阅读说明：本技术 一种酶法制备磷酸酯淀粉的方法 (Method for preparing phosphate starch by enzyme method ) 是由 洪雁 吴梦晗 姜成辰 顾正彪 程力 李兆丰 李才明 班宵逢 于 2021-01-18 设计创作，主要内容包括：本发明公开了一种酶法制备磷酸酯淀粉的方法,属于农产品深加工以及变性淀粉制备方法领域。本发明所述的方法,包括如下步骤：(1)将淀粉和磷酸盐缓冲液混合均匀,得到淀粉乳；(2)将磷酸酶加入磷酸盐缓冲液中溶解,并加入催化离子,混合均匀、活化得到酶液；(3)将酶液分次加入淀粉乳中进行反应；(4)反应结束之后调节体系为中性,之后洗涤、离心、干燥、粉碎,得到磷酸酯淀粉；其中,步骤(1)中淀粉与步骤(2)中酶的质量比为30～300：1；步骤(1)所述淀粉乳的固形物浓度为20％～60％。本发明的方法反应温度低于40℃,反应时间小于5h,反应效率高,而且制备的磷酸酯淀粉可以高达0.806。(The invention discloses a method for preparing phosphate starch by an enzymatic method, belonging to the field of deep processing of agricultural products and preparation methods of modified starch. The method comprises the following steps: (1) uniformly mixing starch and phosphate buffer solution to obtain starch milk; (2) adding phosphatase into phosphate buffer solution for dissolving, adding catalytic ions, uniformly mixing, and activating to obtain enzyme solution; (3) adding the enzyme solution into the starch milk for reaction; (4) after the reaction is finished, adjusting the system to be neutral, and then washing, centrifuging, drying and crushing to obtain phosphate starch; wherein the mass ratio of the starch in the step (1) to the enzyme in the step (2) is 30-300: 1; the solid concentration of the starch milk in the step (1) is 20-60%. The reaction temperature of the method is lower than 40 ℃, the reaction time is less than 5h, the reaction efficiency is high, and the prepared phosphate starch can reach 0.806.)

1. A method for preparing phosphate starch by an enzymatic method is characterized by comprising the following steps:

(1) uniformly mixing starch and phosphate buffer solution to obtain starch milk;

(2) adding phosphatase into phosphate buffer solution for dissolving, adding catalytic ions, uniformly mixing, and activating to obtain enzyme solution;

(3) adding the enzyme solution obtained in the step (2) into the starch milk obtained in the step (1) for reaction;

(4) after the reaction is finished, adjusting the system to be neutral, and then washing, centrifuging, drying and crushing to obtain phosphate starch;

wherein the mass ratio of the starch in the step (1) to the phosphatase in the step (2) is 30-300: 1.

2. the method of claim 1, wherein the catalytic ions of step (2) comprise Cu²⁺、Zn²⁺、Fe^{2 +}、Ca²⁺、Mg²⁺、Fe³⁺(ii) a The concentration of the catalytic ions in the enzyme solution is 0.001-1M.

3. The method according to claim 1 or 2, wherein the reaction in step (3) has a pH of 7 to 10.

4. The method according to any one of claims 1 to 3, wherein the temperature of the reaction in the step (3) is 20 to 40 ℃.

5. The method according to any one of claims 1 to 4, wherein the reaction time in step (3) is 1 to 5 hours.

6. The method according to any one of claims 1 to 5, wherein the molar concentration of the phosphate buffer solution in step (1) and step (2) is 0.1 to 1.0M; wherein the phosphate comprises orthophosphate, polyphosphate, metaphosphate and pyrophosphate.

7. The method according to any one of claims 1 to 6, wherein the phosphatase in the step (2) comprises acid phosphatase or alkaline phosphatase.

8. The method according to any one of claims 1 to 7, wherein the concentration of starch in the reaction system in the step (3) is 25 to 35% (w/w).

9. Phosphate starch prepared by the process according to any one of claims 1 to 8.

10. Use of the phosphate starch according to claim 9 in the fields of food, cosmetics, medicine, paper, building materials, textiles, agriculture, and the like.

Technical Field

The invention relates to a method for preparing phosphate starch by an enzymatic method, belonging to the field of deep processing of agricultural products and preparation methods of modified starch.

