WO2024082385A1 - 一种乳酸化改性小麦面筋蛋白的方法 - Google Patents

一种乳酸化改性小麦面筋蛋白的方法 Download PDF

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WO2024082385A1
WO2024082385A1 PCT/CN2022/135377 CN2022135377W WO2024082385A1 WO 2024082385 A1 WO2024082385 A1 WO 2024082385A1 CN 2022135377 W CN2022135377 W CN 2022135377W WO 2024082385 A1 WO2024082385 A1 WO 2024082385A1
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wheat gluten
gluten protein
protein
lactic acid
modifying
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French (fr)
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李言
王宇
钱海峰
王立
樊铭聪
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Jiangnan University
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Jiangnan University
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/14—Vegetable proteins
    • A23J3/18—Vegetable proteins from wheat

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  • the invention belongs to the technical field of wheat protein processing, and particularly relates to a method for lactic acid-modified wheat gluten protein.
  • Wheat is one of the world's largest, most widely planted, and earliest cultivated food crops. In my country, wheat planting area is second only to rice, and it plays a very important role in people's food structure and consumption.
  • Wheat protein is a by-product of wheat starch production, also known as wheat gluten or wheat gluten. In the food industry, the application of wheat gluten is mainly reflected in two aspects: one is as a protein nutritional supplement, and the other is as a dough strengthener to improve the rheological properties of dough and product quality.
  • wheat gluten protein can be divided into four proteins: albumin, globulin, gliadin and glutenin.
  • the latter two proteins are the main components of wheat gluten protein.
  • Glutenin is a naturally occurring heterogeneous macromolecular polymer protein, which can be divided into two glutenin subunits, namely high molecular weight glutenin subunit (HMW-GS) and low molecular weight glutenin subunit (LMW-GS). These two subunits are connected by polypeptide bonds through intermolecular disulfide bonds.
  • HMW-GS high molecular weight glutenin subunit
  • LMW-GS low molecular weight glutenin subunit
  • Gliadin is a protein with a smaller molecular weight, which can bind to glutenin through non-covalent bonds and embed into the three-dimensional network structure of glutenin, resulting in extremely poor processing performance (solubility, dispersibility, etc.) of wheat gluten protein.
  • wheat gluten protein has the characteristics of amino acid sequence diversity and overall structural complexity, which greatly limits the application of wheat gluten protein. Therefore, it is necessary to reasonably modify wheat gluten protein to increase its solubility and improve other functional properties.
  • Physical modification method refers to the use of physical effects such as heat, pressure, radiation, sound waves, etc. to change the aggregation mode and secondary and tertiary structures of protein molecules.
  • Chemical modification refers to the use of chemical methods to cause partial breakage of the peptide chain of the protein, or the introduction of new active groups, thereby changing the protein structure, hydrophobic groups, charge characteristics, etc., and ultimately causing changes in its spatial structure and functional properties.
  • Enzymatic modification refers to the use of proteases to hydrolyze the peptide bonds or (and) amide bonds of proteins to a limited extent, or to cause covalent cross-linking between molecules, thereby directional changes in its functional properties.
  • chemical modification has become the mainstream technology for protein modification because it has the advantages of fast reaction speed, high efficiency, good performance, obvious modification effect and easy cross-linking compared to other modification methods.
  • Wheat gluten protein is a high-quality plant protein with high nutritional value. However, due to its own structural characteristics, wheat gluten protein has poor water solubility, which leads to unsatisfactory functional properties and hinders the application of wheat gluten protein in food and other industries.
  • the invention provides a method for lactic acid modification of wheat gluten protein, which can change the internal structure of gluten protein molecules and improve the functional properties of wheat gluten protein such as solubility and emulsification.
  • the method steps include the following:
  • Precipitating proteins adjusting the pH of the lactated wheat gluten protein treatment solution to neutral with a pH adjuster to precipitate the gluten protein, centrifuging, collecting the precipitated protein and washing with water; removing the residual pH adjuster and some free lactic acid and sodium lactate that failed to participate in the reaction by washing with water;
  • Dialysis The washed precipitated protein is redissolved and dialyzed in deionized water to remove salt ions to obtain a lactic acid purified solution of wheat gluten protein; the dialysis is to remove hydrogen ions, sodium ions, free lactic acid and sodium lactate;
  • Vacuum freeze drying vacuum freeze drying the lactated purified wheat gluten protein to obtain lactated modified wheat gluten protein.
  • the mass of wheat gluten protein is 25 g, and the lactic acid solution is 250 mL (the solid-liquid ratio is 1:10), and the concentration of the lactic acid solution is 0.1 mol/L; or, the mass of wheat gluten protein is 25 g, and the aqueous solution of sodium lactate is 250 mL, wherein the mass of sodium lactate is 5 g, that is, the mass ratio of sodium lactate to wheat gluten protein is 1:5.
