阅读说明：本技术 一种膜法提取桑叶蛋白的工艺方法 (Technological method for extracting mulberry leaf protein by membrane method ) 是由 王璐莹 吴雅茹 雷建都 李赛赛 陈贵茹 于 2021-10-12 设计创作，主要内容包括：本发明公开了一种膜法提取桑叶蛋白工艺方法,具体实验步骤如下,将桑叶清洗干燥后粉碎,按照一定料液比,用去离子水浸泡并搅拌后离心,收集上清液,离心后的滤渣加水搅拌,离心弃去滤渣,得到的上清液与第一次离心后的上清液合并。上清液经过超滤膜进行超滤得到蛋白浓缩液后冷干,冷干后即可得到桑叶蛋白。本方法操作简便,原料分布广,成本低,绿色环保。所得蛋白截留率高达97％以上,提取率4％,所得的叶蛋白纯度约为30％。(The invention discloses a process method for extracting mulberry leaf protein by a membrane method, which comprises the following specific experimental steps of cleaning and drying mulberry leaves, crushing the mulberry leaves, soaking the mulberry leaves in deionized water according to a certain material-liquid ratio, stirring, centrifuging, collecting supernatant, adding water into the centrifuged filter residue, stirring, centrifuging, discarding the filter residue, and combining the obtained supernatant with the supernatant obtained after the first centrifugation. And (3) carrying out ultrafiltration on the supernatant through an ultrafiltration membrane to obtain a protein concentrated solution, then carrying out cold drying, and carrying out cold drying to obtain the mulberry leaf protein. The method is simple and convenient to operate, wide in raw material distribution, low in cost and environment-friendly. The retention rate of the obtained protein is as high as more than 97%, the extraction rate is 4%, and the purity of the obtained leaf protein is about 30%.)

1. A process for extracting mulberry leaf protein by a membrane method is characterized by comprising the following specific steps

(1) Washing impurities on the surface of fresh mulberry leaves, drying at 40-60 ℃, crushing and sieving with a 40-70 mesh sieve;

(2) according to a certain material-liquid ratio: mulberry leaf powder: deionized water 1: soaking and stirring the materials for 15-30 g/ml, performing ultrasonic treatment for 10-30 min, performing water bath, placing the materials in a refrigerator for refrigeration for 10-30 min at 20-50 ℃, centrifuging, and collecting centrifuged juice and waste residues respectively;

(3) adding deionized water into the waste residue, stirring, centrifuging again to obtain juice, discarding the waste residue, combining the juice obtained in the two steps, performing suction filtration by using gauze, and centrifuging 3000 r-5000 r of the filtrate obtained after suction filtration for 10 min; removing waste residues to obtain a crude extract of the protein;

(4) ultrafiltering the centrifuged juice to obtain folium Mori protein concentrate, collecting the filtrate, and measuring protein content;

(5) and (3) carrying out cold drying on the concentrated solution to obtain leaf protein powder, wherein the cold drying condition is that the condensation temperature is as follows: the vacuum degree is 24Pa at-57 to-30 ℃.

2. The method for extracting mulberry leaf protein by using the membrane method as claimed in claim, wherein the membrane for membrane separation is polyacrylonitrile membrane, polysulfone membrane and polyethersulfone membrane.

3. The method for extracting mulberry leaf protein by using a membrane method of claim, wherein the molecular weight cut-off of the ultrafiltration membrane in the ultrafiltration process is as follows: WMCO: 10000-300000.

Technical Field

The invention relates to the technical field of extracting protein from mulberry leaves, in particular to a technological method for extracting mulberry leaf protein by a membrane method.

Background

The mulberry has wide planting area and even distribution in China, a certain amount of mulberry leaves are exported to foreign countries every year, and in addition, a large amount of mulberry leaves cause waste. The leaf protein plant leaf protein is one of the largest green renewable protein resources in the world, contains trace elements required by a human body, does not contain cholesterol, and has great economic value. The mulberry leaves are rich in nutrition, have high protein content, especially have the highest content of glutamic acid and aspartic acid, and can be used as important protein supplement for human diet. In conclusion, the mulberry leaf protein is a plant protein resource with great development value, and can be widely applied along with the continuous improvement of the production process and the continuous improvement of the extraction rate of the mulberry leaf protein, so that a road for a mulberry leaf high-purity protein process which is simple to operate and environment-friendly is explored, and great economic benefits and social benefits are achieved.

The existing method for extracting the mulberry leaf protein comprises a heating method, an acid-base precipitation method, an acid-heating method, an alkali-heating method, a salting-out method, an organic solvent method and the like, and the methods have a plurality of defects. The yield of the protein by the direct heating method is not high, and the protein is easy to denature due to overhigh temperature; the pH value is not easy to control in the acid-base precipitation operation process, and waste liquid generated by using a large amount of acid or base is not environment-friendly; the salting-out method generally needs ammonium sulfate removal treatment by adding ammonium sulfate, so that the extraction process of the mulberry leaf protein becomes more complicated; the organic solvent method uses a large amount of organic solvent, and has problems such as difficulty in recovery and residue.

Disclosure of Invention

The invention aims to solve the problems of mulberry leaf resource waste and the defects of the technology. We invent a method for extracting mulberry leaf protein by a membrane method to solve the problems. The method has the advantages of low cost, simple operation, low energy consumption, environment-friendly and nuisanceless production process, high purity and green and nontoxic products. The invention adopts the following process method to achieve the aim.

