously different, specifically, as shown in fig. 1, the smaller the molecular weight is, the higher the digestibility is, namely, the digestibility is the highest for the small peptide with less than 1kDa, which can reach 95%, the digestibility is about 87% for the 1kDa-1.5k Da polypeptide, and the digestibility is about 68% for the 1.5kDa-2.5k Da polypeptide.