阅读说明：本技术 一种利用山核桃饼粕制备山核桃蛋白的方法 (Method for preparing hickory protein by using hickory cake meal ) 是由 何志平 吴峰华 刘兴泉 郭宜 何浙华 余琳 于 2021-08-27 设计创作，主要内容包括：本发明公开一种利用山核桃饼粕制备山核桃蛋白的方法,包括山核桃饼粕预处理、酶辅助碱提取、糖化酶酶解纯化、乙醇水溶液洗脱除杂、蛋白质喷雾干燥。本发明利用物理方法对山核桃饼粕进行脱皮、脱壳,操作简单快捷,绿色环保,同时减少了山核桃饼粕蛋白损失。本发明利用酶辅助碱提取山核桃蛋白,不但可以提高碱提液中蛋白质的提取率还可以同时分离山核桃饼粕中残存的油脂。本发明利用糖化酶酶解结合乙醇溶液洗脱工艺纯化提取的山核桃蛋白,步骤简单,纯化周期短,成本低。本发明利用喷雾干燥方法将所得蛋白质进行干燥处理,延长蛋白产品的保存期,且与冷冻干燥相比干燥速率快,更适合工业化生产。(The invention discloses a method for preparing hickory protein by using hickory cake, which comprises the steps of hickory cake pretreatment, enzyme-assisted alkali extraction, saccharification enzyme enzymolysis purification, ethanol aqueous solution elution and impurity removal, and protein spray drying. The method for peeling the pecan cakes utilizes a physical method to peel and unshelling, is simple and rapid to operate, is green and environment-friendly, and reduces the protein loss of the pecan cakes. The method utilizes enzyme-assisted alkali to extract the hickory protein, can improve the extraction rate of the protein in the alkali extract and can separate the residual oil in the hickory cake pulp. The invention utilizes the saccharification enzymatic hydrolysis and ethanol solution elution processes to purify the extracted hickory nut protein, and has simple steps, short purification period and low cost. The invention utilizes the spray drying method to dry the obtained protein, prolongs the storage life of the protein product, has higher drying rate compared with freeze drying, and is more suitable for industrial production.)

1. A method for preparing hickory protein by using hickory cake pulp is characterized by comprising the following steps:

step one, pretreatment of hickory cake dregs

Drying the hickory cake, crushing the dried hickory cake for 4-7 s at 30000-35000 r/min by using a crusher, sieving the crushed material with a 40-60 mesh sieve, discarding undersize, crushing oversize at 30000-35000 r/min for 35-40 s, sieving the powder with a 80-100 mesh sieve, discarding oversize, and taking undersize as a raw material for preparing hickory protein;

step two, enzyme-assisted alkali extraction

Adding water into the hickory nut protein raw material obtained in the first step according to the ratio of 1g to 15ml to 1g to 25ml, adjusting the pH of the solution to 10.0-10.5, adding alkaline protease according to the amount of 1000-1500U/g based on the mass of the hickory nut protein raw material, stirring and extracting at constant temperature of 55-60 ℃ for 1-6 h, wherein the pH of the solution is adjusted to 10.0-10.5 again at 0.5-1 h, centrifuging after extraction is finished, removing an upper emulsion layer, and collecting a middle water layer; collecting the lower-layer precipitated residue, adding water into the precipitated residue according to the material-liquid ratio of 1g:15 ml-1 g:25ml based on the mass of the hickory nut protein raw material, adjusting the pH of the solution to 10.0-10.5, stirring and washing the residue at constant temperature of 55-60 ℃ for 15-30 min, centrifuging after washing the residue, removing the upper-layer emulsion layer and the lower-layer precipitate, and collecting a middle water layer; mixing the intermediate water layers obtained by the first and second centrifugations to obtain a hickory nut protein extracting solution;

step three, enzymatic hydrolysis and purification of saccharification enzyme

Adjusting the pH value of the hickory nut protein extracting solution obtained in the second step to 4.0-4.5, taking the quality of the hickory nut protein raw material as a reference, adding saccharifying enzyme into the hickory nut protein extracting solution according to the amount of 100-130U/g, stirring and performing enzymolysis for 2-6 hours at constant temperature of 55-65 ℃, after the enzymolysis is completed, inactivating the enzyme, cooling to room temperature, adjusting the pH value of an enzymolysis solution to 3.5-4.0, standing for 60-120 min, centrifuging, removing a supernatant, collecting a precipitate, and obtaining a hickory nut extracted protein crude extract;

step four, eluting and removing impurities by using ethanol water solution

Adding 75-85% v/v ethanol aqueous solution into the crude extract of the extracted protein of the hickory nut obtained in the step three according to the proportion of 1g:12 ml-1 g:15ml by taking the mass of the hickory nut protein raw material as a reference, stirring and eluting for 45-90 min at the constant temperature of 50-60 ℃, centrifuging after the elution is finished, discarding the supernatant, collecting the lower-layer precipitate, adding 5-8 times of volume of water to redissolve the precipitate, and adjusting the pH of the solution to 7.0 to obtain a hickory nut protein solution;

step five, protein spray drying

And D, spray drying the hickory protein solution obtained in the step four to obtain hickory protein powder, namely the hickory protein.

