Vegetable protein isproteinFromBotany, comprehensive nutrition, andanimal protein Similar, easy to be digested and absorbed by the human body, with a variety of physiological health care functions.[1]
Plant protein is an important source of human dietary protein.Cereals generally contain 6% - 10% protein, but it containsessential amino acid Incomplete category.Potatoes contain 2% - 3% protein.Some nuts, such as peanuts, walnuts, almonds and lotus seeds, contain high protein (15% - 30%).The protein content of legumes, such as some dried beans, can be as high as about 40%.Especially soybean is more prominent in beans.It is not only high in protein content, but also high in quality. It is a good source of human food protein.Vegetable protein is the main source of protein in vegetarian diet, which can be used to make meat like products similar to corresponding animal food in shape, taste and taste.Vegetarian foodIncomplete protein, will occurNutritional deficiency, must eat concurrentlysoybean protein Quality.[2]
Plant protein is mainly from rice, flourpeas and beans, butRice NoodlesLeguminousproteinThe nutritional value is different.Lack of protein from rice and flourLysine(an essential amino acid), so itsAmino acid scoreLow, only 0.3~0.5, thisProteoidThe degree of absorption and utilization by the human body will also be poor.[2]
classification
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In terms of nutrition, plant proteins can be roughly divided into two categories: one isComplete protein, such as soybean protein;The second is incomplete protein. The vast majority of plant proteins belong to this category.[2]All proteins containing various essential amino acids and with appropriate proportion are called complete proteins;A protein lacking any essential amino acid is called incomplete protein.Incomplete protein alone cannot maintain the nitrogen balance and growth and development requirements of the body.[3]
The nutritional classification of protein is as follows[3]
Nutritional classification of proteins
Complete protein
Casein, lactalbumin, albumin, ovalbumin, soy protein
Incomplete protein
Glue protein, soybean globulin, corn protein
Glue protein, barley protein, kidney bean protein
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Legumes, wheat (flour) and rice generally contain more plant protein (soybean seeds contain up to 40% protein).But the protein nutritional value of rice, flour and beans is different.[2]
Cereals generally contain 6% - 10% protein, which is not high, but it is still the main source of protein because it is people's staple food.Beans are rich in protein, especially the protein content of soybeans is as high as 36% - 40%. The amino acid composition is also relatively reasonable, and the utilization rate in the body is high. It is a very good protein in plant protein.Some nuts, such as peanuts, walnuts, almonds and lotus seeds, contain high protein (15% - 30%).[2]
extract
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The most common methods to separate the target protein from non protein impurities and non target protein by using the difference of plant protein areAlkali dissolving and acid precipitation methodEnzyme extraction, organic solvent extraction, salt solution extraction, reverse micelle extraction, etc.[4]
Alkali dissolving and acid precipitation method
Alkali dissolution and acid precipitation is the most commonly used method for extracting plant protein. The principle is that plant protein is easily soluble in alkaline environment and precipitates under acidic isoelectric point conditions.The advantages of alkali solution and acid precipitation method are high protein extraction rate and purity, easy operation and low cost;The disadvantage is that too high concentration of lye will cause Maillard reaction of the extracted protein and affect the nutritional characteristics of the protein.Yuan Shihan[5]The best extraction rate of yellow rice protein was 86.93% through orthogonal test optimization.Xu Jiangbo[6]The flaxseed protein was extracted from flaxseed by alkali solution and acid precipitationSingle factor experimentBased on the optimization of response surface methodology, the optimum extraction rate of flaxseed protein was 79.26%, and the purity was 92.34%.
