CN112725312A - Preparation method of complex enzyme and resistant dextrin - Google Patents
Preparation method of complex enzyme and resistant dextrin Download PDFInfo
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Abstract
The invention discloses a complex enzyme and a method for preparing resistant dextrin by matching with synchronous extrusion, belonging to the technical field of deep processing of starch. The invention selects the proportion of the compound amylase based on different glycosidic bond action sites, carries out high-shear co-extrusion processing on the compound enzyme and the starch, can finely regulate and control the breaking sequence and degree of the amylose and the branch chain of the starch through thermo-mechanical multi-physical field coupling (mum-level non-directional shearing) in an extrusion cavity and synergistic enzymolysis fixed-point depolymerization (nm-level specific shearing),to form a differential chain length distribution. The method takes a built-in double-screw cavity as a reactor for high-efficiency starch enzymolysis, reduces the dosage of enzyme liquid under the condition of increasing the axial diffusion rate, the residence time and the action area, and forms high-crystallinity resistant dextrin (M)w: 0.5 to 8 kDa). The invention simplifies the enzymolysis steps before and after extrusion, and further realizes a starch micro-domain regulation and control method with fine regulation and control, high efficiency, continuity, water saving and economy.
Description
Technical Field
The invention relates to preparation of polysaccharide products with high resistance (digestion resistance) structures, in particular to a complex enzyme and a processing method for regulating and controlling the ordered degradation degree of starch chains through multi-physical field coupling and synergetic directional enzymolysis under the extrusion environment with high shearing, heat energy and mechanical energy input, thereby efficiently forming resistant dextrin.
Background
Resistant Dextrin (RD) is a small molecular water-soluble dietary fiber with the structure recombined after gelatinization and melting of starch or glycosylation transfer, contains structures such as alpha-1, 2 glycosidic bond, alpha-1, 3 glycosidic bond, dextran and beta-1, 6 glucoside and the like in the molecule, and has the effects of preventing colon cancer, reducing cholesterol level, regulating blood sugar metabolism, promoting Ca metabolism2+、Fe3+Mineral absorption and the like. However, since enzyme resistance generally derives from a highly dense crystalline lamellar arrangement structure, modified products thereof often have limitations of high molecular weight, high hardness, low palatability and the like, and thus market application thereof is limited. Therefore, the significance of preparing the edible safe low molecular weight RD and the derived functional food thereof according to the microstructure regulation strategy is great.
At present, the RD is mainly prepared by a physical method, a chemical method, a biological enzyme method and the like. However, chemical methods tend to induce the starch structure to be non-homogeneous by chemical agents, and the chemical agent residue and production wastewater discharge of the obtained product are still problems. Thus, the advantages of physical and biological enzymatic means in the preparation of destructured starch are particularly pronounced. However, although the biological enzymes are safe and reliable for human health, high enzyme amount or long enzymolysis time is often required to improve the yield of the modified product, which results in expensive preparation cost and low efficiency, so that the limitation in practical production is obvious. Extrusion is a common processing mode of starch-based materials, and is a continuous physical processing technology integrating units such as transportation, mixing, heating, shearing, molding and the like. The extrusion cavity is used as an enzyme reactor, and can provide an efficient and short-time enzyme reaction micro-mixing environment. In recent years, enzymatic extrusion has been attempted for use in the production of yellow wine, white spirit, starch sugar, and the like. However, in practical applications, the enzymolysis pretreatment time of the above-mentioned combination means is still long, the precise collocation of the complex enzyme is not considered, and it is difficult to reasonably cooperate with the multi-physical-field coupled high-efficiency processing environment, so a new idea needs to be further developed on the basis of the prior art for the bio-enzyme-extrusion processing, so as to obtain RD with controllable Chain Length Distribution (CLDs), branching degree or molecular weight.
Disclosure of Invention
The invention provides a complex enzyme, wherein the action site of the complex enzyme comprises alpha-1, 4 glycosidic bond or beta-1, 4 glycosidic bond, alpha-1, 6 glycosidic bond, and the goal of directional melting of starch chains is achieved by regulating and controlling the compounding ratio of the complex enzyme, so that a retrogradation matrix with low polymerization Degree (DP) CLDs is formed.