2. The method for preparing the hickory nut protein by using the hickory nut cake as claimed in claim 1, wherein the first step is to bake the hickory nut cake in an oven at 40-50 ℃ for 12-24 h.

3. The method for preparing the hickory nut protein by using the hickory nut cake as claimed in claim 1, wherein the pH is adjusted by using 35-45% w/v sodium hydroxide solution in the second step, the pH is adjusted by using 8-12% v/v hydrochloric acid solution in the third step, and the pH is adjusted by using 35-45% w/v sodium hydroxide solution in the fourth step.

4. The method for preparing the hickory nut protein by using the hickory nut cake as claimed in claim 1, wherein after the second extraction step, the second extraction step is performed for 15-20 min under 3800-4000 r/min, and after the second residue washing step, the second extraction step is performed for 15-20 min under 3800-4000 r/min; standing for 60-120 min, and centrifuging for 5-10 min at 1500-2000 r/min; and step four, after the elution is finished, centrifuging for 5-10 min at 1500-2000 r/min.

5. The method for preparing the hickory nut protein by using the hickory nut cake as claimed in claim 1, wherein the enzyme is inactivated for 10-15 min at 85-90 ℃.

6. The method for preparing the hickory nut protein by using the hickory nut cake as claimed in claim 1, wherein in the fifth step, the inlet temperature of the spray dryer is adjusted to 150-180 ℃, the rotation speed of the peristaltic pump is adjusted to 2-5 r/min, and the hickory nut protein solution obtained in the fourth step is spray-dried under the condition.

7. Use of hickory nut protein produced according to any one of claims 1 to 6 as a starting material for the production of active peptides.

8. Use of a hickory nut protein prepared according to any one of claims 1 to 6 as a vegetable protein additive for food processing.

9. Use of a hickory nut protein prepared according to any one of claims 1 to 6 as a functional food ingredient.

10. Use of hickory nut protein produced according to any one of claims 1 to 6 as supplementary feed for animal husbandry protein.

Technical Field

The invention relates to the technical field of agricultural and sideline product processing, and particularly relates to a method for preparing hickory nut protein by using hickory nut cake meal.

Background

Hickory nuts (Carya cathayensis Sarg.) are plants of the genus hickory of the family juglandaceae, and are distributed in Zhejiang and Anhui provinces in China, and the main production area is in the area around Tianmu mountain at the boundary of Zhejiang Anhui. The hickory kernel in the hickory nut is an edible part, has relatively richer nutritional value and highest oil content accounting for 65.8-74.1% of the dry mass of the hickory kernel, has 7.8-9.6% of protein content, and also contains rich mineral elements and vitamins.

Because the hickory is a high-quality oil crop, the deep processing and utilization of the hickory by people are still concentrated on the hickory oil at present, and the preparation method of the hickory oil on the market is mostly a squeezing method at present, so a large amount of hickory cake byproducts are also generated while the hickory oil is produced. However, the pecan cake still contains a large amount of organic matters, which can cause environmental pollution if not disposed properly. At present, the utilization of the hickory cake pulp is mostly directly used as feed or fertilizer, but the hickory is not completely shelled before oil extraction, so that a large amount of hickory shells remain in the hickory cake pulp, and the difficulty in utilization of the hickory cake pulp is increased. In fact, the pecan cake contains a large amount of high-quality protein, and how to more efficiently utilize the protein in the pecan cake is one of the problems to be solved in the current pecan industry chain.

Patent CN 104818312A discloses a preparation method of walnut active polypeptide extract, which comprises the steps of soaking walnut kernels in alkali liquor, peeling, crushing, preparing walnut protein by an alkali extraction and acid precipitation method, adding protease into the protein for enzymolysis, separating the enzymolysis liquid by gel chromatography, and freeze-drying the walnut kernels with the molecular weight of 3000-1000Da as polypeptide powder. The method needs a large amount of alkali liquor in the step of peeling the walnut kernels, and needs long-time soaking, so that a large amount of alkali waste liquor is generated, and walnut protein resources can be lost.