  • step (2) is 3 h
  • the reaction temperature is 25° C.
  • the stirring speed is 100 rpm.
  • the alkaline solution in step (2) is 2 mol/L NaOH, and the pH value is 10.0.
  • the neutral pH in step (3) is 7.0
  • the pH regulator used is 2 mol/L NaOH
  • the centrifugal speed is 4500 r/min
  • the centrifugal time is 15 min
  • the precipitate is washed with water three times.
  • the dialysis time in step (4) is 72 hours.
  • the wheat gluten protein solution After the wheat gluten protein solution is treated with lactic acid and sodium lactate, the wheat gluten protein undergoes lactic acid modification, and the solubility and emulsification properties are significantly improved.
  • the solubility of the gluten protein is increased from the original 0.05 mg/mL to 0.19 mg/mL and 0.21 mg/mL, respectively, and the emulsification activity is increased from the original 16.66 m2 /g to 19.27 m2 /g and 19.58 m2 /g, respectively.
  • Lactic acid as a natural organic weak acid, can be used as a pH regulator and has a certain antiseptic effect. It is a widely used food additive.
  • the present invention utilizes lactic acid to effectively improve the solubility and emulsification properties of wheat gluten protein, and is expected to expand the application scope of wheat gluten protein, and apply it to protein beverages, edible packaging films, non-dairy creamers, ice cream, vegetable meat and other systems.
  • the present invention connects lactic acid groups to the amino acid side chains of proteins to change the structure of proteins and thus improve the functional properties. This can be seen from the fact that lactate, i.e. sodium lactate, can also modify gluten protein.
  • Figure 7 Fluorescence spectrum of lactated wheat gluten.
  • Raw is untreated wheat gluten protein
  • Lactate is wheat gluten protein treated with lactic acid
  • Sodium lactate is wheat gluten protein treated with sodium lactate.
  • Sample preparation After determining the concentration of the protein stored in the drying dishes of Example 1 and Example 2 with the BCA protein analysis reagent, adjust the protein to the same concentration (1 mg/mL) and add it to the protein loading buffer. After mixing, heat at 100°C for 10 minutes to denature the protein.
  • the composition of the protein loading buffer is: 50 mM Tris-HCl, 0.1% bromophenol blue, 10% glycerol (v/v) and 2% SDS (w/v), pH 6.8; Electrophoresis: Prepare a 10% SDS polyacrylamide gel and load 10 ⁇ L of protein per sample.
  • Lane 1 is untreated wheat gluten protein
  • lane 2 is wheat gluten protein treated with lactic acid
  • lane 3 is wheat gluten protein treated with sodium lactate.
  • the western blot results show that the band intensity at 15-20 and 30-50 kDa in lanes 2 and 3 is significantly deepened.
  • Western blot uses specific antibodies to stain proteins treated by gel electrophoresis, and obtains the expression of specific proteins by analyzing the position and depth of staining.
  • the lactic acid pan-antibody used in this experiment is an antibody that can recognize specific proteins with lactic acid groups connected to amino acid side chains.
  • Lane 1 is untreated gluten protein, so there will be no obvious bands.
  • Lanes 2 and 3 are wheat gluten proteins treated with lactic acid and sodium lactate, respectively, and both produce obvious bands, which indicates that the treatment with lactic acid and sodium lactate causes obvious lactic acid modification of gluten protein.
  • the molecular weight of wheat alcohol-soluble protein is about 22-58 kDa
  • the molecular weight of high molecular weight subunit of glutenin is between 80-130 kDa
  • the molecular weight of low molecular weight subunit of glutenin is between 10-70 kDa.
  • the band intensity of lanes 2 and 3 at 15-20 and 30-50 kDa is significantly deepened, so it is speculated that the modified sites are mainly concentrated in alcohol-soluble protein and low molecular weight glutenin subunits.
  • the solubility of wheat gluten protein modified by lactic acid is significantly improved in the pH range of 3.0 to 10.0.
  • the newly introduced lactic acid group causes the gluten protein structure to unfold, which enhances the interaction between the protein side chain and water, which is consistent with the results of the fluorescence spectrum below.
  • the introduction of lactic acid groups increases the negative charge and thus leads to improved solubility.
  • Oil holding capacity determination Accurately weigh 0.3g of wheat gluten protein in a 15mL centrifuge tube and record the total weight of the centrifuge tube and the sample, and add 2mL of sunflower oil to the centrifuge tube. After the sample is fully mixed with a vortex mixer, it is allowed to stand at room temperature for 60min, centrifuged at 4000r/min for 20min, the upper layer of liquid is carefully removed, and its mass is accurately weighed. The oil holding capacity of the sample is expressed as the mass of oil absorbed per gram of protein.