The invention further provides a method for efficiently preparing the low molecular weight RD by using the complex enzyme, which is characterized in that a regenerated CLDS foundation is formed under the induction of the complex enzyme by utilizing the temperature zone distribution of gradient temperature rise according to the limited gelatinization enzymolysis sequence of branched chain unwinding and straight chain classification, and then the RD with a target structure is obtained by recrystallization.
As one aspect of the invention, the invention provides a complex enzyme, which comprises A-type amylase and B-type amylase, and the preparation method comprises the following steps: mixing the A-type amylase and the B-type amylase in acetate buffer solution (pH 5.2), and pre-activating in water bath at 50 deg.C for 30 min. The ratio of the total enzyme activity of the A-type amylase to the total enzyme activity of the B-type amylase is 1: 1.5-6.
Furthermore, the A-type amylase is selected from one or more of alpha-amylase and beta-amylase according to any mixture ratio.
Further, the B-type amylase is one or more of pullulanase and isoamylase.
As another aspect of the invention, the complex enzyme provided by the invention is applied to the preparation of RD.
As another aspect of the present invention, the present invention provides a method for preparing RD, the method comprising: mixing the complex enzyme and starch, and introducing the mixed material into the cavity after each set temperature zone loaded by the extrusion equipment is stable. Cooling and regenerating after screw shearing and co-extrusion treatment to form the low molecular weight RD with high crystallinity. The screw shearing and co-extrusion treatment adopts a temperature field with gradient temperature rise, namely at least comprises a front section of low-temperature screw shearing and co-extrusion treatment and a rear section of high-temperature screw shearing and co-extrusion treatment; the treatment temperature of the former stage is 70 ℃ or lower, and the treatment temperature of the latter stage is higher than that of the former stage and at least 60 ℃ or higher.
During extrusion, the low temperature field causes the class B amylase to first hydrolyze at the alpha-1, 6 glycosidic bond sites, such that the branched double helix outside the starch crystal cluster melts. The A-type amylase is pre-activated again through a low-temperature region, and is matched with axial mixing and conveying of a screw kneading region and a reverse blocking region, so that the A-type amylase can effectively hydrolyze alpha-1, 4 glycosidic bonds or beta-1, 4 glycosidic bonds in a high-temperature region, and under the condition of increasing the contact area of the A-type amylase and starch granules, the straight chain is more easily degraded. The inside of the extruding machine cavity is essentially in the environment of high substrate of enzyme, and the reaction coordination and replacement speed of the enzyme center and the substrate is accelerated under the enzymolysis form that the substrate contains enzyme and the center radiates to the periphery, thereby greatly improving the action efficiency of the enzyme. In addition, the extrusion is mostly a non-molecular level structural modification means, and for molecular level enzyme, the activity reduction is mostly caused by the application of extreme high temperature and high pressure conditions, however, the gradient temperature rise program adopted by the invention enables the pressure in the cavity of the extruder to be lower than 0.8Mpa, so that the amylase can fully exert the enzyme activity in a short time, and the extruded material enters a die head area for gelatinization.
Furthermore, the enzyme activity of the A-type amylase is 5-20U/g, the enzyme activity of the B-type amylase is 7.5-120U/g, and the enzyme activity is calculated on a material dry basis (g).
Further, the screw shearing and co-extrusion treatment comprises five-section temperature zone extrusion, wherein the temperature is sequentially 20-50 ℃ (zone I), 40-70 ℃ (zone II), 60-90 ℃ (zone III), 80-110 ℃ (zone IV) and 100-130 ℃ (die head zone), and the temperature of the five-section temperature zone is sequentially increased; the rotating speed of the screw is 150-400 r/min.
Further, the extrusion material is one or more of corn starch, high amylose corn starch, waxy corn starch, potato starch, wheat starch, tapioca starch, sweet potato starch, and rice starch.
As a preferable scheme, before extrusion, the water content of the mixed material of the complex enzyme and the starch is adjusted to be 20-40 wt%.
Preferably, the cooling regeneration is as follows: and (3) placing the structural recombinant amyloid obtained after the screw shearing co-extrusion treatment at the temperature of 0-10 ℃, and cooling and recrystallizing for 2-8 days.