Patent CN 110643661A discloses a method for rapidly separating and purifying walnut protein from walnut, which comprises the steps of crushing walnut raw materials into particles by using balls to obtain walnut powder, degreasing by using petroleum ether, and volatilizing organic solvent; adopting an alcohol solution as an extraction solvent, and adding proteolytic enzyme for enzymolysis; removing the alcohol solvent by rotary evaporation, extracting protein in the walnut by adopting a supercritical fluid, and adsorbing the extract by using macroporous resin; and (3) eluting the filtrate adsorbed on the macroporous resin by adopting alcoholic solutions with different concentrations to obtain an eluent, combining the eluents, concentrating under reduced pressure, and freeze-drying to obtain the walnut protein solid. The method uses petroleum ether to degrease walnut powder, so that organic solvent residue is easily caused, and the method is not beneficial to human health.

Patent CN 106632583A discloses a method for extracting walnut crude protein from walnut meal, which comprises the steps of taking walnut meal, soaking the walnut meal in ethanol to remove grease, and filtering to obtain degreased walnut meal; drying and crushing the degreased walnut pulp to obtain crushed walnut pulp; mixing and leaching the walnut pulp crushed material and a sodium hydroxide aqueous solution to obtain a walnut pulp leaching solution; centrifugally separating the walnut meal leaching liquor, and taking supernatant liquor for later use; and (3) regulating the pH of the supernatant by using an acid-base agent, standing, then centrifugally separating the supernatant, and taking the lower-layer precipitate to obtain the crude protein. The method removes residual oil in the cake by soaking in ethanol, which causes the loss of walnut prolamin in the walnut cake, and the obtained protein is not dried, thereby greatly reducing the storage time of the protein product.

Disclosure of Invention

The invention aims to provide a method for preparing hickory protein by using hickory cake meal, so as to solve the defects of the prior art.

The invention adopts the following technical scheme:

a method for preparing hickory protein by using hickory cake meal comprises the following steps:

step one, pretreatment of hickory cake dregs

Drying the hickory cake, crushing the dried hickory cake for 4-7 s at 30000-35000 r/min by using a crusher, sieving the crushed material with a 40-60 mesh sieve, discarding undersize, crushing oversize at 30000-35000 r/min for 35-40 s, sieving the powder with a 80-100 mesh sieve, discarding oversize, and taking undersize as a raw material for preparing hickory protein;

step two, enzyme-assisted alkali extraction

Adding water into the hickory nut protein raw material obtained in the first step according to the ratio of 1g to 15ml to 1g to 25ml, adjusting the pH of the solution to 10.0-10.5, adding alkaline protease according to the amount of 1000-1500U/g based on the mass of the hickory nut protein raw material, stirring and extracting at constant temperature of 55-60 ℃ for 1-6 h, wherein the pH of the solution is adjusted to 10.0-10.5 again at 0.5-1 h, centrifuging after extraction is finished, removing an upper emulsion layer, and collecting a middle water layer; collecting the lower-layer precipitated residue, adding water into the precipitated residue according to the material-liquid ratio of 1g:15 ml-1 g:25ml based on the mass of the hickory nut protein raw material, adjusting the pH of the solution to 10.0-10.5, stirring and washing the residue at constant temperature of 55-60 ℃ for 15-30 min, centrifuging after washing the residue, removing the upper-layer emulsion layer and the lower-layer precipitate, and collecting a middle water layer; mixing the intermediate water layers obtained by the first and second centrifugations to obtain a hickory nut protein extracting solution;

step three, enzymatic hydrolysis and purification of saccharification enzyme

Adjusting the pH value of the hickory nut protein extracting solution obtained in the second step to 4.0-4.5, taking the quality of the hickory nut protein raw material as a reference, adding saccharifying enzyme into the hickory nut protein extracting solution according to the amount of 100-130U/g, stirring and performing enzymolysis for 2-6 hours at constant temperature of 55-65 ℃, after the enzymolysis is completed, inactivating the enzyme, cooling to room temperature, adjusting the pH value of an enzymolysis solution to 3.5-4.0, standing for 60-120 min, centrifuging, removing a supernatant, collecting a precipitate, and obtaining a hickory nut extracted protein crude extract;

step four, eluting and removing impurities by using ethanol water solution

Adding 75-85% v/v ethanol aqueous solution into the crude extract of the extracted protein of the hickory nut obtained in the step three according to the proportion of 1g:12 ml-1 g:15ml by taking the mass of the hickory nut protein raw material as a reference, stirring and eluting for 45-90 min at the constant temperature of 50-60 ℃, centrifuging after the elution is finished, discarding the supernatant, collecting the lower-layer precipitate, adding 5-8 times of volume of water to redissolve the precipitate, and adjusting the pH of the solution to 7.0 to obtain a hickory nut protein solution;

step five, protein spray drying

And D, spray drying the hickory protein solution obtained in the step four to obtain hickory protein powder, namely the hickory protein.

