WO2019091471A1 - 一种基于胃肠消化的大分子多肽的制备及其过程原位实时监测方法 - Google Patents

一种基于胃肠消化的大分子多肽的制备及其过程原位实时监测方法 Download PDF

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WO2019091471A1
WO2019091471A1 PCT/CN2018/114949 CN2018114949W WO2019091471A1 WO 2019091471 A1 WO2019091471 A1 WO 2019091471A1 CN 2018114949 W CN2018114949 W CN 2018114949W WO 2019091471 A1 WO2019091471 A1 WO 2019091471A1
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hydrolysis
enzymatic hydrolysis
milk
enzymatic
gastrointestinal digestion
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马海乐
李云亮
任晓锋
杨雪
黄姗芬
王禹程
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Jiangsu University
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Jiangsu University
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Priority claimed from CN201711111327.6A external-priority patent/CN107858390A/zh
Priority claimed from CN201711111354.3A external-priority patent/CN108107018B/zh
Priority claimed from CN201711111381.0A external-priority patent/CN107858391A/zh
Priority claimed from CN201711111383.XA external-priority patent/CN108004287A/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G01N21/359—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using near infrared light

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  • the invention relates to the field of polypeptide preparation and real-time monitoring of protein enzymatic hydrolysis process, in particular to a preparation of a macromolecular polypeptide based on gastrointestinal digestion and an in-situ real-time monitoring method thereof.
  • the active peptides are prepared by enzymatic hydrolysis, mainly in the preparation of small molecular peptides (molecular weight less than 500-3000 Da), because the body transports small peptides faster than the amino acids through the small intestinal wall, and the preparation process is based on the desired target small molecule polypeptides.
  • the direct absorption of the digestive tract in the body is the basic criterion, that is, the concept of “small molecule preparation for small molecule absorption”, because the degree of enzymatic hydrolysis is too high (if the preparation of di- and tri-peptide is the ultimate goal, its hydrolysis degree is above 33.3%), follow-up
  • the processes of centrifugation, membrane separation and concentration also make the preparation process complicated and costly.
  • small molecule polypeptides are digested by proteases in the gastrointestinal tract, which also causes excessive degradation (Katherina Fernández, Simulated digestion of proanthocyanidins in grape skin and seed extracts and the effects of digestion on the angiotensin I-converting enzyme (ACE) inhibitor activity , 2013).
  • proteases in the gastrointestinal tract, which also causes excessive degradation (Katherina Fernández, Simulated digestion of proanthocyanidins in grape skin and seed extracts and the effects of digestion on the angiotensin I-converting enzyme (ACE) inhibitor activity , 2013).
  • the raw materials of the polypeptide prepared by enzymatic hydrolysis mainly include milk protein, casein, zein, rice protein, gluten meal, etc.
  • Most of the proteins are not ideally water-soluble under normal conditions, if directly subjected to low-level short-term enzymatic hydrolysis Due to the large particle size, the insoluble protein may cause different degrees of protein hydrolysis inside and outside the particle, that is, the external protein of the particle is excessively digested, and the internal protein is not hydrolyzed, resulting in low uniformity of the enzymatic product.
  • the degree of hydrolysis of the protein has a significant effect on the biological activity of the product. Therefore, it is important to improve the homogeneity of the enzymatic hydrolysate to improve the quality of the macromolecular polypeptide product.
  • solubility of soy protein increases with temperature, but the temperature exceeds
  • prolonged enzymatic hydrolysis will cause a significant decrease in protease activity and may even cause inactivation.
  • solubility of substrate proteins and the catalytic activity of enzymes the preparation of highly homogenous gastrointestinal digestion-based macromolecular active peptides has not been reported.
  • the degree of proteolysis in the preparation of macromolecular polypeptides is low, which can greatly shorten the enzymatic hydrolysis time, reduce the amount of enzyme used, simplify the process, reduce energy consumption, and achieve the purpose of significantly reducing the production cost.
  • the detection index of the preparation of macromolecular peptides based on gastrointestinal digestion still needs to be determined offline (after online sampling, and then biologically active after digestion by gastrointestinal simulation), which is not only time-consuming, large in workload, but also has hysteresis in measurement data. .
  • the present invention provides a method for preparing a highly uniform gastrointestinal digestion-based macromolecular macromolecular active polypeptide and in-situ real-time monitoring thereof.
  • the above protein may be a protein that is not easily soluble in water, such as casein, zein, and a protein having a greater degree of gelation and viscosity after being dissolved in water, such as soy protein isolate, or may be dissolved. Protein in water, such as milk protein.
  • the above-described solubilization pretreatment means includes, but is not limited to, adjusting the ratio of non-polar solvent, temperature or pH.
  • the above protein is not seriously gelatinized if it is soluble, such as whey protein, egg white albumin, etc., and no solubilization pretreatment may be employed.
  • the protein may be pretreated with heat and/or ultrasound.
  • the above protease is preferentially a protease having a strong complementarity to a hydrolysis site of a pepsin (preferably hydrolyzes a peptide bond formed by two hydrophobic amino acid residues, such as Phe-Phe), such as neutral.
  • the method for preparing casein macromolecular polypeptide is carried out according to the following steps: taking a certain amount of casein, preheating in a water bath at 50 ° C for 10 min, adjusting the pH to 8.0-8.5, completely dissolving, and adjusting the protein concentration to 50 g/L, according to 5% (E/S) is added to the neutral protease.
  • E/S 5%
  • the pH drops to 6.5-7.5
  • the NaOH is continuously added dropwise to maintain the pH constant.
  • the reaction is to a certain degree of hydrolysis (10%-20%), the enzymatic hydrolysis is completed, and the enzyme is eliminated. After cooling, it is dried to prepare a finished product.
  • the preparation method of the zein macromolecule polypeptide is carried out according to the following steps: dissolving the zein with 60%-70% ethanol, preheating in a water bath at 50 ° C for 10 min, slowly adding isothermal water, and diluting the ethanol concentration of the solution to 15%-25%, the protein concentration was adjusted to 15g/L, and the zein was pretreated by concentrating ultrasonic (cell disrupter).
  • the ultrasonic treatment parameters were as follows: ultrasonic time 5-60min, ultrasonic power 40-500W/L.