Background

The phosphate starch is a starch derivative obtained by esterification reaction of starch and phosphate, and has excellent use characteristics of easy gelatinization, high transparency, high viscosity, good freeze-thaw stability and the like, so that the phosphate starch is widely applied to food additives and non-food additives. The phosphate starch can be used as an emulsifier, a thickening stabilizer and applied to various foods, such as cream, cheese, jam, frozen food and the like, and in the non-food industry, the phosphate starch can be used as a flocculating agent and an ion exchanger, and can also be used as a hydrogel and a high water absorbent in cosmetics, medicines and agriculture.

The phosphatase is an important regulation enzyme which is widely existed in plants, animals and microorganisms and can catalyze the hydrolysis and transfer reactions of various phosphate groups; by controlling the enzyme reaction conditions, the enzyme reaction effect can be converted between hydrolysis and synthesis; compared with kinase using ATP (adenosine triphosphate) as a donor, phosphatase has certain economic advantages compared with phosphate as a phosphorus donor.

Disclosure of Invention

[ problem ] to

The current methods for producing phosphate starch mainly comprise the following steps: dry, wet, semi-dry, extrusion, microwave. Wherein, the wet method has low product yield, and the dry method and the semi-dry method are not easy to be uniform in stirring, so that the product quality is unstable. In addition, the starch produced by the chemical method has the problems of complex product, more byproducts, difficult separation and the like.

[ solution ]

In order to solve at least one of the above problems, the present invention provides a method for enzymatically preparing phosphate starch. Compared with starch phosphate produced by a chemical method, the reaction efficiency and yield are improved by the biological enzyme method; the quality of the obtained product is uniform due to the selectivity of the enzyme; in addition, the enzymatic reaction conditions are mild, the process is simple, the reaction efficiency is high, the number of used equipment is small, and the method has an industrial production prospect.

The first object of the present invention is to provide a method for preparing phosphate starch by an enzymatic method, comprising the steps of:

(1) uniformly mixing starch and phosphate buffer solution to obtain starch milk;

(2) adding phosphatase into phosphate buffer solution for dissolving, adding catalytic ions, uniformly mixing, and activating to obtain enzyme solution;

(3) adding the enzyme solution obtained in the step (2) into the starch milk obtained in the step (1) for reaction;

(4) after the reaction is finished, adjusting the system to be neutral, and then washing, centrifuging, drying and crushing to obtain phosphate starch;

wherein the mass ratio of the starch in the step (1) to the phosphatase in the step (2) is 30-300: 1; the volume ratio of the phosphate buffer solution in the step (1) to the phosphate buffer solution in the step (2) is 1.0-2.5: 1; the solid concentration of the starch milk in the step (1) is 20-60% (m/v).

In one embodiment of the invention, the mass ratio of starch in step (1) to enzyme in step (2) is 30: 0.1 to 0.5.

In one embodiment of the present invention, the concentration of starch in the reaction system in step (3) is 25 to 35% (w/w), and more preferably 30%.

In one embodiment of the present invention, the starch in step (1) comprises potato starch, common corn starch, waxy corn starch, tapioca starch, rice starch, pea starch, sweet potato starch, mung bean starch.

In one embodiment of the present invention, the molar concentration of the phosphate buffer solution in step (1) and step (2) is 0.1-1.0M; wherein the phosphate comprises orthophosphate, polyphosphate, metaphosphate and pyrophosphate.

In one embodiment of the present invention, the starch milk in step (1) needs to be maintained at 20-40 ℃, so that the optimal reaction temperature of the starch milk and the optimal reaction temperature of the phosphatase are consistent, and the reaction is convenient.

In one embodiment of the present invention, the temperature of the activation in the step (2) is 20 to 40 ℃ and the time is 10 to 50 min.

In one embodiment of the present invention, the phosphatase in the step (2) comprises acid phosphatase or alkaline phosphatase.

In one embodiment of the present invention, the catalytic ion in step (2) includes but is not limited to Cu²⁺、Zn²⁺、Fe^{2 +}、Ca²⁺、Mg²⁺、Fe³⁺(ii) a The concentration of the catalytic ions in the enzyme solution is 0.001-1M; more preferably, the catalytic ions are zinc chloride and magnesium chloride, and the concentrations thereof in the enzyme solution are 0.1M, respectively.

In one embodiment of the invention, the adding in the step (2) is divided into 2-5 times, and the adding is performed for multiple times to continuously promote the forward reaction.

In one embodiment of the invention, the pH of the reaction in the step (3) is 7-10, and the pH is adjusted by adding an alkaline compound into the starch milk, wherein the alkaline compound includes but is not limited to carbonate and hydroxide.

In one embodiment of the present invention, the reaction time in step (3) is 1-5 h.