Further, in the first step, the pecan cake is dried in an oven at 40-50 ℃ for 12-24 hours.

Further, the pH value is adjusted by 35-45% w/v sodium hydroxide solution in the second step, the pH value is adjusted by 8-12% v/v hydrochloric acid solution in the third step, and the pH value is adjusted by 35-45% w/v sodium hydroxide solution in the fourth step.

Further, centrifuging for 15-20 min at 3800-4000 r/min after extraction in the second step, and centrifuging for 15-20 min at 3800-4000 r/min after slag washing; standing for 60-120 min, and centrifuging for 5-10 min at 1500-2000 r/min; and step four, after the elution is finished, centrifuging for 5-10 min at 1500-2000 r/min.

And further, inactivating the enzyme at the temperature of 85-90 ℃ for 10-15 min.

Further, in the fifth step, the inlet temperature of the spray dryer is adjusted to 150-180 ℃, the rotating speed of a peristaltic pump is adjusted to 2-5 r/min, and the hickory nut protein solution obtained in the fourth step is spray-dried under the condition.

The hickory protein prepared by the method is applied to preparing active peptide raw materials.

The prepared hickory protein is applied to being used as a vegetable protein additive for food processing.

The prepared hickory protein is applied to functional food ingredients.

The prepared hickory protein is applied to animal husbandry protein supplementary feed.

The invention has the beneficial effects that:

1. the method removes peel and residual fruit shell in the hickory cake by using a physical sieving method, solves the problems of loss of hickory cake protein in the soaking process by using an alkali liquor soaking and peeling method and environmental pollution caused by a large amount of alkaline waste liquid generated after soaking, saves the time for pretreating raw materials, and is simple and rapid to operate, green and environment-friendly.

2. The invention utilizes alkaline protease to assist alkali to extract the hickory protein, the alkaline protease can carry out enzymolysis on insoluble hickory protein into soluble peptide fragments, the extraction rate of the hickory protein is increased, meanwhile, the alkaline protease can carry out enzymolysis on lipoprotein complexes existing in the hickory protein, and the fat content of the extracted protein can be reduced to a certain extent, so that the crude fat content of the extracted hickory protein is less than 5 percent and is lower than that of common similar products by 8 to 10 percent. Therefore, the alkaline protease is used for assisting alkali extraction of the hickory protein, so that the extraction rate of the protein in the alkali extraction liquid can be improved, the residual oil in the hickory cake can be separated, and the problems of solvent residue and hickory cake protein loss caused by extraction of the hickory cake residual oil by using an organic solvent are solved.

3. The method can hydrolyze insoluble starch in the extracted hickory protein into soluble sugar by a purification step of saccharifying enzyme, so that the soluble sugar is dissolved in water and then removed, thereby achieving the purification effect; the step of eluting with the ethanol water solution can dissolve the pigment, the phenolic polymer and the soluble sugar in the extracted hickory protein into the ethanol solution, so as to achieve the purification effect, and finally obtain the light brown hickory protein with the purity of more than 50% (determined by Kjeldahl method in GB5009.5-2016, calculated by dry basis and protein conversion coefficient of 6.25), and meet the purity requirement of the vegetable hydrolyzed protein in the vegetable protein for processing GB 20371-2016 food. Compared with the prior art, the invention has the advantages of simple process steps, short purification period and low cost, and can purify the extracted hickory protein quickly and in an energy-saving way.

4. The invention utilizes the spray drying method to dry the obtained protein, prolongs the storage life of the protein product, has higher drying rate compared with freeze drying, and is more suitable for industrial production.

5. According to the method, the sediment residues obtained after the first centrifugal operation in the enzyme-assisted alkali extraction step are further cleaned, so that the proteins remaining in the sediment residues in the centrifugal process can be dissolved in water, the loss of the protein extracting solution in the centrifugal process is reduced, and the utilization rate of the proteins in the hickory nut protein raw material is improved.

Drawings

FIG. 1 is a schematic flow chart of the method of the present invention.

Detailed Description

The invention is explained in more detail below with reference to exemplary embodiments and the accompanying drawings. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.