  • the preparation method of the soybean protein macromolecular polypeptide is carried out according to the following steps: taking a certain amount of soy protein isolate, adding water to form a protein concentration of 50 g/L, preheating in a water bath at 63-68 ° C for 10 min, adjusting the pH to 8.0-8.5, adding 5% (E/S) neutral protease was enzymatically digested.
  • E/S 5% neutral protease
  • the preparation method of the milk protein macromolecular polypeptide is carried out according to the following steps: taking milk, heating and denaturation in an oil bath, cooling to room temperature, pretreatment of milk (protein concentration 34 g/L) by concentrating ultrasonic (cell disruptor), ultrasound
  • the processing parameters are as follows: ultrasonic time 5 ⁇ 60min, ultrasonic power 40 ⁇ 500W / L. After the pretreatment, the temperature is adjusted to 40-60 ° C, 2% ⁇ 10% (E / S) neutral protease is added, enzymatic hydrolysis to a certain degree of hydrolysis (2% ⁇ 20%), after the end of enzymatic digestion, extinction
  • the enzyme dried and dried, is prepared into a finished product.
  • the invention provides a method for in situ real-time monitoring of macromolecular polypeptide preparation based on gastrointestinal digestion, which is carried out according to the following steps:
  • the index of enzymatic hydrolysis is the blood pressure reduction of milk and the antihypertensive activity of the enzymatic hydrolyzed product after gastrointestinal digestion (The ACE inhibition rate is an indicator).
  • the simulated gastrointestinal digestion is carried out according to the following steps: the enzymatic hydrolyzate is added to the pepsin according to the amount of 2% (E/S) enzyme, and the enzymatic hydrolysis is carried out for 2 h, and after the end of the enzymatic hydrolysis, the enzyme is added according to 4% (E/S). Trypsin, enzymatically hydrolyzed for 4 h, and the enzyme was sterilized at 100 ° C for 15 min after the reaction.
  • the source of the protein described above is not limited to milk, but also includes other proteins which are better soluble.
  • soy protein egg white or whey protein.
  • the present invention unifies the solubility of the protein substrate and the catalytic activity of the enzyme, so that the substrate is always in a dissolved state during the enzymatic hydrolysis process (or does not form large particles).
  • the present invention is an in-situ real-time monitoring method for preparing milk macromolecular polypeptide based on gastrointestinal digestion, and online monitoring and preparation of milk macromolecular hypotensive peptide enzymatic hydrolysis process and its enzymatic hydrolyzed product after gastrointestinal digestion Blood pressure lowering activity.
  • milk as a sample, the in-situ real-time spectra were prepared during the enzymatic hydrolysis of macromolecular hypotensive peptides.
  • the joint index and the least squares method were used to establish the model and the spectrogram. The deviation between the measured value and the predicted value was used as the index.
  • the correlation coefficient R of the hydrolysis degree prediction model of milk hydrolysate was 0.962, the standard deviation was 1.12%; the correlation coefficient R of the hypotensive activity prediction model of digestive products after gastrointestinal digestion was 0.972, and the standard deviation was 2.88%.
  • the in-situ detection of the degree of hydrolysis and the bioactivity of the enzymatic hydrolysate after digestion in the process of enzymatic hydrolysis of the prepared macromolecule polypeptide was carried out.
  • Figure 1 is a flow chart of in-situ on-line monitoring and quantitative modeling of the enzymatic hydrolysis process of milk macromolecular peptides.
  • 2 is a diagram of an apparatus for in situ real-time monitoring of milk macromolecular polypeptides prepared by gastrointestinal digestion.
  • 1 acid-base burette
  • 2 sandwich beaker
  • 3 magnetic stirrer
  • 4 immersed fiber optic probe
  • 5 is super incubator
  • 6 tungsten light source
  • 7 micro-miniature near-infrared spectrometer
  • 8 information acquisition system
  • 9 is an automatic titrator.
  • protease and casein of this and the comparative examples were purchased from Sigma.
  • the degree of hydrolysis of the present example and the comparative example was determined by formaldehyde titration after the neutralization of the neutral protease was started until the pH reached the set pH value, and the pH-stat method was used after the pH was reached.
  • the total degree of hydrolysis is the sum of the two.
  • In vitro simulated gastrointestinal digestion of the present example and the comparative example were carried out by taking two parts of the enzymatic hydrolyzed product, adding pepsin according to a certain amount of 2% (E/S) enzyme amount, enzymatic hydrolysis for 2 hours, and end of enzymatic hydrolysis. Thereafter, one portion is used for enzyme digestion, centrifugation, and blood pressure lowering activity of the gastric digestion product is measured. The other part was added with trypsin according to 4% (E/S) enzyme amount, and enzymatically hydrolyzed for 4 hours. After the end of enzymatic hydrolysis, the enzyme was inactivated and centrifuged to measure the blood pressure lowering activity of the gastrointestinal digestion product.
  • E/S 2%
  • the antihypertensive activity of the present example and the control example were determined by angiotensin converting enzyme (ACE) inhibition rate, and the specific operation was carried out with reference to Ding Qingzhi (pulse ultrasonic assisted enzymatic hydrolysis method for preparation of ACE yellow active peptide). 2008) method.
  • ACE angiotensin converting enzyme
  • the method for determining the protein which was not digested in the present example and the comparative example was as follows: a trichloroacetic acid precipitation method (the final concentration of trichloroacetic acid was 10%).
  • the unenzymatic protein was 9.64 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 31.23%.
  • the unenzymatic protein was 11.30 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 46.23%.
  • the unenzymatic protein was 10.99 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 32.26%.
  • the unenzymatic protein was 8.74 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 51.37%.
  • the unenzymatic protein was 4.53 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 56.74%.
  • the unenzymatic protein was 4.21 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 39.62%.
  • the unenzymatic protein was 4.76 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 44.35%.
  • the unenzymatic protein was 1.92 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 65.17%.
  • the unenzymatic protein was 3.47 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 54.68%.
  • ultrasonic processing parameters are as follows: ultrasonic time 5min, ultrasonic power 40W / L. After the pretreatment, the temperature was adjusted to 37 ° C and pH 7.5, 337.5 mg trypsin was added for enzymatic hydrolysis, and the pH was kept constant with 1 mol/L sodium hydroxide solution. When the degree of hydrolysis was 10%, the pH was adjusted to 5, and the ethanol was rotary evaporated. After that, the enzyme is hydrated, spray dried, and prepared into a finished product.
  • the unenzymatic protein was 1.78 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 58.47%.
