阅读说明：本技术 核桃肽钙螯合物及其制备方法 (Walnut peptide calcium chelate and preparation method thereof ) 是由 王丰俊 阮国瑞 金凤 张国红 于 2019-09-30 设计创作，主要内容包括：本发明提供了一种核桃肽钙螯合物及其制备方法。该制备方法包括：(1)将核桃粕经蛋白酶进行酶解反应,以便获得酶解产物；(2)将所述酶解产物进行发酵处理,以便获得发酵产物；(3)对所述发酵产物进行分离处理,以便获得分子量在5kDa以下的核桃肽；(4)将所述核桃肽和钙离子混合,进行螯合反应,以便制备得到所述核桃肽钙螯合物。由该方法制得的产品口感良好,螯合率高,获得的核桃肽钙螯合物可被人体高效的吸收利用,在补钙的同时还可发挥核桃多肽的生物活性功能,具有良好的应用前景。(The invention provides a walnut peptide calcium chelate and a preparation method thereof. The preparation method comprises the following steps: (1) carrying out enzymolysis reaction on the walnut meal by using protease so as to obtain an enzymolysis product; (2) carrying out fermentation treatment on the enzymolysis product so as to obtain a fermentation product; (3) separating the fermentation product to obtain walnut peptide with molecular weight below 5 kDa; (4) and mixing the walnut peptide and calcium ions, and carrying out a chelation reaction so as to prepare the walnut peptide-calcium chelate. The product prepared by the method has good taste and high chelating rate, the obtained walnut peptide calcium chelate can be efficiently absorbed and utilized by human bodies, the biological activity function of walnut peptide can be exerted while calcium is supplemented, and the method has good application prospect.)

1. A preparation method of a walnut peptide calcium chelate is characterized by comprising the following steps:

(1) carrying out enzymolysis reaction on the walnut meal by using protease so as to obtain an enzymolysis product;

(2) carrying out fermentation treatment on the enzymolysis product so as to obtain a fermentation product;

(3) separating the fermentation product to obtain walnut peptide with molecular weight below 5 kDa;

(4) and carrying out chelation reaction on the walnut peptide and calcium ions so as to prepare the walnut peptide-calcium chelate.

2. The method according to claim 1, wherein the protease in step (1) is an alkaline protease;

optionally, the mass of the alkaline protease accounts for 4% -6%, preferably 5% of the mass of the walnut pulp;

optionally, the temperature of the enzymolysis reaction is 50-60 ℃, preferably 55 ℃, and the pH of the enzymolysis reaction is 8-9, preferably 8.7;

optionally, the enzymolysis reaction is carried out for 3 hours at the constant temperature of 55 ℃.

3. The production method according to claim 1, wherein the fermentation treatment is carried out using Aspergillus niger in step (2);

optionally, the aspergillus niger is inoculated into the enzymolysis product in the form of bacterial suspension, the inoculum size of the bacterial suspension accounts for 8-20%, preferably 10% of the enzymolysis product, the temperature of the fermentation treatment is 30-35 ℃, preferably 30 ℃, and the pH is 6.0-7.0.

4. The method according to claim 1, wherein the fermentation product is subjected to separation treatment using an ultrafiltration membrane having a molecular weight of 5kDa in step (3).

5. The method according to claim 1, wherein the calcium ion in step (4) is derived from anhydrous calcium chloride;

optionally, the chelation reaction is carried out by mixing the walnut peptide and the anhydrous calcium chloride according to a mass ratio of 1:1-5:1, preferably 3: 1;

optionally, the temperature of the chelation reaction is 40-50 degrees celsius, preferably 45 degrees celsius, the pH of the chelation reaction is 7.0-9.0, preferably 8.0, and the time of the chelation reaction is 30-50 minutes, preferably 40 minutes.

6. The method of claim 1, further comprising, before step (1):

carrying out degreasing treatment on the walnut meal by using an organic solvent, sieving the obtained degreased walnut meal, and then carrying out enzymolysis reaction.

7. The method according to claim 1, wherein, before the step (3), the method further comprises:

and (3) carrying out boiling water bath treatment on the enzymolysis product, wherein the water bath treatment time is 5-15 minutes, and preferably 10 minutes.