A method for preparing fructus Juglandis protein from fructus Juglandis cake as shown in figure 1 comprises the following steps:

step one, pretreatment of hickory cake dregs

Baking the hickory cake pulp in an oven at 40-50 ℃ for 12-24 h, crushing the dried hickory cake pulp by a crusher at 30000-35000 r/min for 4-7 s, sieving the crushed material by a 40-60 mesh sieve, discarding undersize (mainly a layer of reddish brown thin skin covered on the pulp of the hickory kernel), crushing oversize at 30000-35000 r/min for 35-40 s, sieving the powder by an 80-100 mesh sieve, discarding oversize (mainly hickory nut wooden shell), and taking the undersize as a raw material for preparing hickory protein;

step two, enzyme-assisted alkali extraction

Adding water into the hickory nut protein raw material obtained in the first step according to the ratio of 1g to 15ml to 1g to 25ml, adjusting the pH of the solution to 10.0 to 10.5 by using 35 to 45 percent w/v sodium hydroxide solution, adding alkaline protease according to the amount of 1000 to 1500U/g based on the mass of the hickory nut protein raw material, stirring and extracting at the constant temperature of 55 to 60 ℃ and 400 to 600r/min for 1 to 6 hours, adjusting the pH of the solution to 10.0 to 10.5 by using 35 to 45 percent w/v sodium hydroxide solution again when 0.5 to 1 hour is finished, centrifuging for 15 to 20 minutes under the conditions of 3800 to 4000r/min after extraction is finished, removing an upper emulsion layer, and collecting a middle water layer; collecting the lower-layer precipitated residue, adding water into the precipitated residue again according to the proportion of 1g:15 ml-1 g:25ml of the hickory nut protein raw material by using the mass as the reference, adjusting the pH of the solution to 10.0-10.5 by using 35-45% w/v sodium hydroxide solution, stirring and washing the residue for 15-30 min at the constant temperature of 55-60 ℃ and 400-600 r/min, centrifuging for 15-20 min at 3800-4000 r/min after washing the residue, discarding the upper-layer emulsion layer and the lower-layer precipitate, and collecting an intermediate water layer; mixing the intermediate water layers obtained by the first and second centrifugations to obtain a hickory nut protein extracting solution;

step three, enzymatic hydrolysis and purification of saccharification enzyme

Adjusting the pH of the hickory nut protein extracting solution obtained in the second step to 4.0-4.5 by using 8-12% v/v hydrochloric acid solution, taking the quality of the hickory nut protein raw material as a reference, adding saccharifying enzyme into the hickory nut protein extracting solution according to the amount of 100-130U/g, stirring at constant temperature of 55-65 ℃ and 400-600 r/min for enzymolysis for 2-6 h, inactivating the enzyme at 85-90 ℃ for 10-15 min after the enzymolysis is finished, cooling to room temperature, adjusting the pH of an enzymolysis solution to 3.5-4.0 by using 8-12% v/v hydrochloric acid solution, standing for 60-120 min, centrifuging at 1500-2000 r/min for 5-10 min, removing supernatant, collecting precipitate, and obtaining a hickory nut protein extract crude extract;

step four, eluting and removing impurities by using ethanol water solution

Adding 75-85% v/v ethanol aqueous solution into the crude extract of the extracted protein of the hickory nut obtained in the step three according to the proportion of 1g:12 ml-1 g:15ml by taking the mass of the hickory nut protein as a reference, stirring and eluting for 45-90 min at a constant temperature of 50-60 ℃ and 400-600 r/min, centrifuging for 5-10 min at 1500-2000 r/min after the elution is finished, discarding the supernatant, collecting the lower precipitate, adding 5-8 times of water to redissolve the precipitate, and adjusting the pH of the solution to 7.0 by using 35-45% w/v sodium hydroxide solution to obtain a hickory nut protein solution;

step five, protein spray drying

And fifthly, adjusting the inlet temperature of a spray dryer to 150-180 ℃, adjusting the rotating speed of a peristaltic pump to 2-5 r/min, and spray drying the hickory nut protein solution obtained in the fourth step under the condition to obtain the hickory nut protein powder, namely the hickory nut protein.

The prepared hickory protein can be used as an active peptide raw material, or used as a vegetable protein additive for food processing, or used as a functional food ingredient, or used as a protein supplementary feed for animal husbandry.

The hickory cake meal related to the following examples is prepared by the following steps: after the pecans are hulled, grease is removed by a normal temperature squeezing mode, the squeezed pecan cakes have partial shells, the residual oil content of the cakes is about 34% (measured by a Soxhlet extraction method in GB 5009.6-2016), the crude protein content is about 14% (measured by a Kjeldahl method in GB5009.5-2016, and the result shows that the nitrogen conversion coefficient is 6.25 on a dry basis), and the water content is about 5 wt% (measured by a direct drying method in GB 5009.3-2016).

Example 1

1. Baking the pecan cake pulp in a 45 ℃ oven for 24h, crushing the dried pecan cake pulp for 5s at 32000r/min by using a swing type multifunctional crusher (platinum European hardware factory, 400Y), sieving the crushed material by a 40-mesh sieve, discarding undersize, crushing oversize at 32000r/min for 35s, sieving the powder by a 80-mesh sieve, discarding oversize, and taking undersize as a raw material for preparing pecan protein.