  • ultrasonic processing parameters are as follows: ultrasonic time 60min, ultrasonic power 500W / L. After the pretreatment, the temperature was adjusted to 37 ° C and pH 7.5, 337.5 mg trypsin was added for enzymatic hydrolysis, and the pH was kept constant with 1 mol/L sodium hydroxide solution. When the degree of hydrolysis was 10%, the pH was adjusted to 4, and the ethanol was rotary evaporated. After that, the enzyme is hydrated, spray dried, and prepared into a finished product.
  • the unenzymatic protein was 1.69 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 58.47%.
  • ultrasonic processing parameters are as follows: ultrasonic time 30min, ultrasonic power 100W / L. After the pretreatment, the temperature was adjusted to 37 ° C and pH 7.5, 337.5 mg trypsin was added for enzymatic hydrolysis, and the pH was kept constant with 1 mol/L sodium hydroxide solution. When the degree of hydrolysis was 10%, the pH was adjusted to 4, and the ethanol was rotary evaporated. After that, the enzyme is hydrated, spray dried, and prepared into a finished product.
  • the unenzymatic protein was 1.71 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 60.32%.
  • the unenzymatic protein was 12.63 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 29.38%.
  • the unenzymatic protein was 14.21 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 31.47%.
  • the unenzymatic protein was 6.41 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 36.97%.
  • the unenzymatic protein was 7.28 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 35.18%.
  • the unenzymatic protein was 6.51 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 44.61%.
  • the unenzymatic protein was 7.17 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 52.30%.
  • the ultrasonic treatment parameters are as follows: ultrasonic time 20 min, ultrasonic power 300 W/L.
  • the ultrasonically pretreated milk was placed in a water bath, the temperature was adjusted to 50 ° C, 680 ⁇ L of neutral protease was added, and the enzyme was decomposed to a degree of hydrolysis of 18%.
  • the enzyme was deactivated at 100 ° C for 15 min, cooled and dried to prepare. Into the finished product.
  • the unenzymatic protein was 6.83 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 60.2%.
  • the unenzymatic protein was 6.12 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 58.8%.
  • the ultrasonic treatment parameters were as follows: ultrasonic time 20 min, ultrasonic power 300 W/L.
  • the ultrasonically pretreated milk was placed in a water bath, the temperature was adjusted to 50 ° C, 680 ⁇ L of alkaline protease was added, and the enzyme was decomposed to a degree of hydrolysis of 18%.
  • the enzyme was deactivated at 100 ° C for 15 min, cooled and dried to prepare. Into the finished product.
  • the unenzymatic protein was 4.61 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 56.4%.
  • the unenzymatic protein was 3.90 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 64.6%.
