CN112725312B - 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 synchronous extrusion, belonging to the technical field of starch deep processing. The invention selects the proportion of the compound amylase based on different glycosidic bond acting sites, carries out high-shearing co-extrusion processing on the compound enzyme and starch, and can finely regulate and control the breaking sequence and degree of starch straight chain and branched chain by thermo-mechanical multi-physical field coupling (mu m-level non-directional shearing) and synergistic enzymolysis fixed-point depolymerization (nm-level specific shearing) in an extrusion cavity so as to form differential chain length distribution. The method takes the built-in double screw cavity as a reactor for efficiently hydrolyzing starch, reduces the enzyme liquid dosage under the condition of increasing the axial diffusion rate, the detention time and the action area of the reactor, and forms the high-crystallinity resistant dextrin (M) w : 0.5-8 kDa). The invention simplifies enzymolysis steps before and after extrusion, and further realizes a fine, efficient, continuous, water-saving and economical starch micro-structural domain regulation method.
Description
Technical Field
The invention relates to a method for preparing polysaccharide products with high-resistance (digestion-resistant) structures, in particular to a processing method for efficiently forming resistant dextrin by regulating and controlling the ordered degradation degree of starch chains through multi-physical field coupling and synergetic directional enzymolysis under the extrusion environment of high shear and heat energy and mechanical energy input.
Background
Resistant dextrin (Resistant dextrin, RD) is a micromolecular water-soluble dietary fiber with gelatinized starch for melting or structure recombination after glycosylation transfer, and the micromolecular water-soluble dietary fiber contains alpha-1, 2 glycosidic bond, alpha-1, 3 glycosidic bond, dextran, beta-1, 6 glucoside and other structures, and has the functions of preventing colon cancer, reducing cholesterol level, regulating blood sugar metabolism and promoting Ca 2+ 、Fe 3+ Mineral absorption and other functions. However, since enzyme resistance generally derives from a highly densely crystallized lamellar arrangement structure, the modified product thereof often has limitations such as high molecular weight, high hardness, low palatability, and the like, which limit its market application. Therefore, the significance of preparing the low molecular weight RD and the derived functional food thereof which are safe to eat according to the microstructure regulation strategy is great.
The prior RD preparation method mainly comprises a physical method, a chemical method, a biological enzyme method and the like. However, chemical methods tend to induce starch structures to assume non-homogeneous states by chemical agents, and chemical agent residues and production wastewater discharge of the resulting products remain problematic. Thus, the advantages of physical and biological enzymatic means in the preparation of modified starches are particularly pronounced. However, though the biological enzyme is safe and reliable for human health, high enzyme amount or long enzymolysis time is often required to improve the yield of the modified product, so that the preparation cost is expensive and the efficiency is low, and the limitation in practical production is obvious. Extrusion is a common starch-based material processing mode, and is a continuous physical processing technology integrating units of transportation, mixing, heating, shearing, forming 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 to be applied to the production process of products such as yellow wine, white spirit, starch sugar and the like. However, in practical application, the enzymolysis pretreatment time of the combined means is still longer, the precise collocation of the complex enzyme is not considered, and the efficient processing environment of the complex enzyme coupled by multiple physical fields is difficult to reasonably cooperate, so that the bio-enzyme-extrusion processing still needs to further develop a new idea on the basis of the prior art so as to obtain RD with controllable chain length distribution (Chain length of distributions, CLDs), branching degree or molecular weight.
Disclosure of Invention
The invention provides a complex enzyme, the action site of which comprises alpha-1, 4 glycosidic bond or beta-1, 4 glycosidic bond and alpha-1, 6 glycosidic bond, and the aim of directional melting of starch chain is achieved by regulating and controlling the compounding proportion of the complex enzyme, so as to form a retrogradation matrix with low polymerization degree (Degree of polymerization, DP) CLDs.
The invention further provides a method for efficiently preparing the low-molecular-weight RD by adopting the complex enzyme, which utilizes the temperature zone distribution of gradient heating to form a retrograded CLDs foundation under the induction of the complex enzyme according to the limited pasting enzymolysis sequence of branched-chain unwinding and linear-chain grading, and then recrystallizing to obtain the RD with a target structure.