(1) Washing impurities on the surface of fresh mulberry leaves, drying at 60 ℃, crushing and sieving with a 40-60-mesh sieve;

(2) according to a certain material-liquid ratio: mulberry leaf powder: deionized water 1: soaking and stirring the materials for 15-30 g/ml, performing ultrasonic treatment for 10-30 min, performing water bath, placing the materials in a refrigerator for refrigeration for 10-30 min at 20-40 ℃, centrifuging, and collecting centrifuged juice and waste residues respectively;

(3) adding deionized water into the waste residue, stirring, centrifuging again to obtain juice, discarding the waste residue, combining the juice obtained in the two steps, passing the juice through a gauze, centrifuging the obtained juice at a high speed of 4000-5000 r for 10 mm, and removing the waste residue to obtain a crude extract of the protein.

(4) Performing membrane separation on the centrifuged juice to obtain a mulberry leaf protein concentrated solution;

(5) and (3) carrying out cold drying on the concentrated solution to obtain a leaf protein solid, wherein the cold drying condition is that the condensation temperature is as follows: the vacuum degree is 24Pa at-57 to-30 ℃.

Wherein, the membrane material for membrane separation in the process (4) can be a polyacrylonitrile membrane, a polysulfone membrane and a polyethersulfone membrane, and is preferably a polyacrylonitrile membrane.

Wherein, in the process (4), the molecular weight cut-off of the ultrafiltration membrane in the ultrafiltration process is as follows: WMCO: 10000-300000, preferably polyacrylonitrile membrane (WMCO: 100000).

The invention has the beneficial effects that: the process method has the biggest characteristics of low cost, simple operation, low energy consumption, environment-friendly and pollution-free production process, suitability for large-scale production, high yield and green and nontoxic products. The mulberry leaf powder is added with water twice, stirred and centrifuged to increase the dissolution rate of protein, and the crude extract is centrifuged for 10min at 5000r and then ultrafiltered by an ultrafiltration membrane, so that suspended particles can be better removed to prevent the membrane from being blocked, and the purity of a product obtained by intercepting through the ultrafiltration membrane can be improved.

Drawings

FIG. 1 is a process flow chart of the mulberry leaf protein extraction method by a membrane method.

Detailed Description

The present invention will be further described with reference to the following specific operation examples, it should be noted that the examples are only illustrative of the extraction process of mulberry leaf protein of the present invention, and do not represent the whole patent of the present invention, and are not to be understood as limiting the scope of the present invention.

Preprocessing mulberry leaves: respectively weighing 3 parts of 4g of mulberry leaf powder according to a material-liquid ratio of 1: adding deionized water 20(g/ml), mixing, stirring, ultrasonic treating for 15min, stirring at 40 deg.C and 400r for 20min in water bath, cooling, centrifuging at 3000r, collecting juice, adding water 30ml into waste residue, mixing, stirring, filtering with gauze, mixing the filtrate with the juice obtained at the last time, centrifuging at 5000r, discardingMixing the three obtained juices, reserving 4ml, and measuring protein concentration by Coomassie brilliant blue method and recording as C_{Crude extract}. The calculation formulas of the protein retention rate, the protein purity and the protein extraction rate are as follows:

EXAMPLE 1 Membrane extraction of Mulberry leaf protein (Polyacrylonitrile membrane)

Ultrafiltering 50ml of the pretreated juice with polyacrylonitrile ultrafiltration membrane, pressurizing with nitrogen in ultrafiltration cup at 0.6MPa, stirring at room temperature at 1000r, collecting 33ml of filtrate, and measuring protein concentration C by Coomassie brilliant blue method_FiltrateAnd carrying out cold drying after intercepting to obtain a protein concentrated solution, wherein the condensation temperature is-44 ℃, and the vacuum degree is 24 Pa. The mass M of the crude protein obtained after the cold drying for 48 hours_{Cold drying}The protein retention rate, protein purity and protein extraction rate are shown in table 1.

Example 2 Membrane method for extracting Mulberry leaf protein (polysulfone membrane)

Taking 50ml of the pretreated juice, adopting a polysulfone ultrafiltration membrane, pressurizing the ultrafiltration cup with nitrogen at 0.6MPa, stirring at room temperature of 1000r, collecting 35ml of filtrate, and measuring the protein concentration C by adopting a Coomassie brilliant blue method_FiltrateAnd carrying out cold drying after intercepting to obtain a protein concentrated solution, wherein the condensation temperature is-44 ℃, and the vacuum degree is 24 Pa. The mass M of the crude protein obtained after the cold drying for 48 hours_{Cold drying}The protein retention rate, protein purity and protein extraction rate are shown in table 1.

EXAMPLE 3 Membrane extraction of Mulberry leaf protein (polyethersulfone Membrane)

Collecting the above pretreated juice, subjecting 50ml of the juice to polysulfone ultrafiltration membrane, pressurizing with nitrogen in ultrafiltration cup at 0.6MPa, stirring at room temperature for 1000r, and collecting34ml of filtrate, and protein concentration C was measured by Coomassie brilliant blue method_FiltrateAnd carrying out cold drying after intercepting to obtain a protein concentrated solution, wherein the condensation temperature is-44 ℃, and the vacuum degree is 24 Pa. The mass M of the crude protein obtained after the cold drying for 48 hours_{Cold drying}The protein retention rate, protein purity and protein extraction rate are shown in table 1.

TABLE 1