  • the unenzymatic protein was 3.76 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 62.3%.
  • the unenzymatic protein was 5.82 g, and the blood pressure lowering activity of the gastrointestinal digestion product was determined to be 57.51%.
  • the blood pressure lowering activity of the milk hydrolyzed product after digestion with gastrointestinal protease was determined.
  • the method for measuring blood pressure lowering activity was carried out with reference to the method of Ding Qingzhi and slightly modified (Ding Qingzhi. Preparation of ACE yellow active peptide by pulverized ultrasound assisted enzymatic hydrolysis method [D]. Jiangsu University, 2008.).
  • Add 50 ⁇ L to each of ACE and FAPGG add 100 ⁇ L to HEPES buffer, and add 100 ⁇ L to the sample.
  • the InGaAs detector was used to acquire 900-2500nm spectrum (using 50°C distilled water as background, using transflective mode; optical path 4mm; scanning times 16 times, resolution 6.4nm, totaling 90 variables. Each sample was collected 3 times in succession. The spectrum is taken as the average of the sample.

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Abstract

一种基于胃肠消化的大分子多肽的制备及其过程原位实时监测方法，涉及多肽制备及在线监测领域。在制备多肽时采用可溶化技术处理蛋白，加入特定的蛋白酶，使反应体系接近最佳催化体系，维持至预定的酶解水解度，使得未被酶解的蛋白含量显著下降，酶解产物均一性高，经胃肠消化后的可快速吸收性、生物学活性显著提高。在制备过程中在线监测水解度及酶解产物经胃肠消化后的降血压活性，预测模型和实测模型非常接近，可实现有效原位检测。

Description

一种基于胃肠消化的大分子多肽的制备及其过程原位实时监测方法 技术领域
本发明涉及多肽制备和蛋白质酶解过程实时监测领域，特别是指一种基于胃肠消化的大分子多肽的制备及其过程原位实时监测方法。
背景技术
我国蛋白质资源丰富，每年有9000万吨的蛋白类农产品加工副产物，但是其综合利用率只有40％，不到发达国家的一半，造成资源的浪费和经济损失。主要原因在于目前我国蛋白质的加工方式粗放，缺乏高集成度的相关技术。因此，开展蛋白资源精深综合加工产业提质增效的关键技术研究非常迫切，符合国家现代农业发展要求。
大量科学研究表明，通过选择适当的蛋白酶，水解的蛋白质可以得到大量的具有各种生物功能的生物活性肽，这些活性肽不仅具有极其广泛的活性和多样性，而且其来源丰富、成本低、安全性好。操作简单、便于工业化生产，因此已成为研究的新热点(庞广昌等，生物活性肽的研究进展理论基础与展望，2001)。其相关产品已广泛地应用于营养食品、特医食品、保健品、化妆品等领域。
目前酶解法制备活性肽，主要以制备小分子多肽为主(分子量小于500～3000Da)，因为机体转运小肽通过小肠壁的速度比氨基酸更快，其制备工艺均是基于期望目标小分子多肽被体内消化道直接吸收为基本准则，即“小分子制备小分子吸收”的理念，由于其酶解程度过高(若以制备二、三肽为终极目标，其水解度达33.3％以上)，后续的离心、膜分离和浓缩等环节也致使其制备过程复杂，成本过高。另外小分子多肽经过胃肠道内的蛋白酶消化，还会产生过度降解的问题(Katherina Fernández,Simulated digestion of proanthocyanidins in grape skin and seed extracts and the effects of digestion on the angiotensin I-converting enzyme(ACE)inhibitory activity,2013)。从该角度考虑，利用酶解法制备基于胃肠消化的大分子活性肽(分子量大于6000Da，水解度在2％-20％)具有很大优势，该方案是基于“大分子制备小分子吸收”的理念，将蛋白在体外经过低程度酶解制备大分子多肽，口服进入肠胃后，利用体内胃肠的蛋白酶解能力，形成至少达到与传统“小分子制备小分子吸收”类似效果的小分子活性肽被机体吸收，同时还能起到类似缓释的效果。
目前酶解法制备的多肽的原料主要有牛奶蛋白、酪蛋白、玉米蛋白、大米蛋白、谷朊粉等，其中大部分蛋白在正常情况下水溶性均不理想，如果直接进行低程度短时间地酶解，不溶性的蛋白由于颗粒度较大，会导致颗粒内外蛋白水解程度不一，即颗粒外部蛋白被过度酶 解，内部蛋白未被酶解，造成酶解产物的均一度低。通常蛋白的水解程度对其产物的生物活性有显著的影响。因此，提高酶解产物的均一性提高大分子多肽产品质量具有重要意义。
提高蛋白溶解性的方法有很多，如：(1)改变温度，如大豆蛋白溶解于较高温度的水(pH8.0)中，不仅溶解度大幅提高，而且蛋白溶液胶体化程度和粘稠度会显著下降；(2)改变pH值，如酪蛋白极难溶于中性或者酸性的水中，在pH大于8时，溶解较好；(3)改变非极性溶液比例，如玉米醇溶蛋白难溶于水中，但是易溶于60％乙醇溶液。这些方法虽然能有效解决蛋白的溶解问题，但是这些方法对应的体系并不一定是常用蛋白酶的最佳催化体系，比如在一定温度范围内，大豆蛋白溶解度随温度升高而升高，但是温度超过蛋白酶最佳催化温度时，长时间酶解，会引起蛋白酶酶活大幅下降甚至会引起失活。综合考虑底物蛋白的溶解性和酶的催化活性，制备高均一性的基于胃肠消化的大分子活性多肽的研究还未见报道。