  • the effect of lactic acid modification on the water and oil holding capacity of wheat gluten protein is shown in Figure 3.
  • the reduction in water holding capacity may be due to the increase in solubility. It has been reported that highly water-soluble proteins have poor water absorption capacity.
  • the increase in oil holding capacity can be attributed to the fact that lactic acid modification unfolds the protein structure, and the exposed hydrophobic groups further form protein-oil complexes with oil.
  • A500 is the absorbance value measured at 500nm (0min); n is the dilution factor; C is the protein mass concentration; volume fraction of dispersed phase; L is the light path of the colorimetric cell; A0 and At are the absorbance values measured at 0 and 10 min; ⁇ T is 10 min.
  • Proteins with good emulsifying ability can effectively reduce surface tension, quickly absorb into the oil-water interface and continuously expand, combining the oil-water system.
  • the solubility of protein plays an important role in the emulsification process. Solubility is a prerequisite for protein to have good emulsifying properties. Only proteins with good solubility can easily diffuse to the oil-water interface. The good emulsifying activity and emulsifying stability of wheat gluten protein after lactic acidification can be attributed to the improvement of protein solubility.
  • the rheological properties of wheat gluten protein were determined using a DHR-3 rheometer with a 40 mm plate. The sample was mixed with deionized water and relaxed for 30 min. The edge of each sample was coated with silicone oil to prevent water loss. A strain of 1% was selected in the linear viscoelastic region, and the frequency range was set to 0.1 Hz to 10 Hz during the frequency scan. The equilibrium time of wheat gluten protein before the test was set to 300 s, and the test temperature was set to 25 °C.
  • the secondary structure of wheat gluten protein was determined by Fourier transform infrared method. 1 mg of wheat gluten protein and 100 mg of potassium bromide were quantitatively weighed and ground into powder, which was then pressed into a translucent uniform sheet under a press. The sample was placed in a Fourier transform infrared spectrometer for scanning and determination, with the atmosphere as the scanning background. The scanning frequency was 4000-400 cm -1 , the number of scans was 32, and the resolution was 4 cm -1 . Finally, Peakfit was used to analyze the secondary structure content of the sample.
  • lactic acid modification causes the ⁇ -turn of the secondary structure of wheat gluten to be partially converted into ⁇ -fold.
  • ⁇ -fold is a relatively stable secondary structure, while ⁇ -helix, ⁇ -turn and random coil are relatively flexible.
  • the significant increase in ⁇ -fold structure indicates that the stability of wheat gluten after lactic acid treatment is enhanced.
  • the increase in ⁇ -fold structure significantly changes the interaction between proteins, which is beneficial to the viscoelasticity of gluten, which is consistent with the previous rheological results.
  • the arrangement of peptide chains is reversed through the ⁇ -turn located on the surface of the protein, so that the proteins are closely connected.
  • the ⁇ -turn content of wheat gluten after lactic acid modification is significantly reduced, indicating that the protein molecules are further unfolded.
  • Amino acids such as tryptophan in gluten protein can emit fluorescence.
  • the measurement of protein fluorescence spectrum can reflect the amino acid microenvironment in the protein, and then infer the changes in the tertiary structure of the protein.
  • the maximum absorption fluorescence peak of wheat gluten protein after lactic acid modification is red-shifted, which indicates that the amino acid side chain group is exposed to a more hydrophilic environment. The reason is that lactic acid modification causes the unfolding of the wheat gluten protein structure.
  • the exposed amino acids did not increase the fluorescence intensity, which may be due to the fluorescence quenching of tryptophan.
  • UV-1800 UV spectrophotometer was used to record the UV absorption spectrum. Wheat gluten protein was dissolved in phosphate buffer (0.05 mol/L pH 7.0), and the protein concentration was measured using a BCA kit and the final sample concentration was adjusted to 0.4 mg/mL. The UV spectrophotometer had a scanning range of 200-400 nm, an optical path of 1 cm, and a response time of 0.1 s.
  • Ultraviolet absorption spectra are also commonly used to infer changes in the tertiary structure of proteins. As shown in Figure 8, the maximum absorption peak of wheat gluten protein after lactic acid treatment has a blue shift phenomenon, which indicates that the microenvironment of tryptophan and tyrosine in the wheat gluten protein after treatment has changed, and the maximum ultraviolet absorption peak after treatment has also increased significantly, further proving that lactic acid treatment has a great influence on the tertiary conformation of wheat gluten protein.
  • SDS-PAGE analysis The sample solution (1 mg/mL) was added to the protein loading buffer, mixed and heated at 100°C for 10 min.