The beneficial technical effects of the invention are as follows:
1. compared with the traditional enzymatic extrusion processing technology, the invention reasonably utilizes the synchronous action of screw extrusion (mum-level non-directional shearing) and enzyme (nm-level specific shearing) to regulate and control the degradation sequence and degree of starch ordered (single/double helix), unordered (amorphous area) and molecular structure, thereby forming the difference CLDS, and forming resistance helical structures which are mainly made of different DP chains on the basis of the difference CLDS by crystallization, wherein the resistance helical structures comprise branched double helix, straight chain single helix, straight chain-branched chain hybrid helix and the like.
2. The CLDS regulation and control strategy adopted by the invention enables the compound enzyme method extrusion to become a high-efficiency starch micro-domain degradation and recombination means, so that on one hand, the pretreatment time of enzyme liquid is shortened, and a plurality of processes of enzyme method auxiliary extrusion are simplified, and on the other hand, the relation between the starch CLDS and the resistant fine structure thereof can be established for guiding production practice.
Drawings
FIG. 1 is a process flow diagram of a RD prepared according to the present invention;
fig. 2 is an X-ray diffraction (XRD) pattern of RD prepared in the examples of the present invention and the comparative example, which is used to illustrate the change of crystalline form of RD prepared in the present invention.
Detailed Description
The invention is further illustrated by the following examples, which are intended to be illustrative and not limiting in scope.
Example 1
A method for preparing RD by synchronous extrusion and restriction enzymolysis comprises the following steps:
(1) pre-activating mixed enzyme liquid: according to the moisture content and the dry basis weight of starch of the pre-adjusted mixed material, the concentration of enzyme solution for preparing the A-type amylase (high temperature resistant alpha-amylase) is as follows: 5U/g; the concentration of the enzyme solution for preparing the B-type amylase (pullulanase) is as follows: 30U/g. And mixing the enzyme solutions to obtain the mixed enzyme solution of high-temperature resistant alpha-amylase and pullulanase in a ratio of 1: 6. Preactivating for 30min in 50 deg.C water bath before extrusion;
(2) temperature-controlled high-shear extrusion: adjusting the water content of common corn starch to 40 wt%, and synchronously adding the mixed enzyme liquid compounded in the step (1) during extrusion. The system parameters of the double-screw extruder barrel are as follows: the temperature zones are distributed at 40 ℃, 60 ℃, 80 ℃, 100 ℃ and 120 ℃ (the die head zone) in sequence; the rotating speed of the screw is 250r/min, and amyloid is extruded in one step;
(3) and (3) rejuvenation control: the extruded amyloid is placed at 0 ℃ and cooled for 2 days. After vacuum freeze drying, grinding and sieving by a 200-mesh sieve to obtain the RD;
(4) and (3) detecting the molecular weight: adopting a high performance liquid phase molecular exclusion chromatography and a system (HPSEC-MALLS-RI) for combining a multi-angle light scattering instrument and a refraction detector, and calculating and calibrating by using a Mark-Houwink parameter to obtain the weight average molecular weight (Mw) of a sample;
(5) in vitro simulated digestion: the samples were digested at 37 ℃ with the enzyme mix prepared in situ. The enzyme activity ratio of the mixed enzyme solution is as follows: pancreatin (500U/ml), glucosidase (700U/ml) and invertase (400U/ml). The absorbance at Free-sugar Glucose (FSG), Glucose after digestion for 20min (Glucose of 20, G20), Glucose after digestion for 120min (Glucose of 120, G120) and Total sugar (TG) was measured using a Glucose oxidase assay kit (GOPOD-FORMAT) and the yield (%) of Resistant Starch (RS) was calculated as follows:
RS(％)＝(TG-G120)×0.9/TS×100
wherein, the polysaccharide glucose is converted into starch values of different digestion components by using a conversion coefficient of 0.9, and TS is total starch mass (g).
(6) Detection of CLDS: the number distribution of amylopectin chains is determined in N-CHO coated capillary by gel-assisted sugar electrophoresis (PA-800 Plus Face system), i.e. N is obtained by using detector signalde(X)。
The RD produced in this example has a Mw of 6.128kDa, a RD content of 46.60% in starch and a low DP amylopectin (DP <6) content of 58.24%.