2. Adding water into the hickory nut protein raw material obtained in the step 1 according to the ratio of 1g to 16ml of material to liquid, adjusting the pH of the solution to 10.0 by using 40% w/v sodium hydroxide solution, adding alkaline protease (Solarbio, B8360) according to the amount of 1000U/g on the basis of the mass of the hickory nut protein raw material, stirring and extracting for 1.5h at the constant temperature of 60 ℃ and 500r/min, wherein the pH of the solution is adjusted to 10.0 by using 40% w/v sodium hydroxide solution again when 0.5h is carried out, centrifuging for 15min at 3800r/min after the extraction is finished, discarding the upper emulsion layer, and collecting a middle water layer; collecting the lower layer of precipitation residue, adding water into the precipitation residue again according to the proportion of material-liquid ratio of 1g:16ml based on the mass of the hickory nut protein raw material, adjusting the pH of the solution to 10.0 by using 40% w/v sodium hydroxide solution, stirring and washing the residue at constant temperature of 60 ℃ and 500r/min for 30min, centrifuging for 15min at 3800r/min after washing the residue, discarding the upper layer of emulsifying layer and the lower layer of precipitation, and collecting the middle water layer; and (3) combining the intermediate water layers obtained by the first centrifugation and the second centrifugation to obtain a hickory nut protein extracting solution, wherein the extraction rate of the hickory nut protein raw material in the extracting solution reaches 89.7% (determined by Kjeldahl method in GB5009.5-2016, and the nitrogen conversion coefficient is 6.25). And (3) simultaneously serving as a control group, changing the addition amount of the alkaline protease in the step (2) to 0U/g, and carrying out the same operation on the rest parts, wherein the extraction rate of the hickory nut protein raw material in the extracting solution is 59.0% (determined by Kjeldahl method in GB5009.5-2016, and the nitrogen conversion coefficient is 6.25). After the hickory nut protein extract obtained in step 2 of this example and the hickory nut protein extract obtained in the control were respectively concentrated to 1/5 of the original volume, the obtained crude fat content of the powder (determined by soxhlet extraction method in GB 5009.6-2016) was determined after spray-drying according to the conditions shown in step 5, wherein the crude fat content of the powder obtained from the extract obtained in step 2 of this example was 4.9%, and the crude fat content of the powder obtained from the extract obtained in the control was 26.8%, which makes it possible to determine that the alkali extraction assisted by alkaline protease is better than the general alkali extraction and to reduce the fat content of the extracted protein to a certain extent.

3. Adjusting the pH of the hickory nut protein extracting solution obtained in the step 2 to 4.0 by using 10% v/v hydrochloric acid solution, adding saccharifying enzyme (Solarbio, T8500) into the hickory nut protein extracting solution according to the amount of 100U/g by taking the quality of the hickory nut protein raw material as a reference, stirring at constant temperature at 60 ℃ and 500r/min for enzymolysis for 2.5h, inactivating enzyme at 90 ℃ for 15min after the enzymolysis is finished, cooling to room temperature, adjusting the pH of an enzymolysis solution to 3.7 by using 10% v/v hydrochloric acid solution, standing for 60min, centrifuging for 5min at 2000r/min, discarding supernatant, collecting precipitate, and obtaining the hickory nut extracted protein crude extract.

4. Adding 80% v/v ethanol water solution into the crude extract of the extracted protein of the hickory nut obtained in the step 3 according to the proportion of 1g to 12ml by taking the mass of the hickory nut protein raw material as a reference, stirring and eluting for 45min at the constant temperature of 50 ℃ and 500r/min, centrifuging for 5min at 2000r/min after the elution is finished, discarding the supernatant, collecting the lower-layer precipitate, adding 5 times of water to redissolve the precipitate, and adjusting the pH of the solution to 7.0 by using 40% w/v sodium hydroxide solution to obtain the hickory nut protein solution.