8. The method of claim 1, wherein step (4) further comprises:

and washing the product after the chelation reaction by using absolute ethyl alcohol, centrifuging to obtain a precipitate, adding a calcium indicator into the supernatant to stop discoloration, and obtaining the walnut peptide calcium chelate.

9. A preparation method of a walnut peptide calcium chelate is characterized by comprising the following steps:

1) carrying out degreasing treatment on walnut meal, and carrying out enzymolysis reaction on the degreased walnut meal by using alkaline protease so as to obtain an enzymolysis product;

2) carrying out fermentation treatment on the enzymolysis product of aspergillus niger so as to obtain a fermentation product;

3) carrying out ultrafiltration treatment on the fermentation product by using an ultrafiltration membrane with the molecular weight of 5kDa so as to obtain walnut peptide;

4) mixing the walnut peptide and anhydrous calcium chloride according to the mass ratio of 1:1-5:1, preferably 3:1, and carrying out chelation reaction at the temperature of 40-50 ℃, the pH of 7.0-9.0, preferably at the temperature of 45 ℃ and the pH of 8.0 so as to prepare the walnut peptide calcium chelate.

10. A walnut peptide calcium chelate, which is prepared by the preparation method of any one of claims 1 to 8 or the preparation method of claim 9.

Technical Field

The invention relates to the field of food, in particular to a walnut peptide calcium chelate and a preparation method thereof.

Background

The abundant polypeptide substances contained in protein resources play an important role for human bodies. There are different ways of processing when obtaining polypeptide substances from protein resources. The method for obtaining the polypeptide from the protein resource by the enzymolysis method has the advantages of simple operation process, short period, low cost and the like, but the obtained polypeptide has bitter odor. The protein resource is fermented by microorganisms, and the generated terminal peptidase can modify bitter groups at the tail end of a peptide chain, so that bitter can be removed, and the polypeptide with good taste and flavor can be obtained.

The only route of calcium intake in bone is dietary intake, by which the most common forms of calcium are ionized calcium, calcium carbonate and calcium gluconate. Calcium carbonate needs to be digested and absorbed by means of relevant vitamins and enzymes, and excessive intake of calcium carbonate may cause intestinal side effects such as constipation, flatulence and the like; the ionized calcium is easy to form calcium hydroxide precipitate with oxalic acid, phytic acid and the like, and the absorption utilization rate is low; compared with ionized calcium, the calcium existing in a chelated form has the advantages of high bioavailability, low biotoxicity and the like, can be applied to development of functional foods, and can also be applied to industrial production as a bacteriostatic agent, an antioxidant, a calcium supplement and the like.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention aims to provide a walnut peptide calcium chelate and a preparation method thereof.

The walnut meal obtained by squeezing oil from walnut kernels is rich in protein and amino acid which has important functions for human bodies, so that the walnut meal can be developed. The walnut pulp is processed to obtain the active polypeptide substance in the walnut kernel, and then the obtained walnut polypeptide is chelated with calcium ions to form a walnut peptide calcium chelate serving as a calcium supplement, so that the absorption and utilization of calcium ions by a human body can be further improved, the method has guiding significance for the research and development and production of calcium preparations, and has important significance for the development of high value-added products of the walnut.

The inventor finds out in the research process that: in the process of obtaining the walnut polypeptide, the walnut meal can be subjected to enzymolysis treatment, and then the polypeptide obtained by combining a fermentation method has the advantages of enzymolysis treatment and fermentation treatment, small molecular weight, good taste and flavor, controllable operation and simple process. For example, after the walnut meal is subjected to enzymolysis for a period of time, the enzymolysis system can be directly inoculated with microorganisms for fermentation treatment without any treatment, so as to obtain an enzymolysis fermentation product. In the process, simultaneous enzymolysis and microbial fermentation are not excluded, but the enzymolysis and fermentation are carried out, the operation process is simple, the obtained polypeptide has good taste and flavor, small molecular weight and high activity, and the utilization rate of the walnut meal is improved. Further, the bioavailability of calcium ions in the human body can be improved by chelating with calcium ions under appropriate conditions. The obtained walnut peptide calcium chelate has high chelating rate and can be used as a calcium supplement and a polypeptide nutrient with high biological absorptivity.