Example 2
A method for preparing RD by synchronous extrusion and restriction enzymolysis comprises the following steps:
(1) pre-activating mixed enzyme liquid: according to the moisture content and the dry base quality of starch of the pre-adjusted mixed material, the concentration of enzyme solution for preparing the A-type amylase (moderate temperature alpha-amylase) is as follows: 10U/g; the concentration of the enzyme solution for preparing the B-type amylase (pullulanase) is as follows: 30U/g. And mixing the enzyme solutions to obtain a mixed enzyme solution which is compounded into medium-temperature alpha-amylase and pullulanase in a ratio of 1: 3. Preactivating for 30min in 50 deg.C water bath before extrusion;
(2) temperature-controlled high-shear extrusion: adjusting the water content of common corn starch to 30 wt%, and synchronously adding the mixed enzyme liquid compounded in the step (1) during extrusion. The system parameters of the double-screw extruder barrel are as follows: the temperature zones are distributed at 30 ℃, 50 ℃, 70 ℃, 90 ℃ and 110 ℃ (the die head zone) in sequence; the rotating speed of the screw is 200r/min, and amyloid is extruded in one step;
(3) and (3) rejuvenation control: the extruded amyloid is placed at 5 ℃ and cooled for 5 days. After vacuum freeze drying, grinding and sieving by a 200-mesh sieve to obtain the RD;
(4) and (3) detecting the molecular weight: adopting a high performance liquid phase molecular exclusion chromatography and a system (HPSEC-MALLS-RI) for combining a multi-angle light scattering instrument and a refraction detector, and calculating and calibrating by using a Mark-Houwink parameter to obtain the weight average molecular weight (Mw) of a sample;
(5) in vitro simulated digestion: the samples were digested at 37 ℃ with the enzyme mix prepared in situ. The enzyme activity ratio of the mixed enzyme solution is as follows: pancreatin (500U/ml), glucosidase (700U/ml) and invertase (400U/ml). The absorbances at FSG, G20, G120 and TG were measured using a glucose oxidase detection kit (GOPOD-FORMAT) and the RS yield (%) was calculated as follows:
RS(％)＝(TG-G120)×0.9/TS×100
wherein, the conversion coefficient of 0.9 is used for converting glucose into starch values of different digestion components, and TS is total starch mass (g).
(6) Detection of CLDS: the number distribution of amylopectin chains is determined in N-CHO coated capillary by gel-assisted sugar electrophoresis (PA-800 Plus Face system), i.e. N is obtained by using detector signalde(X)。
The RD produced in this example has a Mw of 2.835kDa, a RD content of 58.27% in starch and a low DP amylopectin (DP <6) content of 64.73%.
Example 3
A method for preparing RD by synchronous extrusion and restriction enzymolysis comprises the following steps:
(1) pre-activating mixed enzyme liquid: according to the moisture content and the dry basis weight of starch of the pre-adjusted mixed material, the concentration of enzyme solution for preparing the A-type amylase (beta-amylase) is as follows: 20U/g; the concentration of the enzyme solution for preparing the B-type amylase (isoamylase) is as follows: 30U/g. And mixing the enzyme solutions to prepare a mixed enzyme solution of beta-amylase and isoamylase in a ratio of 1: 1.5. Preactivating for 30min in 50 deg.C water bath before extrusion;
(2) temperature-controlled high-shear extrusion: adjusting the water content of common corn starch to 20 wt%, and synchronously adding the mixed enzyme liquid compounded in the step (1) during extrusion. The system parameters of the double-screw extruder barrel are as follows: the temperature zone distribution is 20 ℃, 40 ℃, 60 ℃, 80 ℃ and 100 ℃ (die head zone) in sequence; the rotating speed of the screw is 150r/min, and amyloid is extruded in one step;
(3) and (3) rejuvenation control: the extruded amyloid is placed at 10 ℃ and cooled for 8 days. After vacuum freeze drying, grinding and sieving by a 200-mesh sieve to obtain the RD;
(4) and (3) detecting the molecular weight: adopting a high performance liquid phase molecular exclusion chromatography and a system (HPSEC-MALLS-RI) for combining a multi-angle light scattering instrument and a refraction detector, and calculating and calibrating by using a Mark-Houwink parameter to obtain the weight average molecular weight (Mw) of a sample;
(5) in vitro simulated digestion: the samples were digested at 37 ℃ with the enzyme mix prepared in situ. The enzyme activity ratio of the mixed enzyme solution is as follows: pancreatin (500U/ml), glucosidase (700U/ml) and invertase (400U/ml). The absorbances at FSG, G20, G120 and TG were measured using a glucose oxidase detection kit (GOPOD-FORMAT) and the RS yield (%) was calculated as follows:
RS(％)＝(TG-G120)×0.9/TS×100
wherein, the conversion coefficient of 0.9 is used for converting glucose into starch values of different digestion components, and TS is total starch mass (g).