In one embodiment of the present invention, the temperature of the reaction in step (3) is 20 to 40 ℃.

In one embodiment of the present invention, the adjusting the system to be neutral in step (4) specifically comprises: the pH of the system is neutralized to 7 by using an acid solution, wherein the acid solution used includes, but is not limited to, a hydrochloric acid solution, a carbonic acid solution, an acetic acid solution, and the like, and it is further preferable that the pH of the starch milk is adjusted to 7 by using the hydrochloric acid solution.

In one embodiment of the present invention, the washing in step (4) is water washing.

In one embodiment of the invention, the centrifugation in the step (4) is 3500 rpm-8000 rpm for 5-20 min.

In one embodiment of the present invention, the drying in step (4) can be performed by various conventional apparatuses, and may be, but not limited to, freeze drying, vacuum drying, forced air drying, and spray drying.

In one embodiment of the present invention, the pulverization in step (4) may be carried out by a pulverizer, and it is required that the starch after sieving is passed through a 100-mesh sieve.

The second object of the invention is phosphate starch prepared by the method of the invention.

The third purpose of the invention is the application of the phosphate starch in the fields of food, cosmetics, medicine, paper making, building materials, textile, agriculture and the like.

In one embodiment of the invention, the application in the food field comprises using phosphate starch as an emulsifier, a thickening stabilizer in food products, such as cream, cheese, jam, yoghurt, meat products, and the like.

In one embodiment of the invention, the phosphate starch is used as a flocculant, an ion exchanger and a high water absorbent in the fields of building materials, textiles, papermaking, cosmetics, medicines and agriculture.

[ advantageous effects ]

(1) Compared with the chemical production process, the method has the advantages that the reaction temperature is lower than 40 ℃, the reaction time is less than 5 hours, the energy consumption in the reaction process is reduced, and the reaction efficiency is high.

(2) The phosphate starch prepared by the method has high substitution degree, and when the phosphate donor is single and the molar concentration is below 1.0M, the substitution degree of the prepared phosphate starch reaches more than 0.011 and can reach 0.806.

(3) The phosphate starch prepared by the method has stable quality, and the enzyme reaction has selectivity, so that the product of the enzyme reaction is simpler than a chemical method under the condition of controlling the reaction temperature and the reaction pH, and the side reaction is less, thereby being convenient for product application.

Drawings

FIG. 1 is a process flow diagram of the method for preparing phosphate starch by an enzymatic method according to the present invention.

FIG. 2 is a graph showing gelatinization characteristics of a common corn native starch and a phosphate starch of example 1.

FIG. 3 is a graph showing gelatinization characteristics of a common corn native starch and phosphate starch of example 2.

FIG. 4 is a graph showing gelatinization characteristics of a common corn native starch and a phosphate starch of example 3.

FIG. 5 is a graph showing gelatinization characteristics of a common corn native starch and phosphate starch of example 4.

FIG. 6 is a graph showing gelatinization characteristics of a common corn native starch and phosphate starch of example 5.

FIG. 7 is a graph showing gelatinization characteristics of a common corn native starch and phosphate starch of example 6.

FIG. 8 is a graph showing gelatinization characteristics of a common corn native starch and phosphate starch of example 7.

FIG. 9 is a graph showing gelatinization characteristics of a conventional corn native starch and phosphate starch of comparative examples 1 and 2.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The test method comprises the following steps:

1. phosphate starch degree of substitution determination

Determination of bound phosphorus: according to the first method in the national standard GB 5009.87-2016, molybdenum blue spectrophotometry; the resulting phosphorus content was calculated as the combined phosphorus mass fraction.

The method for measuring the free phosphorus comprises the following steps: weighing 2g of sample, dissolving with 1M dilute hydrochloric acid, fixing the volume, and developing color by the method combined with the phosphorus determination method.

The degree of substitution is calculated as follows:

2. determination of gelatinization Properties

Testing the gelatinization properties of the samples using a rapid visco-analyzer (RVA) to prepare a 6% (w/w) sample concentration suspension in an RVA aluminum box; the measurement was carried out according to the standard procedure 2 specified in AACC.