另外，大分子多肽的制备过程中蛋白水解程度低，可以大幅地缩短酶解时间、减少用酶量、简化工艺、降低能耗，达到显著降低制备成本的目的。但是目前基于胃肠消化的大分子多肽制备的检测指标仍然需要离线测定(在线取样后，经胃肠模拟消化后再测定其生物学活性)，不仅耗时长、工作量大而且测定数据具有滞后性。因此需要一种快速、连续的方法获得基于胃肠消化的大分子多肽制备酶解反应中的重要参数，用以判断酶解反应终点。近年来，光谱监测手段已应用于多肽的制备过程中(基于原位实时光谱在线监测蛋白质酶解过程的装置和方法，公开号：CN105628644A)。原位实时在线光谱技术能对过程中化学成分的变化进行连续性测量，而且该技术由于采用了微小型光纤光谱仪，因此其具有方便携带，成本低廉，速度快，无污染，实时连续检测的优点。目前已实现光谱与酶解产物活性定量模型的建立，但是能否实现光谱与酶解产物经胃肠消化后的活性定量模型的建立还未见报道。
发明内容
本发明的目的为了克服传统技术的不足，是提供了一种高均一性的基于胃肠消化的大分子大分子活性多肽的制备方法及其过程原位实时监测。
为了实现上述发明目的，其具体的技术方案如下：
称取蛋白，进行可溶化预处理，加入特定的蛋白酶，借助体系内部或外部因素控制，使反应体系随着酶解反应的进行接近(或达到)蛋白酶最佳催化体系，并维持至达到预定的酶解水解度，灭酶，冷却后干燥，制备成成品。
其中上述的蛋白，可以为不易溶于水的蛋白，如酪蛋白、玉米醇溶蛋白，和溶于水后凝 胶化程度和粘稠度较大的蛋白，如大豆分离蛋白，也可以是溶于水的蛋白，如牛奶蛋白。
其中上述的可溶化预处理手段包括且不局限于调节非极性溶剂比例、温度或pH。
其中上述的蛋白如果可溶性较好凝胶化不严重，如乳清蛋白、卵清白蛋白等，可以不采用可溶化预处理。
其中上述的蛋白如果溶于水，可采用热和/或超声预处理蛋白。
其中上述的蛋白酶，优先为其蛋白水解位点与胃蛋白酶的水解位点(优先水解由两个疏水性氨基酸残基形成肽键，如Phe-Phe)具有较强互补性的蛋白酶，如中性蛋白酶、胰蛋白酶、胰酶(主要为胰蛋白酶)、木瓜蛋白酶、菠萝蛋白酶、无花果蛋白酶等。
其中酪蛋白大分子多肽制备方法，按照下述步骤进行：取一定量的酪蛋白，50℃水浴预热10min，调节pH至8.0-8.5，使之完全溶解，调节蛋白浓度为50g/L，按5％(E/S)加入中性蛋白酶，待pH下降至6.5-7.5时，不断滴加NaOH维持pH恒定，待反应到一定水解度(10％-20％)，酶解结束，灭酶，冷却后干燥，制备成成品。
其中玉米醇溶蛋白大分子多肽制备方法，按照下述步骤进行：用60％-70％的乙醇溶解玉米醇溶蛋白，50℃水浴预热10min，缓慢加入等温水，将溶液的乙醇浓度稀释为15％-25％，蛋白浓度调节至15g/L，采用聚能式超声(细胞破碎仪)预处理玉米醇溶蛋白，超声处理参数如下：超声时间5-60min，超声功率40-500W/L。预处理结束后，调节温度和pH，加入5％(E/S)胰酶进行酶解，采用1mol/L氢氧化钠溶液维持pH恒定，反应到一定水解度(5％-15％)，酶解结束，调pH为4-5，旋转蒸发掉乙醇后，补水灭酶，干燥，制备成成品。
其中大豆蛋白大分子多肽制备方法，按照下述步骤进行：取一定量的大豆分离蛋白，加水配成蛋白浓度为50g/L，63-68℃水浴预热10min，调节pH至8.0-8.5，加入5％(E/S)中性蛋白酶进行酶解，待pH下降6.5-7.5时，不断滴加NaOH维持pH恒定，再降低温度至50-55℃。反应到一定水解度(10％-20％)，酶解结束，灭酶，冷却后干燥，制备成成品。
其中牛奶蛋白大分子多肽制备方法，按照下述步骤进行：取牛奶，油浴加热变性，冷却至室温后，采用聚能式超声(细胞破碎仪)预处理牛奶(蛋白浓度34g/L)，超声处理参数如下：超声时间5～60min，超声功率40～500W/L。预处理结束后，将温度调至40～60℃，加入2％～10％(E/S)中性蛋白酶，酶解至一定的水解度(2％～20％)，酶解结束后，灭酶，冷却后干燥，制备成成品。
本发明一种基于胃肠消化的大分子多肽制备原位实时监测方法，按照下述步骤进行：
(1)对牛奶进行酶解，酶解过程中定时取样，并对所取的样品进行模拟胃肠消化，酶解过程指标为牛奶水解度及其酶解产物经过胃肠消化后的降血压活性(以ACE抑制率为指 标)。
所述模拟胃肠消化按以下步骤进行：取酶解产物按照2％(E/S)加酶量加入胃蛋白酶，酶解2h，酶解结束后按照4％(E/S)加酶量加入胰酶，酶解4h，反应结束后于100℃灭酶15min。
(2)制备牛奶大分子降血压肽酶解过程中，定时对酶解液进行快速采集原位实时近红外光谱，采用便携式微小型近红外光谱仪采集数据，采集的光谱波长范围：900-2500nm)。
(3)对光谱数据进行预处理，具体为采用多项式卷积平滑(sg)的方法对样品光谱数据进行预处理。
(4)筛选最佳光谱区间，具体为采用联合区间偏最小二乘回归方法筛选牛奶水解度及其酶解产物经过胃肠消化后的降血压活性的最佳光谱区间。也可以采用偏最小二乘法、区间偏最小二乘法等方法。
(5)建立校正和预测模型，具体为采用联合区间最小二乘回归方法，将牛奶水解度及其酶解产物经过胃肠消化后的降血压活性与原位实时光谱数据建立校正和预测模型，其中校正集(70个)和预测集(20个)。
(6)将上述预测模型应用于制备牛奶大分子降血压肽酶解过程中，利用上述预测模型，对建模外的制备牛奶大分子降血压肽的酶解过程进行原位实时在线监测，预测酶解过程中水解度及其酶解产物经过胃肠消化后的降血压活性，并计算预测值和实测值的偏差。
以上所述的蛋白来源不限于牛奶，还包括一些可溶性较好的其他蛋白。例如大豆蛋白、蛋清或乳清蛋白等。
本发明的优点：
(1)本发明与现有技术相比，一方面，使得蛋白底物的溶解性和酶的催化活性得到了统一，使得底物在酶解过程中始终处于溶解状态(或者不形成大的颗粒沉淀或提高蛋白底物的均一性)，从而使产物的均一性得到提高，酶解体系中未被酶解的蛋白含量下降9.34％-54.39％(与直接酶解蛋白制备小分子多肽相比)；另一方面，充分利用人体的胃肠消化功能，使大分子多肽在体内进一步降解，与直接酶解蛋白制备小分子多肽相比，降血压活性(以ACE抑制率为指标)提高了19.74％-81.68％，并且所采用的中性蛋白酶(其酶切位点与胃蛋白酶互补)，其酶解产物经过胃肠消化之后的生物学活性优于碱性蛋白酶。
(2)本发明是一种基于胃肠消化制备牛奶大分子多肽的原位实时监测方法，在线监测制备牛奶大分子降血压肽酶解过程中水解度及其酶解产物经过胃肠消化后的降血压活性。以牛奶为样本，采集制备大分子降血压肽酶解过程中的原位实时光谱，采用联合区间偏最小二 乘法将检测指标和光谱图建立模型，以实测值和预测值的偏差为指标，评价牛奶水解度及其酶解产物经过胃肠消化后的降血压活性的回归模型。牛奶酶解液的水解度预测模型的相关系数R为0.962，标准偏差为1.12％；酶解产物经胃肠消化后的降血压活性预测模型的相关系数R为0.972，标准偏差为2.88％，实现了对制备牛奶大分子多肽酶解过程中水解度、酶解产物经胃肠消化后生物活性的有效原位检测。
下面通过附图和实施例，对本发明的技术方案做进一步的详细描述。
附图说明
图1为牛奶大分子多肽的酶解过程原位在线监测定量建模流程图。