(6) Detection of CLDS: the number distribution of amylopectin chains is determined in N-CHO coated capillary by gel-assisted sugar electrophoresis (PA-800 Plus Face system), i.e. N is obtained by using detector signalde(X)。
The RD produced in this example had a Mw of 0.908kDa, a RD content of 65.43% in starch and a low DP amylopectin (DP <6) content of 69.22%.
Comparative example 1
A method for preparing RD by synchronous extrusion comprises the following steps:
(1) to prove the regulation and control effect of the complex enzyme on the starch micro-domain in the invention, the comparative example is set as no enzyme extrusion, i.e. the complex enzyme is not added;
(2) temperature-controlled high-shear extrusion: adjusting the moisture content of common corn starch to 30 wt%. The system parameters of the double-screw extruder barrel are as follows: the temperature zones are distributed at 30 ℃, 50 ℃, 70 ℃, 90 ℃ and 110 ℃ (the die head zone) in sequence; the rotating speed of the screw is 150r/min, and amyloid is extruded in one step;
(3) and (3) rejuvenation control: the extruded amyloid is placed at 4 ℃ and cooled for 4 days. After vacuum freeze drying, grinding and sieving by a 200-mesh sieve to obtain the RD;
(4) and (3) detecting the molecular weight: adopting a high performance liquid phase molecular exclusion chromatography and a system (HPSEC-MALLS-RI) for combining a multi-angle light scattering instrument and a refraction detector, and calculating and calibrating by using a Mark-Houwink parameter to obtain the weight average molecular weight (Mw) of a sample;
(5) in vitro simulated digestion: the samples were digested at 37 ℃ with the enzyme mix prepared in situ. The enzyme activity ratio of the mixed enzyme solution is as follows: pancreatin (500U/ml), glucosidase (700U/ml) and invertase (400U/ml). The absorbances at FSG, G20, G120 and TG were measured using a glucose oxidase detection kit (GOPOD-FORMAT) and the RS yield (%) was calculated as follows:
RS(％)＝(TG-G120)×0.9/TS×100
wherein, the conversion coefficient of 0.9 is used for converting glucose into starch values of different digestion components, and TS is total starch mass (g).
(6) Detection of CLDS: the number distribution of amylopectin chains is determined in N-CHO coated capillary by gel-assisted sugar electrophoresis (PA-800 Plus Face system), i.e. N is obtained by using detector signalde(X)。
The sample prepared in this example had a Mw of 37.23kDa, a RS content of 42.50% and a low DP branch (DP <6) content of 19.52%, i.e., no successful preparation of small molecular weight RD.