  • the composition of the protein loading buffer was: 50 mM Tris-HCl, 0.1% bromophenol blue, 10% glycerol (v/v) and 2% SDS (w/v), pH 6.8. After the sample was cooled to room temperature, 10 ⁇ L was loaded on the gel and the gel was incubated at 120 V for 60 min. The gel was stained in Coomassie brilliant blue solution for 30 min and destained with 10% acetic acid and 5.5% ethanol at 37°C overnight. Lane 1 is untreated wheat gluten protein, lane 2 is lactic acid-treated wheat gluten protein, and lane 3 is sodium lactate-treated wheat gluten protein.
  • the effect of lactic acid treatment on the molecular weight distribution of gluten protein can be further analyzed by SDS-PAGE.
  • the 40-70 kDa band is significantly deepened, which may be due to the increase in molecular weight after the protein molecules bind to the lactic acid group.
  • Another possibility is that the protein after lactic acid treatment aggregates, resulting in the deepening of the band.

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Abstract

一种乳酸化改性小麦面筋蛋白的方法及得到的小麦面筋蛋白，将小麦面筋蛋白分散于乳酸溶液或含有乳酸钠的水溶液中，对蛋白进行乳酸化改性，改变面筋蛋白分子内部结构，改善溶解性及乳化性。

Description

一种乳酸化改性小麦面筋蛋白的方法 技术领域
本发明属于小麦蛋白加工技术领域，具体涉及一种乳酸化改性小麦面筋蛋白的方法。
背景技术
小麦是全球范围内种植面积最大、种植范围最广、栽培最早的粮食作物之一。在我国，小麦的种植面积仅次于水稻，在人们的食物结构和消费方面占有非常重要的地位。小麦蛋白是生产小麦淀粉的副产物，也称为谷朊粉或小麦面筋蛋白。在食品工业中，小麦面筋蛋白的应用主要体现在两个方面，一是作为蛋白质营养补充剂，二是作为面团增筋剂，改善面团的流变性质和产品品质。
根据小麦面筋蛋白溶解性的不同，小麦面筋蛋白可分为清蛋白、球蛋白、麦醇溶蛋白和麦谷蛋白4种蛋白，后2种蛋白是构成小麦面筋蛋白的主要成分。麦谷蛋白是天然存在的非均质的大分子聚合体蛋白质，其可分为2种麦谷蛋白亚基，分别是高分子量麦谷蛋白亚基(HMW-GS)和低分子量麦谷蛋白亚基(LMW-GS)，这2种亚基是由多肽键通过分子间二硫键连接而成。麦醇溶蛋白则是一种分子量较小的蛋白质，其可以与麦谷蛋白通过非共价键结合，嵌入到麦谷蛋白的三维网络结构中，从而会导致小麦面筋蛋白加工性能(溶解性、分散性等)极差。加之受基因、生长环境和加工工艺等因素影响，小麦面筋蛋白具有氨基酸序列多样性和整体结构复杂性的特点，这在很大程度上限制了小麦面筋蛋白的应用。因此，需要对小麦面筋蛋白进行合理的改性，增加其溶解度，改善其他功能性质。