Specifically, the invention provides the following technical scheme:

in a first aspect of the invention, the invention provides a preparation method of a walnut peptide calcium chelate, which comprises the following steps: (1) carrying out enzymolysis reaction on the walnut meal by using protease to obtain an enzymolysis product; (2) carrying out fermentation treatment on the enzymolysis product so as to obtain a fermentation product; (3) separating the fermentation product to obtain walnut peptide with molecular weight below 5 kDa; (4) and carrying out chelation reaction on the walnut peptide and calcium ions so as to prepare the walnut peptide-calcium chelate.

After the walnut meal is subjected to protease enzymolysis and fermentation treatment, the obtained product has high content of polypeptide with small molecular weight, and the polypeptide in the product has good taste and flavor. The walnut peptide calcium chelate is high in chelating rate through a chelating reaction with calcium ions, can be efficiently absorbed and utilized by a human body, can supplement calcium and simultaneously exert the bioactivity function of walnut peptide, and has a good application prospect.

According to the embodiment of the invention, the preparation method of the walnut peptide calcium chelate described above may further include the following technical features:

in some embodiments of the invention, the protease in step (1) is an alkaline protease. The alkaline protease is capable of efficiently hydrolyzing peptide bonds in proteins, whereby polypeptide substances of molecular weight can be rapidly obtained.

In some embodiments of the present invention, the mass of the alkaline protease accounts for 4% -6%, preferably 5% of the mass of the walnut meal. During the enzymolysis process of the walnut pulp by using the alkaline protease, the walnut pulp can be quickly subjected to enzymolysis by using a proper amount of the alkaline protease. Under the condition of the content, the rapid enzymolysis of the walnut meal can be realized, and the excessive enzymolysis can not be caused, so that unnecessary small molecular peptides which are not beneficial to human bodies are generated.

In some embodiments of the invention, the temperature of the enzymatic reaction is 50-60 ℃, preferably 55 ℃, and the pH of the enzymatic reaction is 8-9, preferably 8.7. Therefore, the rapid enzymolysis of the walnut meal can be realized, and the target polypeptide can be obtained.

In some embodiments of the present invention, the enzymatic hydrolysis is performed by shaking at a constant temperature of 55 ℃ for 3 hours. Therefore, the rapid enzymolysis of the walnut meal can be realized, and the target polypeptide can be obtained.

In some embodiments of the invention, the fermentation treatment in step (2) is performed with aspergillus niger. Aspergillus niger is a commonly used microorganism used for fermentation treatment, and the generated telopeptidase can modify bitter groups at the tail end of a peptide chain, remove bitter taste and obtain polypeptide with good taste and flavor.

In some embodiments of the invention, the aspergillus niger is inoculated into the enzymolysis product in the form of bacterial suspension, the inoculum size of the bacterial suspension accounts for 8-20%, preferably 10%, the temperature of the fermentation treatment is 30-35 ℃, preferably 30 ℃, and the pH is 6.0-7.0. And under the conditions of the temperature and the pH, the aspergillus niger fermentation is carried out, so that the polypeptide with good taste and flavor can be quickly obtained, and the obtained polypeptide is proper in size.

In some embodiments of the invention, the fermentation product is subjected to a separation treatment in step (3) using an ultrafiltration membrane having a molecular weight of 5 kDa. The ultrafiltration membrane is used for separation treatment, the condition is mild, and the walnut polypeptide can not be damaged.

In some embodiments of the invention, the calcium ions in step (4) are derived from anhydrous calcium chloride. The method utilizes anhydrous calcium chloride as a calcium source to provide calcium ions, the anhydrous calcium chloride is easy to dissolve in water, and the formed walnut peptide calcium chelate complex is not easy to dissolve in an organic solvent, so that the method is favorable for the chelating reaction and the separation of the formed walnut peptide calcium chelate complex and free calcium.

In some embodiments of the invention, the chelation reaction comprises: mixing the walnut peptide and the anhydrous calcium chloride according to the mass ratio of the peptide source to the calcium source (1:1) - (5:1), preferably 3:1, to perform the chelation reaction. Therefore, the walnut peptide calcium chelate with high calcium ion content can be obtained.