5. And (2) adjusting the inlet temperature of a spray dryer to 150 ℃, adjusting the rotating speed of a peristaltic pump to 2r/min, and spray-drying the pecan protein solution obtained in the step (4) under the condition, wherein the crude protein content in the finally obtained pecan protein powder is 53.8% (determined by Kjeldahl method in GB5009.5-2016, the result is that the nitrogen conversion coefficient is 6.25 on a dry basis), the water content is 9.5 wt% (determined by direct drying method in GB 5009.3-2016), the crude fat content is 4.6% (determined by Soxhlet extraction method in GB 5009.6-2016), the ash content is 1.0% (determined by GB 5009.4-2016 direct burning method), and the protein utilization rate in the pecan protein raw material is 66.4%. Meanwhile, as a control group, the hickory nut protein extract obtained in the step 2 in the embodiment is adjusted to pH 3.7 by using a 10% v/v hydrochloric acid solution, the hickory nut protein extract is kept stand for 60min and then centrifuged for 5min at 2000r/min, the supernatant is discarded, the lower layer precipitate is collected, 5 times of water is added to redissolve the precipitate, the pH of the solution is adjusted to 7.0 by using a 40% w/v sodium hydroxide solution to obtain an unpurified hickory nut protein solution, and then the unpurified hickory nut protein powder is obtained by spray drying according to the conditions given in the step 5, wherein the crude protein content of the unpurified hickory nut protein powder is only 35.2% (determined by a Kjeldahl method in GB5009.5-2016, the result is calculated on a dry basis, and the nitrogen conversion coefficient is 6.25), so that the method for eluting and purifying the glucoamylase combined with the ethanol aqueous solution can be judged to have a certain purification effect.

Example 2

1. Baking the pecan cake pulp in a 50 ℃ oven for 12h, crushing the dried pecan cake pulp for 7s at 32000r/min by using a swing type multifunctional crusher (platinum European hardware factory, 400Y), sieving the crushed material by a 60-mesh sieve, discarding undersize, crushing oversize at 32000r/min for 40s, sieving the powder by a 100-mesh sieve, discarding oversize, and taking undersize as a raw material for preparing pecan protein.

2. Adding water into the hickory nut protein raw material obtained in the step 1 according to the ratio of 1g to 18ml of material to liquid, adjusting the pH of the solution to 10.3 by using 40% w/v sodium hydroxide solution, adding alkaline protease (Solarbio, B8360) according to the amount of 1100U/g on the basis of the mass of the hickory nut protein raw material, stirring and extracting at the constant temperature of 58 ℃ and 500r/min for 2.5h, wherein the pH of the solution is adjusted to 10.3 by using 40% w/v sodium hydroxide solution again when 1h is finished, centrifuging for 16min under the condition of 4000r/min after the extraction is finished, discarding an upper emulsion layer, and collecting a middle water layer; collecting the lower layer of precipitation residue, adding water into the precipitation residue again according to the proportion of material-liquid ratio of 1g to 18ml based on the mass of the hickory nut protein raw material, adjusting the pH of the solution to 10.3 by using 40% w/v sodium hydroxide solution, stirring and washing the residue at constant temperature of 58 ℃ and 500r/min for 20min, centrifuging for 16min under the condition of 4000r/min after washing the residue, discarding the upper layer of emulsifying layer and the lower layer of precipitation, and collecting a middle water layer; and (3) combining the intermediate water layers obtained by the first centrifugation and the second centrifugation to obtain a hickory nut protein extracting solution, wherein the extraction rate of the hickory nut protein raw material in the extracting solution reaches 89.1% (determined by a Kjeldahl method in GB5009.5-2016, and the nitrogen conversion coefficient is 6.25).

3. Adjusting the pH of the hickory nut protein extracting solution obtained in the step 2 to 4.5 by using 10% v/v hydrochloric acid solution, adding saccharifying enzyme (Solarbio, T8500) into the hickory nut protein extracting solution according to the amount of 130U/g by taking the quality of hickory nut protein raw materials as a reference, stirring at a constant temperature of 58 ℃ and 500r/min for enzymolysis for 3 hours, inactivating enzyme at 85 ℃ for 13 minutes after the enzymolysis is finished, cooling to room temperature, adjusting the pH of an enzymolysis solution to 4.0 by using 10% v/v hydrochloric acid solution, standing for 120 minutes, centrifuging for 8 minutes at 1800r/min, discarding supernatant, collecting precipitate, and obtaining the crude extract of the hickory nut protein.

4. Adding 75% v/v ethanol water solution into the crude extract of the extracted protein of the hickory nut obtained in the step 3 according to the proportion of 1g to 15ml by taking the mass of the hickory nut protein raw material as a reference, stirring and eluting for 90min at the constant temperature of 60 ℃ and 500r/min, centrifuging for 8min at 1800r/min after the elution is finished, discarding the supernatant, collecting the lower-layer precipitate, adding 6 times of volume of water to redissolve the precipitate, and adjusting the pH of the solution to 7.0 by using 40% w/v sodium hydroxide solution to obtain the hickory nut protein solution.