Enzyme extraction
The advantages of enzymatic extraction of plant protein are high efficiency, mild reaction conditions, safe operation, and no harmful substances;The disadvantage is that the requirements for the operating environment are relatively strict, and compared with the alkaline extraction of protein, the steps of enzyme killing need to be added[7]。Tan Ping and others studiedAcid protease、neutral proteaseandAlkaline proteaseThe results showed that alkaline protease had the best extraction effect on tartary buckwheat protein.[8]
Organic solvent extraction method
The organic solvent extraction method is mainly aimed at proteins that are insoluble in water, acid, alkali and dilute salt solutions. Because such proteins are firmly bound to lipids, they are only soluble in organic solvents with strong lipophilicity such as ethanol and acetone, and must be operated at low temperature to preventProtein denaturation。[9]Jiang Fujia et al. used ethanol extraction method to extractGliadinOn the basis of single factor experiment and response surface optimization, the optimal extraction conditions of gliadin from brewer's grains were determined as follows: ethanol concentration 81%, solid-liquid ratio 1 ∶ 21 (g/mL), extraction temperature 48 ℃, extraction time 50 min. Under these conditions, the extraction rate of gliadin was 7.8%.[10]
Salt solution extraction method
Salt solubility can be defined as the phenomenon that a small amount of neutral salts such as ammonium sulfate will increase the charge on the surface of protein molecules, promote the interaction between protein molecules and water molecules, and then increase the solubility of protein molecules in aqueous solutions.The advantage of salt solution method is that it maintains the natural conformation of protein and is not easy to denature, but the extraction rate and purity are low.Bai Zhengchen et al. used broad bean powder as raw material, added 2.5% NaCl solution, and obtained the supernatant after magnetic stirring and freeze centrifugation, which is broad bean salt soluble protein.[11]
Reverse micellar extraction
Reverse micellar extraction technologyIt is a new technology for protein extraction.Reverse micelle refers to that the hydrophilic end of the surfactant molecule faces inward and the hydrophobic end faces outward, forming a "pool" with the ability to solubilize proteins. Its essence isThermodynamic stabilityAnd optical transparency.Liu Haiyuan mainly studied the optimal process conditions, influencing factors and kinetic process of extracting soybean protein with AOT, sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB) three reverse micellar systems, and compared with the soybean protein extracted by alkali solution and acid precipitation. The results showed that the functional properties of soybean protein extracted by reverse micellar system were more advantageous,The reverse micellar extraction technology has little effect on the structural characteristics of soybean protein, and has a certain protective effect.[12]
Other extraction technologies
The above mentioned protein extraction technologies are relatively common. Other extraction technologies are mainly auxiliary extraction methods and compound extraction methods, with the purpose of increasing the extraction rate of protein.Yang Xijuan[13]The ultrasonic assisted method was used to extract highland barley protein. Through orthogonal test, the optimal extraction process conditions of highland barley protein were determined as pH10.5, the ratio of material to liquid 1 ∶ 22 (g/mL), the ultrasonic power 550W, and the extraction time 20min. Under this condition, the optimal extraction rate of highland barley protein was 93.15%, and the purity was 78.67%.The ultrasonic assisted extraction of highland barley protein has good functional properties and can be used in meat products and baked goods.Ma Qian et al. extracted corn yellow flour by microwave assisted methodZeinThrough the orthogonal test, it was found that the extraction rate of zein was 25.73% under the conditions of ethanol concentration 80%, material liquid ratio 1 ∶ 14 (g/mL), particle size 20 mesh, microwave power 420W, and treatment time 360s, and it could be well used in the egg coating preservation process.[14]
Separation and purification
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Utilize the difference in solubility
Different proteins have different solubility, and the solubility of protein molecules in water mainly depends on the thickness of the hydration layer and the number of charges on the surface of protein molecules.[15]Different protein molecules, due to different types, quantities and arrangements of polar groups on their molecular surfaces, have different thickness of hydration layer and different number of charges, resulting in different solubility of proteins.In addition, external environmental factors such as pH value of solution, ionic strength, temperature and dielectric constant can affect the solubility of protein.Therefore, proper change of external factors can reduce the solubility of protein in the mixed solution of protein and improve the separation effect.