图2为基于胃肠消化制备牛奶大分子多肽的原位实时监测的装置图。其中1为酸碱滴定管，2为夹层烧杯，3为磁力搅拌器，4为浸入式光纤探头，5为超级恒温箱，6为钨灯光源，7为微小型近红外光谱仪，8为信息采集系统，9为自动滴定仪。
具体实施方式
在本发明中所使用的术语，除非有另外说明，一般具有本领域普通技术人员通常理解的含义。下面结合具体的实施例，并参照数据进一步详细地描述本发明。应理解，这些实施例只是，对本发明进行进一步说明，不能理解为对本发明保护范围的限定，该领域的技术工程师可根据上述发明的内容对本发明作出一些非本质的改进和调整。
在以下的实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法。所用试剂的来源、商品名以及有必要列出其组成成分者，均在首次出现时标明，其后所用相同试剂如无特殊说明，均以首次标明的内容相同。
本实施例和对照例的蛋白酶和酪蛋白均购于Sigma公司。
本实施例和对照例的水解度在加入中性蛋白酶开始酶解时至pH达到设定pH值之前，酪蛋白的水解度采用甲醛滴定法测定，在达到设定pH以后采用pH-stat法测定，总水解度为两者相加之和。
本实施例和对照例的体外模拟胃肠道消化均采用如下方法：取酶解产物两份，按照一定的2％(E/S)加酶量加入胃蛋白酶，酶解2小时，酶解结束后，一份用于灭酶，离心，测定胃消化产物的降血压活性。另一份按照4％(E/S)加酶量加入胰酶，酶解4小时，酶解结束后，灭酶，离心，测定胃肠消化产物的降血压活性。
本实施例和对照例的降血压活性测定均以血管紧张素转化酶(angiotensin converting enzyme，ACE)抑制率为指标，具体操作参照丁青芝(脉冲超声辅助酶解法制备玉米黄粉 ACEI活性肽的研究，2008)的方法。
本实施例和对照例中未被酶解的蛋白测定方法为：采用三氯乙酸沉淀法(三氯乙酸的终浓度为10％)。
对照例1
称取20g酪蛋白，加入400mL蒸馏水，调节温度至50℃，用3M NaOH调节pH为6.5，平衡15min后，加入1mL中性蛋白酶，不断滴加1M NaOH维持pH为6.5，水解度为26％时，酶解结束，于100℃条件下灭酶15min，离心取上清，并过3000Da超滤膜，将滤过液喷雾干燥，制备成成品。
未酶解蛋白为9.64g，测定其胃肠消化产物的降血压活性为31.23％。
对照例2
称取20g酪蛋白，加入400mL蒸馏水，调节温度至50℃，用3M NaOH调节pH为6.5，平衡15min后，加入1mL中性蛋白酶，不断滴加1M NaOH维持pH为6.5，水解度为20％时，酶解结束，于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为11.30g，测定其胃肠消化产物的降血压活性为46.23％。
对照例3
称取20g酪蛋白，加入400mL蒸馏水，调节温度至50℃，用3M NaOH调节pH为6.5，平衡15min后，加入960mL碱性蛋白酶(与上述所加入的中性蛋白酶酶活相同)，不断滴加1M NaOH维持pH为6.5，水解度为20％时，酶解结束，于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为10.99g，测定其胃肠消化产物的降血压活性为32.26％。
实施例1
称取20g酪蛋白，加入400mL蒸馏水，不断滴加3M NaOH维持pH为8.5，在滴加的过程中不断搅拌，待其全部溶解之后，将温度调节至50℃，加入1mL中性蛋白酶，待pH降至7.5后，不断滴加1M NaOH维持pH为7.5，水解度为10％时，酶解结束，于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为8.74g，测定其胃肠消化产物的降血压活性为51.37％，。
实施例2
称取20g酪蛋白，加入400mL蒸馏水，不断滴加3M NaOH维持pH为8.0，在滴加的过程中不断搅拌，待其全部溶解之后，将温度调节至50℃，加入1mL中性蛋白酶，待pH降至6.5后，不断滴加1M NaOH维持pH为6.5，水解度为20％时，酶解结束，于100℃ 条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为4.53g，测定其胃肠消化产物的降血压活性为56.74％，。
对照例4
称取玉米醇溶蛋白6.75g，加入450mL水，50℃水浴预热10min，并调节pH为8.0，加入337.5mg的胰酶进行酶解，在酶解过程中不断滴加1mol/L NaOH维持pH恒定，水浴温度恒定。反应到水解度为28％，酶解结束，调pH为4，旋转蒸发掉乙醇后，补水于100℃条件下灭酶15min，离心取上清，并过3000Da超滤膜，将滤过液喷雾干燥，制备成成品。
未酶解蛋白为4.21g，测定其胃肠消化产物的降血压活性为39.62％。
对照例5
称取玉米醇溶蛋白6.75g，加入450mL水，50℃水浴预热10min，并调节pH为8.0，加入337.5mg的胰酶进行酶解，在酶解过程中不断滴加1mol/L NaOH维持pH恒定，水浴温度恒定。反应到水解度为15％，酶解结束，调pH为4，旋转蒸发掉乙醇后，补水于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为4.76g，测定其胃肠消化产物的降血压活性为44.35％。
实施例3
称取玉米醇溶蛋白6.75g，加入150mL 60％乙醇完全溶解，50℃水浴预热10min，调节溶液中的乙醇浓度为15％，体系为450mL。立即调节pH为8.0，加入337.5mg的胰酶进行酶解，在酶解过程中不断滴加1mol/L NaOH维持pH恒定，水浴温度恒定。反应到水解度为15％，酶解结束，调pH为4，旋转蒸发掉乙醇后，补水后于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为1.92g，测定其胃肠消化产物的降血压活性为65.17％。
实施例4
称取玉米醇溶蛋白6.75g，加入150mL 70％乙醇完全溶解，50℃水浴预热10min，调节溶液中的乙醇浓度为25％，体系为450mL。立即调节pH为8.0，加入337.5mg的胰酶进行酶解，在酶解过程中不断滴加1mol/L NaOH维持pH恒定，水浴温度恒定。反应到水解度为5％，酶解结束，调pH为5，旋转蒸发掉乙醇后，补水后于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为3.47g，测定其胃肠消化产物的降血压活性为54.68％。
实施例5
称取玉米醇溶蛋白6.75g，加入150mL 60％的乙醇完全溶解，50℃水浴预热10min， 缓慢加入等温水，将溶液的乙醇浓度稀释为15％，蛋白浓度调节至15g/L，采用聚能式超声(细胞破碎仪)预处理玉米醇溶蛋白，超声处理参数如下：超声时间5min，超声功率40W/L。预处理结束后，调节温度37℃和pH 7.5，加入337.5mg胰酶进行酶解，采用1mol/L氢氧化钠溶液维持pH恒定，水解度为10％时，调pH为5，旋转蒸发掉乙醇后，补水灭酶，喷雾干燥，制备成成品。