现今有三种对小麦面筋蛋白改性的技术：物理法、酶法和化学法。物理改性法是指利用热、压力、射线、声波等物理作用定向改变蛋白分子间的聚集方式及其二级、三级结构。化学改性是指采用化学法引起蛋白的肽链部分断裂，或引入新的活性基团，从而使得蛋白结构、疏水基团、电荷特性等改变，最终导致其空间结构和功能性质产生变化的一种改性手段。酶法改性是指利用蛋白酶对蛋白的肽键或(和)酰胺键进行有限度地水解，或发生分子间的共价交联，从而定向改变其功能性质的一种改性方式。其中，化学改性由于相比于其他改性方法具有反应速度快、效率高、性能好、改性效果明显和易发生交联等优势，已成为蛋白质改性的主流技术。
发明内容
小麦面筋蛋白作为一种优质的植物蛋白，具有较高的营养价值，但由于其自身结构特性，小麦面筋蛋白的水溶性较差导致其功能特性发挥不理想，阻碍了小麦面筋蛋白在食品及其他 行业的应用。
本发明提供一种乳酸化改性小麦面筋蛋白的方法，能够改变面筋蛋白分子内部结构，使小麦面筋蛋白的溶解性及乳化性等功能特性得到改善。
所述方法步骤包括如下：
(1)制备蛋白质分散液：将小麦面筋蛋白分散于乳酸溶液或含有乳酸钠的水溶液中，得到小麦面筋蛋白悬浮液；
(2)改性：将小麦面筋蛋白悬浮液搅拌一定的时间，使得乳酸基团与面筋蛋白氨基酸充分接触从而连接到氨基酸上；如果步骤(1)选择的是乳酸钠，则需要在搅拌过程中用碱性溶液维持pH于碱性；
搅拌结束，得到乳酸化小麦面筋蛋白处理液；
(3)沉淀蛋白质：用pH调节剂调节乳酸化小麦面筋蛋白处理液的pH至中性使面筋蛋白沉淀，离心，收集沉淀蛋白并用水洗；通过水洗，除去残留的pH调节剂以及部分未能参与反应的游离乳酸和乳酸钠；
(4)透析：将水洗后的沉淀蛋白复溶，在去离子水中透析以去除盐离子得到小麦面筋蛋白乳酸化纯化液；所述透析是为了除去氢离子、钠离子以及游离乳酸和乳酸钠；
(5)真空冷冻干燥：对所述小麦面筋蛋白乳酸化纯化液进行真空冷冻干燥得到乳酸化改性小麦面筋蛋白。
进一步地，步骤(1)，小麦面筋蛋白质量为25g，乳酸溶液250mL(料液比为1：10)，所述乳酸溶液浓度为0.1mol/L；或者，小麦面筋蛋白质量为25g，乳酸钠的水溶液250mL，其中乳酸钠质量为5g，即乳酸钠与小麦面筋蛋白质量比为1：5。
进一步地，步骤(2)所述反应时间为3h，反应温度为25℃，搅拌转速100rpm。
进一步地，步骤(2)所述碱性溶液为2mol/LNaOH，pH为10.0。
进一步地，步骤(3)所述中性pH为7.0，所用pH调节剂为2mol/LNaOH，离心转速为4500r/min，离心时间为15min，水洗沉淀三次。
进一步地，步骤(4)所述透析时间为72h。
本发明将小麦面筋蛋白溶液经过乳酸和乳酸钠处理之后，小麦面筋蛋白发生了乳酸化修饰，溶解性和乳化特性都有显著提高。其中，当pH约为7时，面筋蛋白溶解性由原来的0.05mg/mL分别提升至0.19mg/mL、0.21mg/mL，乳化活力由原来的16.66m 2/g分别提升至19.27m 2/g、19.58m 2/g。
乳酸作为一种天然有机弱酸，可以用作pH调节剂，具有一定的防腐作用，是一种广泛 使用的食品添加剂。本发明利用乳酸化有效改善小麦面筋蛋白溶解性和乳化性能，有望拓展小麦面筋蛋白的应用范围，将其应用于蛋白饮料、可食性包装膜、植脂末、冰淇淋、植物肉等体系。
不同于其它有机酸通常通过脱酰胺的方式来提高蛋白的功能性质，本发明是将乳酸基团通过连接到蛋白质氨基酸侧链上来改变蛋白的结构进而提高功能性质，这一点从乳酸盐即乳酸钠也能对面筋蛋白起到改性作用可以看出来。
附图说明
图1.western blot分析。
图2.乳酸化小麦面筋蛋白溶解度。
图3.乳酸化小麦面筋蛋白持水(WHC)持油性(OAC)。
图4.乳酸化小麦面筋蛋白乳化能力。
图5.乳酸化小麦面筋蛋白流变特性。
图6.乳酸化小麦面筋蛋白红外图谱。
图7.乳酸化小麦面筋蛋白荧光图谱。
图8.乳酸化小麦面筋蛋白紫外图谱。
图9.乳酸化小麦面筋蛋白SDS-PAGE图谱。
图2至图8中，Raw为未处理的小麦面筋蛋白，Lactate为经乳酸处理的小麦面筋蛋白，Sodium lactate为经乳酸钠处理的小麦面筋蛋白。
具体实施方式
实施例1
(1)称取25g小麦面筋蛋白于250mL 0.1mol/L乳酸溶液中，固液比为1：10(m/v)，得到小麦面筋蛋白悬浮液；
(2)将小麦面筋蛋白悬浮液以100rpm转速搅拌3h，使得乳酸基团与面筋蛋白氨基酸充分反应从而连接到面筋蛋白氨基酸上；