Salting out method
The salting out method is to add a high concentration of neutral salt into the protein solution. The interaction of salt ions and water molecules can reduce the activity of water molecules and the degree of protein hydration, and ultimately lead to the reduction of protein solubility, forming a process of precipitation.The commonly used salting out agent is ammonium sulfate, because of its strong salting out ability, it will not make proteinbiological activityLoss.The advantage of salting out method is that it is easy to operate, can remove more foreign proteins, can protect volatile proteins, and has a certain concentration effect;The disadvantages are poor resolution, low purification times, and the need for dialysis desalination.Chen Xiuqing[16]The effects of acid heating, alkali heating, salting out and organic solvent extraction of protein from the leaves of wedelia chinensis were compared. The results showed that salting out was the most suitable method for extracting protein from the leaves of wedelia chinensis, with high protein extraction rate and good activity.
Isoelectric point precipitation method
Isoelectric point precipitation methodPrinciple: protein isAmphoteric electrolyte, its solubility is related to the number of net charges, when the pH value of the solution is equal toProtein isoelectric pointThe solubility of protein is the lowest and precipitate is formed.Different proteins have different isoelectric points. Using the difference of protein isoelectric points, adjust the pH value of the solution to be equal to the isoelectric point of the target protein to precipitate the target protein. The desired target protein can be obtained by centrifugation.Zhu Xiuling[17]The results showed that the foam stability of sesame protein prepared by isoelectric precipitation was better than that of sesame protein prepared by ultrafiltration.
Organic solvent precipitation method
The principle of organic solvent precipitation method: on the one hand, polar organic solvents can reduce the dielectric constant of water, reduce the degree of hydration of protein molecules, and promote the aggregation and precipitation of protein molecules;On the other hand, polar organic solvents can destroy the hydration layer on the surface of protein molecules and precipitate protein molecules.Ethanol and acetone are the two most commonly used organic solvents and must be used at low temperatures.[4]
Take advantage of size differences
Protein isMacromolecular substancesIt can be considered to use dialysis, ultrafiltration and other methods to remove small molecules, and gel filtration chromatography can also be used to separate the target protein and impurity protein according to the molecular weight of the protein.
Dialysis
Dialysis is a method of separating protein from small molecular impurities by using the interception effect of semipermeable membrane. Protein macromolecules cannot pass through the semipermeable membrane while small molecular impurities can pass through the semipermeable membrane.The protein solution is packed inIntercepted molecular weightThe dialysis bag with semi permeable membrane is placed in distilled water for dialysis. In order to shorten the dialysis time, the water can be changed frequently. After a certain period of time, small molecular impurities are removed through the semi permeable membrane, and the target protein remains in the dialysis bag for preservation.[4]
Ultrafiltration
The ultrafiltration method is mainly used to separate and purify the target protein according to the pore size of the ultrafiltration membrane, that is, the protein molecules larger than the pore size of the ultrafiltration membrane are intercepted and preserved, and the impurity molecules smaller than the pore size of the ultrafiltration membrane are not intercepted and removed.Yang Guolong[18]Production by ultrafiltrationSoy protein concentrateIt is mainly through the flat polyethersulfone ultrafiltration membrane to remove small molecular impurities such as soluble polysaccharides during the production of soybean protein concentrate, so as to obtain soybean protein concentrate with protein content more than 72%.