As one aspect of the invention, the invention provides a complex enzyme comprising a class A amylase and a class B amylase, which is prepared by the following steps: the A-type amylase and the B-type amylase are mixed in acetate buffer solution (pH 5.2) and preactivated for 30min under the water bath condition of 50 ℃ for standby. The ratio of the total enzyme activity of the class A amylase to the total enzyme activity of the class B amylase is 1:1.5-6.
Further, the A-class amylase is selected from one or more of alpha-amylase and beta-amylase according to any proportion.
Further, the B-class 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, which comprises: and mixing the complex enzyme with starch, and introducing a mixed material into the cavity after each set temperature zone loaded by the extrusion equipment is stable. Cooling and regenerating after screw shearing and coextrusion treatment to form the low molecular weight RD with high crystallinity. The screw shearing and co-extrusion treatment adopts a temperature field with gradient heating, namely at least comprises low-temperature screw shearing and co-extrusion treatment of a front section and high-temperature screw shearing and co-extrusion treatment of a rear section; the treatment temperature of the front stage is 70 ℃ or lower, and the treatment temperature of the rear stage is higher than that of the front stage and at least 60 ℃ or higher.
During extrusion, the low temperature field causes the class B amylase to first exert the hydrolysis of the alpha-1, 6 glycosidic bond sites, so that branched double helix chains outside the starch crystalline clusters melt. The alpha-1, 4 glycosidic bond or beta-1, 4 glycosidic bond can be effectively hydrolyzed in a high temperature region by reactivating the class A amylase in a low temperature region and matching with axial mixed conveying of a screw kneading region and a reverse blocking region, and the linear chain is easier to degrade under the condition of increasing the contact area of the enzyme and starch particles. The extruder cavity is substantially in a high substrate environment of the enzyme, and under the enzymolysis mode that the substrate comprises the enzyme 'center to radiate', the reaction coordination and replacement rate of the enzyme center and the substrate are accelerated, so that the action efficiency of the enzyme is greatly improved. In addition, extrusion is a non-molecular structural modification means, and the activity of the enzyme at the molecular level is reduced due to the application of extreme high temperature and high pressure, however, the gradient heating program adopted by the invention promotes the internal pressure of the extruder cavity to be lower than 0.8Mpa, so that the amylase can fully exert the enzyme activity in a short period, and the extruded material enters a die head area for gelatinization.
Further, the enzyme activity of the class A amylase is 5-20U/g, the enzyme activity of the class B amylase is 7.5-120U/g, and the enzyme activities are calculated according to the dry matter (g).
Further, the screw shearing co-extrusion treatment comprises five-stage temperature zone extrusion, the temperatures of which are 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 temperatures of the five-stage temperature zone are sequentially increased; the rotating speed of the screw is 150-400 r/min.
Further, the extruded 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 regulated to be 20-40 wt%.
As a preferred scheme, the cooling regeneration is: and (3) placing the structural recombinant amyloid obtained after the screw shearing and coextrusion treatment at the temperature of 0-10 ℃ for 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 method reasonably utilizes the synchronous action of screw extrusion (mu m-level non-directional shearing) and enzyme (nm-level specific shearing) to regulate and control the order and degree of degradation of ordered (single/double helix), disordered (amorphous region) and molecular structure of starch, thereby forming differential CLDs, and on the basis, the differential CLDs crystallize to form resistant helix structures taking different DP chains as dominant, including branched double helix, linear single helix, linear-branched hetero helix and the like.
2. The CLDs regulation strategy adopted by the invention enables the composite enzyme method extrusion to become an efficient starch micro-structural domain degradation and recombination means, which shortens the enzyme liquid pretreatment time, combines and simplifies a plurality of processes of enzyme method auxiliary extrusion, and can also establish the relationship between the starch CLDs and the resistant fine structure thereof for guiding production practice.
Drawings
FIG. 1 is a process flow diagram of RD made in accordance with the present invention;
FIG. 2 is an X-ray diffraction (XRD) pattern of RD prepared in the examples and comparative examples of the present invention, illustrating the change in crystal form of RD prepared in the present invention.
Detailed Description
The invention is further illustrated by the following examples, which are given for the purpose of illustration and are not intended to limit the scope of the invention.