未酶解蛋白为1.78g，测定其胃肠消化产物的降血压活性为58.47％。
实施例6
称取玉米醇溶蛋白6.75g，加入150mL 60％的乙醇完全溶解，50℃水浴预热10min，缓慢加入等温水，将溶液的乙醇浓度稀释为25％，蛋白浓度调节至15g/L，采用聚能式超声(细胞破碎仪)预处理玉米醇溶蛋白，超声处理参数如下：超声时间60min，超声功率500W/L。预处理结束后，调节温度37℃和pH 7.5，加入337.5mg胰酶进行酶解，采用1mol/L氢氧化钠溶液维持pH恒定，水解度为10％时，调pH为4，旋转蒸发掉乙醇后，补水灭酶，喷雾干燥，制备成成品。
未酶解蛋白为1.69g，测定其胃肠消化产物的降血压活性为58.47％。
实施例7
称取玉米醇溶蛋白6.75g，加入150mL 60％的乙醇完全溶解，50℃水浴预热10min，缓慢加入等温水，将溶液的乙醇浓度稀释为25％，蛋白浓度调节至15g/L，采用聚能式超声(细胞破碎仪)预处理玉米醇溶蛋白，超声处理参数如下：超声时间30min，超声功率100W/L。预处理结束后，调节温度37℃和pH 7.5，加入337.5mg胰酶进行酶解，采用1mol/L氢氧化钠溶液维持pH恒定，水解度为10％时，调pH为4，旋转蒸发掉乙醇后，补水灭酶，喷雾干燥，制备成成品。
未酶解蛋白为1.71g，测定其胃肠消化产物的降血压活性为60.32％。
对照例6
取20g大豆分离蛋白，加入400mL水，50℃水浴预热10min，并调节pH至7.5，加入1mL中性蛋白酶进行酶解，不断滴加1mol/L NaOH维持pH恒定，反应到水解度为25％，酶解结束，于100℃条件下灭酶15min，离心取上清，并过3000Da超滤膜，将滤过液喷雾干燥，制备成成品。
未酶解蛋白为12.63g，测定其胃肠消化产物的降血压活性为29.38％。
对照例7
取20g大豆分离蛋白，加入400mL水，50℃水浴预热10min，并调节pH至7.5，加 入1mL中性蛋白酶进行酶解，不断滴加1mol/L NaOH维持pH恒定，反应到水解度为20％，酶解结束，于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为14.21g，测定其胃肠消化产物的降血压活性为31.47％。
实施例8
取20g大豆分离蛋白，加入400mL水，63℃水浴预热10min，并调节pH至8.5，加入1mL中性蛋白酶进行酶解，待pH下降7.5附近时，不断滴加1mol/L NaOH维持pH恒定，再降低温度至50℃(降温速度为5℃/min)，反应到水解度为20％，酶解结束，于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为6.41g，测定其胃肠消化产物的降血压活性为36.97％。
实施例9
取20g大豆分离蛋白，加入400mL水，68℃水浴预热10min，并调节pH至8.0，加入1mL中性蛋白酶进行酶解，待pH下降6.5附近时，不断滴加1mol/L NaOH维持pH恒定，再降低温度至55℃(降温速度为5℃/min)，反应到水解度为10％，酶解结束，于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为7.28g，测定其胃肠消化产物的降血压活性为35.18％。
对照例8
量取400mL牛奶，将温度调节至50℃，加入680μL中性蛋白酶，酶解至水解度为32％时于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为6.51g，测定其胃肠消化产物的降血压活性为44.61％。
对照例9
量取400mL牛奶，将温度调节至50℃，加入680μL中性蛋白酶，酶解至水解度为18％时于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为7.17g，测定其胃肠消化产物的降血压活性为52.30％。
对照例10
量取400mL牛奶，采用聚能式超声预处理牛奶，超声处理参数如下：超声时间20min，超声功率300W/L。将超声预处理后的牛奶放入水浴锅内，将其温度调节至50℃，加入680μL中性蛋白酶，酶解至水解度为18％时于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为6.83g，测定其胃肠消化产物的降血压活性为60.2％。
对照组11
量取400mL牛奶，于100℃油浴中加热20min后，冷却至室温。将预处理后的牛奶放入水浴锅内，将其温度调节至50℃，加入680μL中性蛋白酶，酶解至水解度为18％于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为6.12g，测定其胃肠消化产物的降血压活性为58.8％。
对照组12
量取400mL牛奶，于100℃油浴中加热20min，冷却至室温后，采用聚能式超声预处理牛奶，超声处理参数如下：超声时间20min，超声功率300W/L。将超声预处理后的牛奶放入水浴锅内，将其温度调节至50℃，加入680μL碱性蛋白酶，酶解至水解度为18％时于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为4.61g，测定其胃肠消化产物的降血压活性为56.4％。
实施例10
量取400mL牛奶，于100℃油浴中加热20min，冷却至室温后，采用聚能式超声(细胞破碎仪)预处理牛奶(蛋白浓度34g/L)，超声处理参数如下：超声时间20min，超声功率300W/L。将超声预处理后的牛奶放入水浴锅内，将其温度调节至50℃，加入680μL中性蛋白酶，酶解至水解度为18％时于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为3.90g，测定其胃肠消化产物的降血压活性为64.6％。
实施例11
量取400mL牛奶，于100℃油浴中加热20min，冷却至室温后，采用聚能式超声(细胞破碎仪)预处理牛奶(蛋白浓度34g/L)，超声处理参数如下：超声时间5min，超声功率40W/L。将超声预处理后的牛奶放入水浴锅内，将其温度调节至40℃，加入2.72μL中性蛋白酶，酶解至水解度为20％时于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为3.76g，测定其胃肠消化产物的降血压活性为62.3％。
实施例12
量取400mL牛奶，于100℃油浴中加热20min，冷却至室温后，采用聚能式超声(细胞破碎仪)预处理牛奶(蛋白浓度34g/L)，超声处理参数如下：超声时间60min，超声功率500W/L。将超声预处理后的牛奶放入水浴锅内，将其温度调节至60℃，加入1360μL中性蛋白酶，酶解至水解度为2％时于100℃条件下灭酶15min，冷却后干燥，制备成成品。
未酶解蛋白为5.82g，测定其胃肠消化产物的降血压活性为57.51％。
实施例13