Gel filtration chromatography
Gel filtration chromatographyAlso calledMolecular sieve chromatographyMolecular exclusion chromatography is one of the most effective methods for separating protein mixtures according to molecular size.When the mixture of protein molecules with different sizes flows through the gel chromatography column, the protein molecules larger than the gel mesh cannot enter the mesh, but move downward along with the buffer outside the mesh, and flow out of the column first;The protein molecules smaller than the gel mesh can enter the mesh smoothly, enter and exit the mesh with different pore sizes according to the molecular weight of the protein, and then flow out of the column with the buffer solution.The protein molecules with larger molecular weight move in a short way and flow out first. The protein molecules with smaller molecular weight move in a long way and flow out finally, so that the proteins with different molecular weights can be separated and purified.Zou Cunyuan and others used neutral protease to enzymatically hydrolyze soybean protein, which was determined by orthogonal testSoybean protein peptideAnd adoptGel filtration chromatographySeparate and collect small peptides with molecular weight less than 1200Da.[19]
Utilize charge difference
Protein molecules contain carboxyl groups, amino groups and other dissociatable groups. Due to the different structures of different protein molecules, the composition of their ionized groups and their exposure on the molecular surface are different, so the properties and net charges of different protein molecules are different.According to the charge difference of protein molecules, electrophoresis andIon exchange chromatographyPerform separation and purification.[4]
Electrophoretic method
In a solution with a certain pH value, protein molecules can be dissociated into molecules with positive and negative charges. In a DC electric field, molecules with positive charges move to the negative pole, and molecules with negative charges move to the positive pole.Different protein molecules are separated from each other due to their different net charge weight and molecular weight.Common electrophoresispolyacrylamide gel electrophoresis, two-dimensional electrophoresisIsoelectric focusing electrophoresisEtc.Magdalena Montowska[20]In order to find out the difference of six meat proteins and obtain meat proteins with heat stability and high quality, two-dimensional electrophoresis was used to analyze the protein expression profile of raw meat. The results showed that two-dimensional electrophoresis could identify regulatory proteins, metabolic enzymes, someMyofibrillar proteinandPlasma proteinAnd observe theprotein structureAnd take the difference as the symbol of meat products.
Ion exchange chromatography
Ion exchange chromatographyIt is a technology to separate charged molecules such as proteins, polypeptides and amino acids with different charged ions according to the charge difference.When the pH value of the solution is greater than the isoelectric point of the protein, the protein molecule is negatively charged and can be combined with the anion exchanger;When the pH value of the solution is less than the isoelectric point of the protein, the protein molecule is positively charged and can be bound to the cation exchanger.Deng Xue[21]The water-soluble protein of camellia was extracted and purified by ammonium sulfate precipitation and DEAE agarose rapid ion exchange chromatography. Through the determination of the ability to adsorb cholate in vitro, the water-soluble protein of camellia has certain blood lipid lowering and health care functions.
Difference in utilization adsorption capacity
Adsorption chromatographyIt is to separate the target by using the difference in the adsorption capacity of the adsorbent to different substances.Generally speaking, protein adsorption on non-polar adsorbents such as activated carbon mainly depends on van der Waals force and hydrophobic effect, while adsorption on polar adsorbents mainly depends on ionic bond and hydrogen bond.[22]Hydrophobic adsorption chromatography has been widely used in the separation and purification of proteins and biomacromolecules.Yao Shanjing et al. studied a new biological separation method, namely mixed mode adsorption chromatography technology, which is mainly electrostatic and hydrophobic interaction. The results show that the separation of target substances can be achieved under the conditions of low salt and high salt, and the separation efficiency is improved, which has certain application value.[23]
Functional characteristics
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Protein has an important impact on the sensory quality of food, mainly on the physical properties of food ingredients during processing and storage.In general, the decisionProtein functional propertiesThe physical and chemical properties of protein include protein structure, size, shape, amino acid composition, net charge distribution, ratio of hydrophobic group to hydrophilic group, interaction force between protein molecules, etc. However, it is difficult to describe which specific protein physical and chemical characteristics are related to the functional properties of food.The sensory quality characteristics of food are obtained through the complex interaction between various functional ingredients.According to the functional characteristics of proteins, the functional characteristics of proteins can be divided into three categories: protein water interaction, such as dispersibility, solubility, viscosity, water holding capacity, etc;Protein protein interaction, such as precipitation and gelation;Protein interface properties, such as emulsification, foaming, oil retention, etc.[24]
The functional properties of protein in the food system are shown in the table below[4]