Example 1

A method for preparing phosphate starch by an enzymatic method is shown in figure 1, and comprises the following steps:

(1) adding 30g of corn starch into 50g of 0.25M sodium pyrophosphate buffer solution, uniformly stirring to prepare starch milk, and keeping the temperature at 37 ℃;

(2) dissolving 0.1g alkaline phosphatase enzyme powder in 20g 0.25M sodium pyrophosphate buffer solution, adding zinc chloride and magnesium chloride, and activating at 37 deg.C for 30 min; obtaining enzyme solution; wherein the concentrations of zinc chloride and magnesium chloride are respectively 0.018M;

(3) uniformly adding the enzyme solution obtained in the step (2) into the starch milk obtained in the step (1) twice for reaction, wherein the mass concentration of starch in a reaction system is 30%, adjusting the pH value to 8, and reacting for 2 hours at 37 ℃;

(4) after the reaction is finished, neutralizing the pH value of the reaction system to 7 by hydrochloric acid solution, washing by water, centrifuging at 4000rpm for 10min to obtain precipitate, drying, crushing, and sieving by a 100-mesh sieve to obtain the phosphate starch (the gelatinization characteristic curve is shown in figure 2).

Example 2

The pH was adjusted to "pH 8" in step (3) of example 1 to "pH not adjusted", and the rest was kept the same as in example 1, to obtain phosphate starch (gelatinization characteristic curve shown in FIG. 3).

Example 3

The amount of alkaline phosphatase enzyme powder used in step (2) of example 2 was adjusted to 0.5g, and the rest was the same as in example 2, to obtain phosphate starch (gelatinization characteristic curve shown in FIG. 4).

Example 4

The reaction conditions in step (3) of example 1 were adjusted to 1 hour, and the rest was kept the same as in example 1, to obtain phosphate starch (gelatinization characteristic curve shown in FIG. 5).

Example 5

The concentrations of zinc chloride and magnesium chloride in step (2) of example 1 were adjusted to 0.1M, respectively, and the other concentrations were kept in agreement with those of example 1, to obtain phosphate starch (gelatinization characteristic curve shown in FIG. 6).

Example 6

The concentrations of zinc chloride and magnesium chloride in step (2) of example 1 were adjusted to 0.2M, respectively, and the other concentrations were kept in agreement with those of example 1, to obtain phosphate starch (gelatinization characteristic curve shown in FIG. 7).

Example 7

The concentrations of zinc chloride and magnesium chloride in step (2) of example 1 were adjusted to 0.05M, respectively, and the other concentrations were kept in agreement with those of example 1, to obtain phosphate starch (gelatinization characteristic curve shown in FIG. 8).

Examples 1-7 were tested for performance and the results were as follows:

as can be seen from tables 1 and 2: during the reaction, the reaction can be controlled by controlling the pH, the enzyme adding amount, the reaction time and the concentration of the catalytic ions in the system. As the degree of substitution increases. The peak viscosity generally increases and then decreases, and the composition of the starch phosphate in the product changes.

Table 1 test results for starches of examples 1-4

Example (b)	Example 1	Example 2	Example 3	Example 4
					Degree of substitution	0.054	0.011	0.088	0.006
Gelatinization temperature/. degree.C	75.4±1.2	75.6±0.8	75.7±0.4	74.7±0.3
					Peak viscosity/(mPa. multidot.s)	587±145.8	609±11.3	442.3±6.1	667±22.6
Final viscosity/(mPas)	658.5±176.4	742.3±26.6	582.3±11.7	765±21.2
					Disintegration value/(mPa. s)	127.2±43	120±5.3	105±2.0	145±12.7
Retrogradation value/(mPa. s)	198.8±75.2	253.3±21.4	245±7.5	243±11.3

Table 2 test results for examples 5-7

Example (b)	Example 5	Example 6	Example 7
				Degree of substitution	0.392	0.806	0.181
Gelatinization temperature/. degree.C	74.4±0.14	75±0.5	73.9±0.1
				Peak viscosity (mPa. s)	473.5±9.2	358.5±3.5	542±11.3
Final viscosity/(mPas)	678.5±12.0	492±4.24	742.5±20.5
				Disintegration value/(mPa. s)	81±1.4	57±1.41	114±7.1
Retrogradation value/(mPa. s)	286±4.2	190.5±2.1	314.5±16.3

Comparative example 1

Phosphate starch was obtained in the same manner as in example 1 except that "the starch milk obtained in step (1) was mixed with 30ml of 0.25M sodium pyrophosphate buffer solution" in the adjustment step (3) in which step (2) in example 1 was omitted.

Comparative example 2

The pH control in example 1 was changed to pH 10.5 instead of 8, and the procedure was otherwise the same as in example 1 to obtain phosphate starch (gelatinization characteristic curve shown in fig. 9).

Table 3 test results for starches of comparative examples 1, 2

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.