Example 1
The preparation method of the RD synchronous extrusion and the restriction enzymolysis comprises the following steps:
(1) Preactivating mixed enzyme solution: according to the water content of the pre-mixed material and the dry basis quality of starch, preparing the enzyme solution concentration of class A amylase (high temperature resistant alpha-amylase) as follows: 5U/g; the enzyme solution concentration of the class B amylase (pullulanase) is prepared as follows: 30U/g. And mixing the enzyme solutions to obtain the high-temperature resistant alpha-amylase-pullulanase=1:6 mixed enzyme solution. Before extrusion, placing the mixture in a water bath at 50 ℃ for preactivation for 30min;
(2) High-temperature-control shearing extrusion: regulating the water content of the common corn starch to 40wt%, and synchronously adding the mixed enzyme solution compounded in the step (1) during extrusion. The system parameters of the twin-screw extruder barrel are: the temperature zone distribution is 40 ℃, 60 ℃, 80 ℃,100 ℃ and 120 ℃ (die head zone) in sequence; the rotating speed of the screw is 250r/min, and the amyloid is extruded in one step;
(3) And (3) retrogradation control: the extruded amyloid is placed at 0 ℃ for 2 days after cooling back. Vacuum freeze drying, grinding, and sieving with 200 mesh sieve to obtain RD;
(4) And (3) molecular weight detection: adopting a high performance liquid chromatography-multi-angle light scattering instrument and refraction detector combined system (HPSEC-MALLS-RI), and calculating and calibrating by using Mark-Houwink parameters to obtain the weight average molecular weight (Mw) of the sample;
(5) In vitro simulated digestion: the sample was digested with the now prepared enzyme mixture at 37℃with time. The enzyme activity ratio of the mixed enzyme solution is as follows: pancreatin (500U/ml): glucosidase (700U/ml): invertase (400U/ml). The absorbance at Free-sugar (FSG), glucose (Glucose of 20, G20) after 20min of digestion, glucose (Glucose of 120, G120) and Total sugar (TG) was measured using a Glucose oxidase test 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 the total starch mass (g).
(6) CLDs detection: the number distribution of amylopectin chains is determined in the capillaries of the N-CHO coating by gel-assisted sugar electrophoresis, i.e.the PA-800Plus Face system, i.e.N is obtained by means of detector signals de (X)。
RD prepared in this example has a Mw of 6.128kDa, a RD content of 46.60% in starch, and a low DP branching (DP < 6) content of 58.24%.
Example 2
The preparation method of the RD synchronous extrusion and the restriction enzymolysis comprises the following steps:
(1) Preactivating mixed enzyme solution: according to the water content of the pre-mixed material and the dry mass of starch, preparing the enzyme solution concentration of class A amylase (medium temperature alpha-amylase) as follows: 10U/g; the enzyme solution concentration of the class B amylase (pullulanase) is prepared as follows: 30U/g. And mixing the enzyme solutions to obtain the medium-temperature alpha-amylase-pullulanase=1:3 mixed enzyme solution. Before extrusion, placing the mixture in a water bath at 50 ℃ for preactivation for 30min;
(2) High-temperature-control shearing extrusion: regulating the water content of the common corn starch to 30wt%, and synchronously adding the mixed enzyme solution compounded in the step (1) during extrusion. The system parameters of the twin-screw extruder barrel are: the temperature zone distribution is 30 ℃, 50 ℃, 70 ℃, 90 ℃ and 110 ℃ (die head zone) in sequence; the rotating speed of the screw is 200r/min, and the amyloid is extruded in one step;
(3) And (3) retrogradation control: the extruded amyloid is placed under 5 ℃ for 5 days after cooling and retrogradation. Vacuum freeze drying, grinding, and sieving with 200 mesh sieve to obtain RD;
(4) And (3) molecular weight detection: adopting a high performance liquid chromatography-multi-angle light scattering instrument and refraction detector combined system (HPSEC-MALLS-RI), and calculating and calibrating by using Mark-Houwink parameters to obtain the weight average molecular weight (Mw) of the sample;
(5) In vitro simulated digestion: the sample was digested with the now prepared enzyme mixture at 37℃with time. The enzyme activity ratio of the mixed enzyme solution is as follows: pancreatin (500U/ml): glucosidase (700U/ml): invertase (400U/ml). Absorbance at FSG, G20, G120 and TG was detected using a glucose oxidase detection kit (glucose-FORMAT), and the yield (%) of RS was calculated as follows:
RS(％)＝(TG-G120)×0.9/TS×100
wherein, glucose is converted into starch values of different digestion components by using a conversion coefficient of 0.9, and TS is the total starch mass (g).