Comparative example 2
A preparation method of RD by synchronous extrusion and single enzymolysis comprises the following steps:
(1) to prove the regulation and control effect of the complex enzyme on the starch micro-domain in the invention, the embodiment is configured as a single amylase combined extrusion processing, that is, the concentration of the enzyme solution for preparing the A-type amylase (beta-amylase) is as follows: 20U/g. Preactivating for 30min in 50 deg.C water bath before extrusion;
(2) temperature-controlled high-shear extrusion: adjusting the moisture content of common corn starch to 30 wt%. The system parameters of the double-screw extruder barrel are as follows: the temperature zone distribution is 20 ℃, 40 ℃, 60 ℃, 80 ℃ and 100 ℃ (die head zone) in sequence; the rotating speed of the screw is 150r/min, and amyloid is extruded in one step;
(3) and (3) rejuvenation control: the extruded amyloid is placed at 4 ℃ and cooled for 4 days. After vacuum freeze drying, grinding and sieving by a 200-mesh sieve to obtain the RD;
(4) and (3) detecting the molecular weight: adopting a high performance liquid phase molecular exclusion chromatography and a system (HPSEC-MALLS-RI) for combining a multi-angle light scattering instrument and a refraction detector, and calculating and calibrating by using a Mark-Houwink parameter to obtain the weight average molecular weight (Mw) of a sample;
(5) in vitro simulated digestion: the samples were digested at 37 ℃ with the enzyme mix prepared in situ. The enzyme activity ratio of the mixed enzyme solution is as follows: pancreatin (500U/ml), glucosidase (700U/ml) and invertase (400U/ml). The absorbances at FSG, G20, G120 and TG were measured using a glucose oxidase detection kit (GOPOD-FORMAT) and the RS yield (%) was calculated as follows:
RS(％)＝(TG-G120)×0.9/TS×100
wherein, the conversion coefficient of 0.9 is used for converting glucose into starch values of different digestion components, and TS is total starch mass (g).
(6) Detection of CLDS: the number distribution of amylopectin chains is determined in N-CHO coated capillary by gel-assisted sugar electrophoresis (PA-800 Plus Face system), i.e. N is obtained by using detector signalde(X)。
The sample prepared in this example had a Mw of 24.86kDa, an RS content of 46.54% and a low DP branch (DP <6) content of 27.13%, i.e., no successful preparation of small molecular weight RD.
Claims (9)
1. The compound enzyme is characterized by comprising a class A amylase and a class B amylase, wherein the action site of the class A amylase is an alpha-1, 4 glycosidic bond or a beta-1, 4 glycosidic bond, and the action site of the class B amylase is an alpha-1, 6 glycosidic bond; the ratio of the total enzyme activity of the A-type amylase to the total enzyme activity of the B-type amylase is 1: 1.5-6.
2. The complex enzyme according to claim 1, wherein the A-type amylase is selected from one or more of alpha-amylase and beta-amylase according to any proportion.
3. The complex enzyme according to claim 1, wherein the B-type amylase is one or more of pullulanase and isoamylase.
4. The use of the complex enzyme of claim 1 in the preparation of resistant dextrin.
5. A preparation method of resistant dextrin is characterized in that: the compound enzyme and starch mixture of claim 1 is processed by screw shearing and co-extrusion, and cooled to form the low molecular weight resistant dextrin with high crystallinity after regeneration. In the mixed material, the enzyme activity of the A-type amylase is 5-20U/g, the enzyme activity of the B-type amylase is 7.5-120U/g, and the enzyme activity is calculated on a material dry basis (g).
6. The preparation method according to claim 5, wherein the screw shearing co-extrusion treatment comprises five-section temperature zone extrusion, the temperature is 20-50 ℃, 40-70 ℃, 60-90 ℃, 80-110 ℃, 100-130 ℃ in sequence, and the temperature of the five-section temperature zone is increased in sequence; the rotating speed of the screw is 150-400 r/min.
7. The method of claim 5, wherein the starch is one or more of corn starch, high amylose corn starch, waxy corn starch, potato starch, wheat starch, tapioca starch, sweet potato starch, rice starch.
8. The preparation method of claim 5, wherein the water content of the mixed material of the complex enzyme and the starch is 20-40 wt%.
9. The method of claim 5, wherein the cooling regeneration is: and (3) placing the structural recombinant amyloid obtained after the screw shearing co-extrusion treatment at the temperature of 0-10 ℃, and cooling and recrystallizing for 2-8 days.
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| CN116158509A (en) * | 2023-03-20 | 2023-05-26 | 浙江大学 | Preparation method of instant brown rice flour |
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| CN116158509A (en) * | 2023-03-20 | 2023-05-26 | 浙江大学 | Preparation method of instant brown rice flour |
| CN116158509B (en) * | 2023-03-20 | 2024-05-28 | 浙江大学 | Preparation method of instant brown rice flour |
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