(3)反应结束后用2mol/LNaOH调节反应体系pH至7.0，4500r/min离心15min后弃上清，水洗沉淀3次蛋白；
(4)透析：将水洗后的沉淀蛋白复溶，并在去离子水中透析72h得到小麦面筋蛋白乳酸化纯化液；
(5)对所述小麦面筋蛋白乳酸化纯化液进行真空冷冻干燥得到乳酸化改性小麦面筋蛋白，将干燥后的小麦面筋蛋白置于干燥皿中保存。
实施例2
(1)称取25g小麦面筋蛋白于250mL去离子水中，固液比为1：10(m/v)，并向其中加入5g乳酸钠得到小麦面筋蛋白悬浮液；
(2)将小麦面筋蛋白悬浮液以100rpm转速搅拌3h，使得乳酸基团与面筋蛋白氨基酸充分反应从而连接到面筋蛋白氨基酸上，搅拌过程中，用2mol/LNaOH调节反应体系pH维持在10.0；
(3)反应结束后用2mol/LNaOH调节反应体系pH至7.0，4500r/min离心15min后弃上清，水洗沉淀3次蛋白；
(4)透析：将水洗后的沉淀蛋白复溶，并在去离子水中透析72h得到小麦面筋蛋白乳酸化纯化液；
(5)对所述小麦面筋蛋白乳酸化纯化液进行真空冷冻干燥得到乳酸化改性小麦面筋蛋白，将干燥后的小麦面筋蛋白置于干燥皿中保存。
检测结果：
一、乳酸化修饰验证
制样：用BCA蛋白分析试剂测定实施例1、实施例2干燥皿中保存的蛋白的浓度后，将蛋白调整至同样浓度(1mg/mL)，并加入蛋白上样缓冲液中，混合后100℃加热10min使蛋白变性。蛋白上样缓冲液的组成为：50mM Tris-HCl，0.1％溴酚蓝，10％甘油(v/v)和2％SDS(w/v)，pH为6.8；电泳：配置10％的SDS聚丙烯酰胺凝胶，以每个样本10μL的蛋白量上样。120V恒压电泳，至前端溴酚蓝跑出胶底部电泳结束；转膜：PVDF膜用甲醇浸泡1min后，然后和胶一起放在转膜液中平衡5min，然后按照阳极-海绵-两层滤纸-PVDF膜-胶-两层滤纸-海绵-阴极的顺序安放好，放到湿转转膜仪中，将转膜仪包埋于冰中，300mA恒流转膜2.5h；封闭：取出PVDF膜，并用5％脱脂牛奶封闭1h；一抗孵育：封闭结束后，加入乳酸化泛抗体4℃水平摇床孵育过夜；洗膜：回收一抗，将膜用TBST洗3次，每次5-10min；二抗孵育：加入用5％脱脂牛奶稀释的相应二抗(1:3000)室温孵育1.5h；洗膜：去掉二抗，将膜用TBST洗3次，每次5-10min；ECL显色：利用数码化学发光成像仪进行拍照。
结果如附图1所示，泳道1为未处理的小麦面筋蛋白，泳道2为经乳酸处理的小麦面筋蛋白，泳道3为经乳酸钠处理的小麦面筋蛋白，western blot结果显示，泳道2和3在15～20，30～50kDa处的条带强度明显加深。Western blot是通过特异性抗体对凝胶电泳处理过的蛋白进行染色，并通过分析着色的位置和着色深度获得特定蛋白质的表达情况。具体地，在本实 验中使用的乳酸化泛抗体是一种能够识别氨基酸侧链连接乳酸基团的特定蛋白质的抗体，泳道1是未经处理的面筋蛋白所以不会有明显的条带，泳道2和3分别是经过乳酸和乳酸钠处理的小麦面筋蛋白，都产生了明显的条带，这说明乳酸和乳酸钠处理使得面筋蛋白发生了明显的乳酸化修饰现象。小麦醇溶蛋白分子量约为22-58kDa，麦谷蛋白的高分子亚基分子量在80-130kDa之间，麦谷蛋白的低分子亚基分子量在10-70kDa之间。从附图1可知，泳道2和3在15-20，30-50kDa处的条带强度明显加深，所以推测修饰的位点主要集中在醇溶蛋白和低分子量麦谷蛋白亚基。
二、乳酸化小麦面筋蛋白功能特性的测定
(1)溶解度测定：将实施例1、实施例2干燥皿中保存的蛋白样品溶解在去离子水中，用HCl和NaOH调节混合物的pH为3.0～10.0，室温下搅拌120min。经10000rpm离心15min后，使用BCA蛋白测定试剂盒测定上清液中的蛋白质含量。
如附图2结果所示，在pH 3.0～10.0范围内，经过乳酸化改性的小麦面筋蛋白溶解度有显著提升。首先，新引入的乳酸基团使得面筋蛋白结构展开，这使得蛋白质侧链和水之间的相互作用力增强，这与下文荧光光谱的结果一致。其次，可能是乳酸基团的引入使得负电荷增加从而导致溶解度提高。