(6) CLDs detection: the number distribution of amylopectin chains is determined in the capillaries of the N-CHO coating by gel-assisted sugar electrophoresis, i.e.the PA-800Plus Face system, i.e.N is obtained by means of detector signals de (X)。
RD prepared in this example has a Mw of 2.835kDa, a RD content of 58.27% in starch, and a low DP branching (DP < 6) content of 64.73%.
Example 3
The preparation method of the RD synchronous extrusion and the restriction enzymolysis comprises the following steps:
(1) Preactivating mixed enzyme solution: according to the water content of the pre-mixed material and the dry mass of starch, preparing the enzyme solution of the class A amylase (beta-amylase) with the concentration of: 20U/g; the enzyme solution concentration of the class B amylase (isoamylase) is prepared as follows: 30U/g. And mixing the enzyme solutions to obtain the mixed enzyme solution of the beta-amylase and the isoamylase=1:1.5. Before extrusion, placing the mixture in a water bath at 50 ℃ for preactivation for 30min;
(2) High-temperature-control shearing extrusion: regulating the water content of the common corn starch to 20wt%, and synchronously adding the mixed enzyme solution compounded in the step (1) during extrusion. The system parameters of the twin-screw extruder barrel are: 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 the amyloid is extruded in one step;
(3) And (3) retrogradation control: the extruded amyloid is placed at 10 ℃ for retrogradation for 8 days after cooling. Vacuum freeze drying, grinding, and sieving with 200 mesh sieve to obtain RD;
(4) And (3) molecular weight detection: adopting a high performance liquid chromatography-multi-angle light scattering instrument and refraction detector combined system (HPSEC-MALLS-RI), and calculating and calibrating by using Mark-Houwink parameters to obtain the weight average molecular weight (Mw) of the sample;
(5) In vitro simulated digestion: the sample was digested with the now prepared enzyme mixture at 37℃with time. The enzyme activity ratio of the mixed enzyme solution is as follows: pancreatin (500U/ml): glucosidase (700U/ml): invertase (400U/ml). Absorbance at FSG, G20, G120 and TG was detected using a glucose oxidase detection kit (glucose-FORMAT), and the yield (%) of RS was calculated as follows:
RS(％)＝(TG-G120)×0.9/TS×100
wherein, glucose is converted into starch values of different digestion components by using a conversion coefficient of 0.9, and TS is the total starch mass (g).
(6) CLDs detection: the number distribution of amylopectin chains is determined in the capillaries of the N-CHO coating by gel-assisted sugar electrophoresis, i.e.the PA-800Plus Face system, i.e.N is obtained by means of detector signals de (X)。
RD prepared in this example has a Mw of 0.908kDa, a RD content of 65.43% in starch, and a low DP branching (DP < 6) content of 69.22%.
Comparative example 1
The preparation method of the RD synchronous extrusion comprises the following steps:
(1) To demonstrate the regulatory effect of the complex enzyme on the starch microdomains in the present invention, this comparative example was set to an enzyme-free extrusion, i.e., without the addition of the complex enzyme;
(2) High-temperature-control shearing extrusion: the water content of the common corn starch is regulated to 30wt%. The system parameters of the twin-screw extruder barrel are: the temperature zone distribution is 30 ℃, 50 ℃, 70 ℃, 90 ℃ and 110 ℃ (die head zone) in sequence; the rotating speed of the screw is 150r/min, and the amyloid is extruded in one step;
(3) And (3) retrogradation control: the extruded amyloid is left to cool at 4 ℃ for 4 days. Vacuum freeze drying, grinding, and sieving with 200 mesh sieve to obtain RD;
(4) And (3) molecular weight detection: adopting a high performance liquid chromatography-multi-angle light scattering instrument and refraction detector combined system (HPSEC-MALLS-RI), and calculating and calibrating by using Mark-Houwink parameters to obtain the weight average molecular weight (Mw) of the sample;
(5) In vitro simulated digestion: the sample was digested with the now prepared enzyme mixture at 37℃with time. The enzyme activity ratio of the mixed enzyme solution is as follows: pancreatin (500U/ml): glucosidase (700U/ml): invertase (400U/ml). Absorbance at FSG, G20, G120 and TG was detected using a glucose oxidase detection kit (glucose-FORMAT), and the yield (%) of RS was calculated as follows:
RS(％)＝(TG-G120)×0.9/TS×100
wherein, glucose is converted into starch values of different digestion components by using a conversion coefficient of 0.9, and TS is the total starch mass (g).