In some embodiments of the invention, the temperature of the chelation reaction is 40 to 50 ℃, preferably 45 ℃, the pH of the chelation reaction is 7.0 to 9.0, preferably 8.0, and the time of the chelation reaction is 30 to 50 minutes, preferably 40 minutes. The prepared walnut peptide calcium chelate has high calcium ion chelation rate, the prepared walnut peptide calcium chelate has obviously improved calcium ion bioavailability, and both calcium ions and walnut polypeptide can be efficiently absorbed by a human body.

In some embodiments of the present invention, before step (1), further comprising: and (3) carrying out degreasing treatment on the walnut meal by using an organic solvent, sieving the obtained degreased walnut meal, and then carrying out the enzymolysis reaction. After degreasing, the excess grease can be removed. Then, sieving treatment is carried out, for example, 60-mesh sieve treatment can be carried out, so that the defatted walnut meal is homogenized, the surface area of subsequent enzymolysis reaction and fermentation reaction is increased, and the high-activity small molecular peptide is quickly obtained.

In some embodiments of the present invention, before step (3), further comprising: and (3) carrying out boiling water bath treatment on the enzymolysis product, wherein the water bath treatment time is 5-15 minutes, and preferably 10 minutes. The enzyme deactivation treatment is carried out under the condition, so that the maximum polypeptide activity can be kept while the inactivation enzymolysis and fermentation reaction are carried out, the excessive enzymolysis is prevented, and the walnut polypeptide with certain activity is obtained.

In some embodiments of the invention, step (4) further comprises: and washing the product after the chelation reaction by using absolute ethyl alcohol, centrifuging to obtain a precipitate, adding a calcium indicator into the supernatant to stop discoloration, and obtaining the walnut peptide calcium chelate. Therefore, free calcium ions and impurities can be removed, and the walnut peptide calcium chelate with high purity is obtained.

In a second aspect of the invention, the invention provides a preparation method of a walnut peptide calcium chelate, which comprises the following steps: 1) carrying out degreasing treatment on walnut meal, and carrying out enzymolysis reaction on the degreased walnut meal by using alkaline protease so as to obtain an enzymolysis product; 2) carrying out fermentation treatment on the enzymolysis product of aspergillus niger so as to obtain a fermentation product; 3) carrying out ultrafiltration treatment on the fermentation product by using an ultrafiltration membrane with the molecular weight of 5kDa so as to obtain walnut peptide; 4) mixing the walnut peptide and anhydrous calcium chloride according to the mass ratio of 1:1-5:1, preferably 3:1, and carrying out chelation reaction at the temperature of 40-50 ℃, the pH of 7.0-9.0, preferably at the temperature of 45 ℃, and the pH of 8.0 so as to prepare the walnut peptide calcium chelate.

In a third aspect of the invention, the invention provides a walnut peptide calcium chelate, which is prepared by the preparation method of any embodiment of the first aspect of the invention or the preparation method of the second aspect of the invention.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

fig. 1 is a schematic flow diagram of a preparation process of a walnut peptide calcium chelate according to one embodiment of the present invention;

FIG. 2 is a graph showing the effect of varying the ratio of peptide to calcium on the calcium ion sequestration rate of walnut peptide calcium chelate, according to an embodiment of the present invention;

FIG. 3 is a graph illustrating the effect of pH change on the calcium ion sequestration rate of walnut peptide calcium chelate, according to one embodiment of the present invention;

FIG. 4 is a graph showing the effect of temperature change on the calcium ion sequestration rate of walnut peptide calcium chelate, according to one embodiment of the present invention;

FIG. 5 is a graph showing the effect of varying chelation reaction time on the calcium ion sequestration rate of walnut peptide calcium chelate, according to an embodiment of the present invention;

FIG. 6 is a graph of response of interaction of factors to the calcium ion sequestration rate of walnut peptide calcium chelate according to one embodiment of the present invention;

FIG. 7 is a UV spectrum of walnut peptide and walnut peptide calcium chelate provided according to one embodiment of the present invention;

FIG. 8 is an infrared spectrum of walnut peptide and walnut peptide calcium chelate provided in accordance with one embodiment of the present invention;

FIG. 9 is an atomic force microscope image of walnut peptides and walnut peptide calcium chelates provided in accordance with one embodiment of the invention;

fig. 10 is a comparison of the solubility of walnut peptide calcium chelate and inorganic calcium salt under different pH conditions, provided according to one embodiment of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings, and it should be noted that the described embodiments are exemplary and are intended to be illustrative of the present invention and should not be construed as limiting the present invention.