(1)取牛奶(蛋白浓度为34g/L和30g/L)400mL于夹层烧杯中，在50℃恒温搅拌平衡 10min，用1mol/L的NaOH调节到pH为7.0。随后加入5％(E/S)的中性蛋白酶进行酶解，整个酶解过程中pH保持恒定，酶解时间90min，每间隔2min取样，同时采集光谱信息，取样后迅速沸水浴灭酶，冷却进行胃肠模拟消化实验。用3mol/L的HCl调节pH为1.60。随后加入2％(E/S)的胃蛋白酶，酶解2h，整个酶解过程中pH保持在1.5-2.0之间。胃蛋白酶酶解结束后，用1mol/L的NaOH调节到pH为8.0，随后加入2％(E/S)胰酶，酶解4h，整个酶解过程中pH保持恒定，酶解结束后取样，取样后迅速用沸水浴灭酶10min，冷却后10000g离心10min，收集上清液储存于4℃待测。共计90个样品。
(3)测定牛奶酶解产物经过胃肠蛋白酶消化后的降血压活性。降血压活性的测定方法参照丁青芝的方法并略作修改(丁青芝.脉冲超声辅助酶解法制备玉米黄粉ACEI活性肽的研究[D].江苏大学，2008.)。ACE、FAPGG各加50μL，HEPES缓冲液加100μL，样品加100μL。
(4)牛奶酶解过程中原位实时光谱的采集。经原位仪器如图2所示，整个系统工作时，在夹层烧杯2中进行牛奶的酶解，开启自动滴定仪、磁力搅拌器、超级恒温箱。将浸入式光纤探头置于牛奶中并固定。由卤钨灯光源6将光源传入浸入式光纤探头4，在酶解反应过程中采集到的相应光谱反映在微小型近红外光谱仪7中，最终通过信息采集系统将光谱信息采集并存储。以InGaAs检测器采集900-2500nm光谱(以50℃蒸馏水为背景，采用透反射方式；光程4mm；扫描次数为16次，分辨率6.4nm，共含90个变量。每个样品连续采集3次光谱，取其平均值作为该样本的原始光谱)。
(5)牛奶酶解过程中原位实时光谱的预处理。采用多项式卷积平滑(sg)预处理方法对酶解过程中采集的原始光谱进行预处理。
(6)校正模型的建立。将90个样品的光谱用sg预处理方法进行预处理，分成校正集(70个)和预测集(20个)两个部分。采用偏最小二乘算法建立酶解过程中牛奶水解度及其酶解产物经过胃肠消化后的降血压活性的定量模型，包括校正模型和预测模型。
(7)酶解过程的预测。牛奶(蛋白浓度34g/L和30g/L)400mL，按照(1)的方法采集光谱，按照(1)、(2)和(3)的方法进行酶解。将采集的光谱带入(7)中所建立的预测模型中，对牛奶水解度及其酶解产物经过胃肠消化后的降血压活性进行预测，并比较其预测值和实测值的残差。残差值小于实测值5％认为该模型预测准确。
表1 牛奶水解度及其酶解产物经胃肠消化后的降血压活性建模结果
Figure PCTCN2018114949-appb-000001
Figure PCTCN2018114949-appb-000002
表2 牛奶大分子肽酶解过程中水解度预测结果
Figure PCTCN2018114949-appb-000003
表3 牛奶酶解产物经胃肠消化后的降血压活性预测结果
Figure PCTCN2018114949-appb-000004

Claims (7)

  1. 一种基于胃肠消化的大分子多肽的制备方法，特征在于，取一定量的酪蛋白，50℃水浴预热10min，调节pH至8.0-8.5使之完全溶解，中性蛋白酶，待pH下降6.5-7.5附近时，不断滴加NaOH维持pH恒定，待反应到一定水解度(10-20％)，酶解结束，灭酶，冷却后干燥，制备成成品。
  2. 一种基于胃肠消化的大分子多肽的制备方法，特征在于，用60％-70％的乙醇溶解玉米醇溶蛋白，50℃水浴预热10min，缓慢加入等温水，将溶液的乙醇浓度稀释为15％-25％，蛋白浓度调节至15g/L，采用聚能式超声(细胞破碎仪)预处理玉米醇溶蛋白，超声处理参数如下：超声时间5-60min，超声功率40-500W/L。预处理结束后，调节温度和pH，加入5％(E/S)胰酶进行酶解，采用1mol/L氢氧化钠溶液维持pH恒定，反应到一定水解度(5％-15％)，酶解结束，调pH为4-5，旋转蒸发掉乙醇后，补水灭酶，干燥，制备成成品。
  3. 一种基于胃肠消化的大分子多肽的制备方法，特征在于，取一定量的大豆分离蛋白，63-68℃水浴预热10min，调节pH至8.0-8.5，加入中性蛋白酶进行酶解，待pH下降6.5-7.5附近时，不断滴加NaOH维持pH恒定，再降低温度至50-55℃；反应到一定水解度(10％-20％)，酶解结束，灭酶，冷却后干燥，制备成成品。
  4. 一种基于胃肠消化的大分子多肽的制备方法，特征在于，取牛奶，油浴加热变性，冷却至室温后，采用聚能式超声(细胞破碎仪)预处理牛奶(蛋白浓度34g/L)，超声处理参数如下：超声时间5～60min，超声功率40～500W/L。预处理结束后，将温度调至40～60℃，加入2％～10％(E/S)中性蛋白酶，酶解至一定的水解度(2％～20％)，酶解结束后，灭酶，冷却后干燥，制备成成品。
  5. 根据权利要求1所述一种基于胃肠消化的大分子多肽的制备过程原位实时监测方法，特征在于，按照下述步骤进行：
    (1)对牛奶进行酶解，酶解过程中定时取样，并对所取的样品进行模拟胃肠消化，酶解过程指标为牛奶水解度及其酶解产物经过胃肠消化后的降血压活性(以ACE抑制率为指标)；
    所述模拟胃肠消化按以下步骤进行：取酶解产物按照2％(E/S)加酶量加入胃蛋白酶，酶解2h，酶解结束后按照4％(E/S)加酶量加入胰酶，酶解4h，反应结束后于100℃灭酶15min；
    (2)制备牛奶大分子降血压肽酶解过程中，定时对酶解液进行快速采集原位实时近红外光谱，采用便携式微小型近红外光谱仪采集数据，采集的光谱波长范围：900-2500nm)；
    (3)对光谱数据进行预处理，具体为采用多项式卷积平滑(sg)的方法对样品光谱数 据进行预处理；
    (4)筛选最佳光谱区间，具体为采用联合区间偏最小二乘回归方法筛选牛奶水解度及其酶解产物经过胃肠消化后的降血压活性的最佳光谱区间；也可以采用偏最小二乘法、区间偏最小二乘法等方法；
    (5)建立校正和预测模型，具体为采用联合区间最小二乘回归方法，将牛奶水解度及其酶解产物经过胃肠消化后的降血压活性与原位实时光谱数据建立校正和预测模型，其中校正集(70个)和预测集(20个)；
    (6)将上述预测模型应用于制备牛奶大分子降血压肽酶解过程中，利用上述预测模型，对建模外的制备牛奶大分子降血压肽的酶解过程进行原位实时在线监测，预测酶解过程中水解度及其酶解产物经过胃肠消化后的降血压活性，并计算预测值和实测值的偏差。
  6. 根据权利要求1所述一种基于胃肠消化的大分子多肽的制备过程原位实时监测方法，其特征在于：将牛奶蛋白酶解过程中的在线光谱信息与牛奶蛋白酶解产物经过胃肠消化后的降血压活性相关联，建立校正和预测模型。
  7. 根据权利要求1所述一种基于胃肠消化的大分子多肽的制备过程原位实时监测方法，其特征在于：利用上述预测模型对建模外的一个制备牛奶蛋白大分子肽的酶解过程进行原位光谱监测，预测酶解过程中牛奶蛋白酶解产物经过胃肠消化后的降血压活性，比较预测值和实测值的残差值。
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