(2)持水性测定：准确称取0.4g小麦面筋蛋白于15mL离心管中并记录离心管与样品总重，向离心管中加入4mL去离子水。样品用旋涡混合器充分混合后在室温下静置60min，经4000r/min离心20min，小心除去上层液体，准确称量其质量。样品的持水性以每克蛋白质吸收水的质量表示。持油性测定：准确称取0.3g小麦面筋蛋白于15mL离心管中并记录离心管与样品总重，向离心管中加入2mL葵花籽油。样品用旋涡混合器充分混合后在室温下静置60min，经4000r/min离心20min，小心除去上层液体，准确称量其质量。样品的持油性以每克蛋白质吸收油的质量表示。
乳酸化修饰对小麦面筋蛋白的持水持油性的影响如附图3所示，持水性的降低可能是由于溶解度的提高，有报道指出高水溶性蛋白吸水能力较差。持油性的提高可归因于乳酸化修饰使得蛋白质结构展开，暴露的疏水基团进一步与油形成蛋白质-油复合物。
(3)乳化能力测定：取20mL 10mg/mL的小麦面筋蛋白溶液(0.05mol/L磷酸盐缓冲液)，加入5mL葵花籽油，使用Ultra-Turrax18型乳化分散机于10000r/min下剪切1min。随后立即从距容器底端约5mm处吸取50μL液体，加入4.95mL 0.1％的SDS溶液，混合均匀，以0.1％SDS溶液作为空白对照于500nm处测吸光值A 0。在室温下静置10min后再次取样，依据A 0的测定步骤来测定吸光值A t。根据下列公式计算乳化能力：
Figure PCTCN2022135377-appb-000001
Figure PCTCN2022135377-appb-000002
A 500为500nm下测定的吸光值(0min)；n是稀释倍数；C是蛋白质质量浓度；
Figure PCTCN2022135377-appb-000003
分散相体积分数；L为比色池光径；A 0和A t为0和10min时测定的吸光值；ΔT为10min。
具有良好乳化能力的蛋白能有效降低表面张力，迅速吸收到油水界面并不断展开，将油水体系结合在一起。蛋白质的溶解度在乳化过程中起着重要作用，溶解度是蛋白质具有良好乳化特性的前提条件，只有溶解度好的蛋白质才容易扩散到油水界面。乳酸化之后的小麦面筋蛋白具有良好的乳化活力和乳化稳定性可以归功于蛋白质溶解度的提高。
(4)流变特性：采用DHR-3流变仪测定小麦面筋蛋白的流变性质，选用40mm平板。样品与去离子水混合后松弛30min，每个样品的边缘涂上硅油防止水分散失。在线性黏弹区选择1％的应变，频率扫描时频率范围设定为0.1Hz～10Hz。设定测试前小麦面筋蛋白平衡时间为300s，测试温度为25℃。
如附图5结果所示，所有的样品都表现出典型的黏弹性和类固体行为，即储能模量大于损耗模量。乳酸和乳酸钠处理均能显著提高小麦面筋蛋白的储存模量和损耗模量，说明乳酸化是提高面筋蛋白黏弹性的有效方法，原因是小麦面筋蛋白经乳酸化处理后溶解度有明显提高，使蛋白质水化更充分。
三、乳酸化小麦面筋蛋白结构特性的测定
(1)二级结构测定：使用傅里叶红外法测定小麦面筋蛋白的二级结构。定量称取1mg小麦面筋蛋白和100mg溴化钾研磨成粉末，置于压力机下压成半透明的均匀薄片。将样品置于傅里叶变换红外光谱仪中扫描测定，以大气为扫描背景。扫描频率为4000～400cm -1，扫描次数为32，分辨率为4cm -1，最后用Peakfit分析样品的二级结构含量。
表1.小麦面筋蛋白二级结构含量
Figure PCTCN2022135377-appb-000004
如表1所示，乳酸化修饰使得小麦面筋蛋白二级结构β-转角部分转化为β-折叠。β-折叠是一种相对稳定的二级结构，α-螺旋、β-转角和无规卷曲较为灵活，β-折叠结构的显著增加表明乳酸化处理后的小麦面筋蛋白稳定性增强。同时，β-折叠结构的增加显著改变了蛋白质与蛋 白质之间的相互作用，有利于面筋蛋白的粘弹性，这与之前的流变学结果一致。此外，肽链的排列通过位于蛋白质表面的β-转角来实现逆转，从而使得蛋白质紧密相连，乳酸化改性后的小麦面筋蛋白β-转角含量显著降低，表明蛋白分子进一步展开。
(2)内源性荧光光谱：使用F-7000荧光光谱仪测定小麦面筋蛋白的荧光光谱。小麦面筋蛋白溶于磷酸盐缓冲液(0.05mol/LpH为7.0)中，用BCA试剂盒测量蛋白浓度并将最终样品浓度调节为0.4mg/mL。荧光光谱仪参数设置如下：激发波长为280nm，发射波长范围为300-500nm，扫描速度为1200nm/min，反应时间为0.5s，电压为600V，激发和发射缝为5nm。