(6) CLDs detection: by gel-assisted sugar electrophoresis, i.e. in the PA-800Plus Face systemDetermination of the number distribution of amylopectin chains in N-CHO-coated capillaries, i.e.N is obtained by means of detector signals de (X)。
The sample prepared in this example had a Mw of 37.23kDa, an RS content of 42.50% and a low DP branching (DP < 6) content of 19.52%, i.e., failed to produce a low molecular weight RD.
Comparative example 2
The preparation method of RD by synchronous extrusion and single enzymolysis comprises the following steps:
(1) In order to prove the regulation effect of the compound enzyme on the starch micro-structural domain, the embodiment is set as the single amylase combined extrusion processing, namely the concentration of the enzyme solution for preparing the class A amylase (beta-amylase) is as follows: 20U/g. Before extrusion, placing the mixture in a water bath at 50 ℃ for preactivation for 30min;
(2) High-temperature-control shearing extrusion: the water content of the common corn starch is regulated to 30wt%. The system parameters of the twin-screw extruder barrel are: 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 the amyloid is extruded in one step;
(3) And (3) retrogradation control: the extruded amyloid is left to cool at 4 ℃ for 4 days. Vacuum freeze drying, grinding, and sieving with 200 mesh sieve to obtain RD;
(4) And (3) molecular weight detection: adopting a high performance liquid chromatography-multi-angle light scattering instrument and refraction detector combined system (HPSEC-MALLS-RI), and calculating and calibrating by using Mark-Houwink parameters to obtain the weight average molecular weight (Mw) of the sample;
(5) In vitro simulated digestion: the sample was digested with the now prepared enzyme mixture at 37℃with time. The enzyme activity ratio of the mixed enzyme solution is as follows: pancreatin (500U/ml): glucosidase (700U/ml): invertase (400U/ml). Absorbance at FSG, G20, G120 and TG was detected using a glucose oxidase detection kit (glucose-FORMAT), and the yield (%) of RS was calculated as follows:
RS(％)＝(TG-G120)×0.9/TS×100
wherein, glucose is converted into starch values of different digestion components by using a conversion coefficient of 0.9, and TS is the total starch mass (g).
(6) CLDs detection: the number distribution of amylopectin chains is determined in the capillaries of the N-CHO coating by gel-assisted sugar electrophoresis, i.e.the PA-800Plus Face system, i.e.N is obtained by means of detector signals de (X)。
The sample prepared in this example had a Mw of 24.86kDa, an RS content of 46.54% and a low DP branching (DP < 6) content of 27.13%, i.e., failed to produce a low molecular weight RD.
Claims (4)
1. A preparation method of resistant dextrin is characterized by comprising the following steps: and (3) shearing and co-extruding the mixed material of the complex enzyme and the starch by a screw, and cooling and regenerating to form the low molecular weight resistant dextrin with high crystallinity. In the mixed material, the enzyme activity of the class A amylase is 5-20U/g, the enzyme activity of the class B amylase is 7.5-120U/g, the enzyme activity is calculated by a dry basis (g) of the material, the screw shearing and co-extrusion treatment comprises five-section extrusion at the temperature of 20-50 ℃, 40-70 ℃, 60-90 ℃, 80-110 ℃, 100-130 ℃ and the temperature of the five-section extrusion is sequentially increased; the rotating speed of the screw is 150-400 r/min;
the complex enzyme comprises class A amylase and 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 class A amylase to the total enzyme activity of the class B amylase is 1:1.5-6; the A-type amylase is selected from one or more of alpha-amylase and beta-amylase according to any proportion; the B-class amylase is one or more of pullulanase and isoamylase.
2. The method according to claim 1, wherein the starch is one or more of corn starch, potato starch, wheat starch, tapioca starch, sweet potato starch, rice starch.
3. The preparation method according to claim 1, wherein the water content of the mixed material of the complex enzyme and the starch is 20-40 wt%.
4. The method of claim 1, wherein the cooling regeneration is: and (3) placing the structural recombinant amyloid obtained after the screw shearing and coextrusion treatment at the temperature of 0-10 ℃ for cooling and recrystallizing for 2-8 days.
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