The invention provides a preparation method of a walnut peptide calcium chelate, which comprises the following steps: (1) carrying out enzymolysis reaction on the walnut meal by using protease so as to obtain an enzymolysis product; (2) carrying out fermentation treatment on the enzymolysis product so as to obtain a fermentation product; (3) separating the fermentation product to obtain walnut peptide with molecular weight below 5 kDa; (4) and carrying out chelation reaction on the walnut peptide and calcium ions so as to prepare the walnut peptide-calcium chelate. The preparation method can improve utilization rate of walnut meal and produce a calcium supplement with high biological absorptivity.

The preparation method of the walnut peptide calcium chelate provided by the invention uses walnut meal which is obtained by pressing oil from walnut kernels as a raw material. The walnut meal can be purchased directly or obtained by pressing walnut kernel oil by conventional technology in the field. Although the variety, quality and oil extraction conditions of walnuts have some influence on the quality of a final product and the calcium ion chelating rate, the method provided by the invention can obtain the peptide calcium chelate which can be chelated with calcium ions and has high bioavailability. In at least some embodiments of the present invention, in order to reduce the influence of oil in the walnut pulp, the walnut pulp used may be defatted with an organic solvent, and the protein content in the walnut pulp after being defatted with the organic solvent is about 60% to 65%, for example, about 63%. The walnut meal with high protein content can be subjected to enzymolysis, combined fermentation and then a large amount of target walnut peptides are obtained.

In the process of degreasing the walnut pulp, some common organic solvents can be used for degreasing, for example, petroleum ether can be used for degreasing the walnut pulp, and the residual oil and fat in the walnut pulp are removed.

In the experiment or production process, walnut meal obtained after oil extraction or defatted walnut meal can be mixed with water to obtain walnut meal mixed liquor, and then protease is used for carrying out enzymolysis reaction. Of course, in order to increase the reaction area of the enzymolysis reaction and the fermentation reaction, the walnut pulp after oil extraction or the walnut pulp after degreasing can be sieved, for example, a 60-mesh sieve can be sieved. When protease is used for enzymolysis reaction, alkaline protease with good effect of walnut protein enzymolysis, easy availability and low price can be selected as hydrolase. The enzymolysis condition can be that the enzymolysis temperature is 50-60 ℃, preferably 55 ℃; the adding amount of the alkaline protease accounts for 4-6% of the mass of the walnut pulp, and is preferably 5%; the pH of the enzymatic hydrolysis is 8-9, preferably 8.7. In at least some embodiments, when the enzymolysis reaction is performed by using the alkaline protease, the walnut meal is used as an enzymolysis substrate, the concentration of the substrate can be 40-60g/1000mL, preferably 50g/1000mL, and the enzymolysis time is about 3 hours. In the enzymolysis process, in order to make the enzymolysis conditions uniform and controllable, the enzymolysis process can be performed with constant temperature shaking treatment and continuously adjusted to pH 8.7 with 1mol/L NaOH.

In at least some embodiments of the present invention, aspergillus niger may be selected for fermentation, and the bitter odor of the walnut peptide may be removed by fermentation using aspergillus niger. The fermentation conditions may be: the fermentation temperature is 30 ℃, the pH value is 6.5, the inoculation amount is 10 percent, and the fermentation time is 60 hours. When Aspergillus niger is used for fermentation, Aspergillus niger suspension can be directly inoculated in an enzymolysis system, so that the walnut meal is continuously fermented to obtain an enzymolysis fermentation product. And after the enzymolysis and fermentation are finished, carrying out enzyme deactivation treatment on the enzymolysis and fermentation system by adopting a boiling water bath to prevent excessive enzymolysis, so that the system contains the walnut polypeptide with certain biological activity. In at least some embodiments, the conditions for deactivating the enzyme in the boiling water bath are 5-10min, preferably 10min of the boiling water bath. After the enzyme deactivation treatment by boiling water bath, adjusting the pH value of the solution to 4.5, centrifuging at 4000r/min for 15min, removing protein, and taking supernatant fluid to obtain polypeptide. And then used for subsequent separation processes.