面筋蛋白中的氨基酸如色氨酸能够发射荧光，通过测定蛋白质荧光光谱能够反映蛋白中氨基酸微环境，进而推测蛋白质三级结构的变化。如附图7所示，乳酸化改性后的小麦面筋蛋白最大吸收荧光峰的红移，这表明氨基酸侧链基团暴露在更亲水的环境中，原因是乳酸化改性导致了小麦面筋蛋白结构的展开。但是，暴露的氨基酸并没有使得荧光强度升高，这可能是由于色氨酸发生了荧光淬灭。
(3)紫外光谱：采用UV-1800紫外分光光度计记录紫外吸收光谱。小麦面筋蛋白溶于磷酸盐缓冲液(0.05mol/LpH为7.0)中，用BCA试剂盒测量蛋白浓度并将最终样品浓度调节为0.4mg/mL。紫外分光光度计扫描范围为200～400nm，光程为1cm，响应时间为0.1s。
紫外吸收光谱通常也用来推测蛋白质三级结构的变化。如附图8所示，乳酸化处理之后的小麦面筋蛋白的最大吸收峰有蓝移现象，这说明处理之后的小麦面筋蛋白中的色氨酸和酪氨酸的微环境发生改变，且处理之后的最大紫外吸收峰值也显著升高，进一步证明乳酸化处理对小麦面筋蛋白三级构象有较大影响。
(5)SDS-PAGE分析：样品溶液(1mg/mL)加入蛋白上样缓冲液中，混合后100℃加热10min。蛋白上样缓冲液的组成为：50mM Tris-HCl，0.1％溴酚蓝，10％甘油(v/v)和2％SDS)w/v)，pH为6.8。样品冷却至室温后，胶中上样10μL，120V电压下60min。胶在考马斯亮蓝溶液中染色30min，用10％醋酸和5.5％乙醇脱色液37℃过夜脱色。泳道1为未处理的小麦面筋蛋白，泳道2为经乳酸处理的小麦面筋蛋白，泳道3为经乳酸钠处理的小麦面筋蛋白。
根据面筋蛋白二级结构和三级结构的变化，通过SDS-PAGE可以进一步分析乳酸化处理对面筋蛋白分子量分布的影响。如附图9所示，乳酸化后，40～70kDa的条带明显加深，可能是蛋白质分子结合乳酸基团后导致分子量的增加，另一种可能是乳酸化处理后的蛋白质发生聚集，从而导致条带加深。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

  1. 一种改性小麦面筋蛋白的方法，其特征在于，包括如下步骤：
    (1)制备蛋白质分散液：将小麦面筋蛋白分散于乳酸溶液或含有乳酸钠的水溶液中，得到小麦面筋蛋白悬浮液；
    (2)改性：搅拌小麦面筋蛋白悬浮液得到乳酸化小麦面筋蛋白处理液；
    (3)沉淀蛋白质：用pH调节剂调节乳酸化小麦面筋蛋白处理液的pH至中性使面筋蛋白沉淀，离心，收集沉淀蛋白并用水洗；
    (4)透析：将水洗后的沉淀蛋白复溶，在去离子水中透析得到小麦面筋蛋白乳酸化纯化液；
    (5)真空冷冻干燥：对所述小麦面筋蛋白乳酸化纯化液进行真空冷冻干燥得到乳酸化改性小麦面筋蛋白。
  2. 根据权利要求1所述的一种改性小麦面筋蛋白的方法，其特征在于，步骤(1)，小麦面筋蛋白与乳酸溶液的料液比为1g：10mL，所述乳酸溶液浓度为0.1mol/L。
  3. 根据权利要求1所述的一种改性小麦面筋蛋白的方法，其特征在于，步骤(1)，小麦面筋蛋白与乳酸钠溶液的料液比为1g：10mL，乳酸钠与小麦面筋蛋白质量比为1：5。
  4. 根据权利要求1所述的一种改性小麦面筋蛋白的方法，其特征在于，步骤(2)搅拌时间为3h，温度为25℃，搅拌转速100rpm。
  5. 根据权利要求1所述的一种改性小麦面筋蛋白的方法，其特征在于，步骤(1)采用乳酸钠水溶液时，步骤(2)搅拌过程中使用碱性溶液控制pH为碱性。
  6. 根据权利要求5所述的一种改性小麦面筋蛋白的方法，其特征在于，所述碱性溶液为2mol/L NaOH，pH为10.0。
  7. 根据权利要求1所述的一种改性小麦面筋蛋白的方法，其特征在于，步骤(3)所述中性的pH为7.0。
  8. 根据权利要求7所述的一种改性小麦面筋蛋白的方法，其特征在于，所用pH调节剂为2mol/L NaOH。
  9. 根据权利要求1所述的一种改性小麦面筋蛋白的方法，其特征在于，步骤(3)的离心转速为4500r/min，离心时间为15min。
  10. 根据权利要求1～9任一所述方法得到的改性小麦面筋蛋白。
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