5. And (2) adjusting the inlet temperature of a spray dryer to 180 ℃, adjusting the rotation speed of a peristaltic pump to 3r/min, and spray-drying the pecan protein solution obtained in the step (4) under the condition, wherein the crude protein content in the finally obtained pecan protein powder is 52.9% (determined by Kjeldahl method in GB5009.5-2016, the result is that the nitrogen conversion coefficient is 6.25 on a dry basis), the water content is 7.2 wt% (determined by direct drying method in GB 5009.3-2016), the crude fat content is 4.3% (determined by Soxhlet extraction method in GB 5009.6-2016), the ash content is 1.6% (determined by GB 5009.4-2016 direct burning method), and the protein utilization rate in the pecan protein raw material is 66.1%.

Example 3

1. Baking the pecan cake pulp in an oven at 40 ℃ for 24h, crushing the dried pecan cake pulp for 4s at 32000r/min by using a swing type multifunctional crusher (platinum European hardware factory, 400Y), sieving the crushed material by a 40-mesh sieve, discarding undersize, crushing oversize at 32000r/min for 38s, sieving the powder by a 80-mesh sieve, discarding oversize, and taking undersize as a raw material for preparing pecan protein.

2. Adding water into the hickory nut protein raw material obtained in the step 1 according to the ratio of 1g to 17ml of material to liquid, adjusting the pH of the solution to 10.1 by using 40% w/v sodium hydroxide solution, adding alkaline protease (Solarbio, B8360) according to the amount of 1500U/g on the basis of the mass of the hickory nut protein raw material, stirring and extracting at the constant temperature of 57 ℃ and 500r/min for 2 hours, wherein the pH of the solution is adjusted to 10.1 by using 40% w/v sodium hydroxide solution again when 0.8 hour is reached, centrifuging for 17min under the condition of 3900r/min after the extraction is finished, discarding the upper emulsion layer, and collecting the middle water layer; collecting the lower layer of precipitation residue, adding water into the precipitation residue again according to the proportion of material-liquid ratio of 1g:17ml based on the mass of the hickory nut protein raw material, adjusting the pH of the solution to 10.1 by using 40% w/v sodium hydroxide solution, stirring and washing the residue at constant temperature of 57 ℃ and 500r/min for 25min, centrifuging for 17min under the condition of 3900r/min after washing the residue, discarding the upper layer of emulsifying layer and the lower layer of precipitation, and collecting the middle water layer; and (3) combining the intermediate water layers obtained by the first centrifugation and the second centrifugation to obtain a hickory nut protein extracting solution, wherein the extraction rate of the hickory nut protein in the extracting solution reaches 89.5% (determined by a Kjeldahl method in GB5009.5-2016, and the nitrogen conversion coefficient is 6.25).

3. Adjusting the pH of the hickory nut protein extracting solution obtained in the step 2 to 4.2 by using 10% v/v hydrochloric acid solution, adding saccharifying enzyme (Solarbio, T8500) into the hickory nut protein extracting solution according to the amount of 110U/g by taking the quality of the hickory nut protein raw material as a reference, stirring at a constant temperature of 62 ℃ and 500r/min for enzymolysis for 2.7h, inactivating enzyme at 88 ℃ for 14min after the enzymolysis is finished, cooling to room temperature, adjusting the pH of an enzymolysis solution to 3.5 by using 10% v/v hydrochloric acid solution, standing for 100min, centrifuging for 7min at 1900r/min, discarding supernatant, collecting precipitate, and obtaining the hickory nut extracted protein crude extract.

4. Adding 85% v/v ethanol water solution into the crude extract of the extracted protein of the hickory nut obtained in the step 3 according to the proportion of 1g to 13ml by taking the mass of the hickory nut protein raw material as a reference, stirring and eluting for 60min at the constant temperature of 55 ℃ and 500r/min, centrifuging for 7min at 1900r/min after the elution is finished, discarding the supernatant, collecting the lower-layer precipitate, adding 7 times of volume of water to redissolve the precipitate, and adjusting the pH of the solution to 7.0 by using 40% w/v sodium hydroxide solution to obtain the hickory nut protein solution.

5. And (2) adjusting the inlet temperature of a spray dryer to 160 ℃, adjusting the rotation speed of a peristaltic pump to 3r/min, and spray-drying the pecan protein solution obtained in the step (4) under the condition, wherein the crude protein content in the finally obtained pecan protein powder is 52.5% (determined by Kjeldahl method in GB5009.5-2016, the result is that the nitrogen conversion coefficient is 6.25 on a dry basis), the water content is 8.5 wt% (determined by direct drying method in GB 5009.3-2016), the crude fat content is 4.0% (determined by Soxhlet extraction method in GB 5009.6-2016), the ash content is 1.3% (determined by GB 5009.4-2016 direct burning method), and the protein utilization rate in the pecan protein raw material is 65.8%.