The pH value of the supernatant is adjusted to be neutral, and the mild neutral environment is more beneficial to the activity of the polypeptide. And (3) carrying out ultrafiltration on the neutral supernatant through a 5kDa ultrafiltration membrane to obtain walnut peptide liquid with the molecular weight less than 5 kDa. The prepared walnut peptide liquid can be directly used for subsequent chelation reaction. The walnut peptide powder can also be stably stored by freeze-drying the walnut peptide liquid, and is not easy to decompose. During subsequent chelation reaction, the prepared walnut peptide powder can be added with water to prepare a walnut peptide solution with a certain concentration, and then the walnut peptide solution and calcium ions are subjected to chelation reaction. In at least some embodiments of the present invention, the concentration of the walnut peptide may be formulated to be 1% when the chelation reaction is performed, and then the walnut peptide and the calcium source are mixed in a mass ratio of 1:1 to 5:1 to perform the chelation reaction.

After the chelation reaction is finished, removing a certain volume of water by using a rotary evaporator; adding the residual mixed solution into a certain volume of organic solvent, standing for a certain time, centrifuging, collecting precipitate, washing with absolute ethyl alcohol until the supernatant is added with a calcium indicator and does not change color; and (4) freeze-drying the collected precipitate to obtain the walnut peptide calcium chelate. In at least some embodiments of the invention, a rotary evaporator may be used to remove two thirds of the water in the chelating system, and the amount of organic solvent used in separating free calcium and peptide calcium chelates may be reduced. In still other embodiments of the present invention, the chelating system after removing water is added with 5-15 times, preferably 10 times, the volume of the anhydrous ethanol, and left for 2-4 hours, preferably 3 hours. The free calcium and the peptide calcium chelate can be separated economically and efficiently by the treatment. Centrifuging the chelate system after standing for 15min at 8000r/min, collecting precipitate, washing with absolute ethanol until the supernatant is discolored after adding calcium indicator; after freeze drying, the walnut peptide calcium chelate with high calcium chelation rate can be obtained.

The walnut peptide calcium chelate prepared by the method provided by the invention is determined to have the calcium ion chelating rate of over 60 percent, and the bioavailability of the walnut peptide calcium chelate is far higher than that of inorganic calcium salt, so the walnut peptide calcium chelate can be used as a calcium supplement. The prepared walnut peptide calcium chelate can be directly packaged to serve as an instant food, and can also be mixed with other edible substances or processed to serve as a food.

In at least some embodiments of the present invention, the present invention provides a method for preparing a walnut peptide calcium chelate, the method comprising: 1) soaking the walnut dregs subjected to hydraulic oil pressing by using petroleum ether to remove grease; 2) sieving the defatted walnut meal with a 60-mesh sieve to obtain walnut defatted powder; 3) preparing walnut defatted powder into a solution, adding alkaline protease, carrying out constant-temperature shaking enzymolysis, and then inoculating aspergillus niger suspension to obtain an enzymolysis fermentation product; 4) performing enzyme deactivation treatment by using boiling water bath, adjusting the pH value of the solution to 4.5, centrifuging, removing precipitate, collecting supernatant, and adjusting the pH value to be neutral; 5) ultrafiltering the supernatant through an ultrafiltration membrane with the molecular weight of 5kDa to obtain walnut peptide liquid with the molecular weight of less than 5kDa, 6) mixing the walnut peptide liquid with anhydrous calcium chloride, and oscillating at constant temperature in a water bath to perform chelation reaction; 7) removing two thirds of water in the chelate system by using a rotary evaporator, adding absolute ethyl alcohol, standing, centrifuging, collecting precipitate, washing by using the absolute ethyl alcohol until the supernatant is added with a calcium indicator, keeping the color unchanged, and freeze-drying to obtain the walnut peptide calcium chelate.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. The alkaline protease used was purchased from Beijing Soilebao Tech Co., Ltd, under the product number B8360, and the Aspergillus niger used was commercially available.