WO2022156211A1 - 一种复合酶及抗性糊精的制备方法 - Google Patents

一种复合酶及抗性糊精的制备方法 Download PDF

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WO2022156211A1
WO2022156211A1 PCT/CN2021/114247 CN2021114247W WO2022156211A1 WO 2022156211 A1 WO2022156211 A1 WO 2022156211A1 CN 2021114247 W CN2021114247 W CN 2021114247W WO 2022156211 A1 WO2022156211 A1 WO 2022156211A1
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starch
amylase
type
enzyme
extrusion
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French (fr)
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徐恩波
刘东红
唐君钰
周建伟
陈健乐
田金虎
程焕
叶兴乾
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浙江大学
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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2425—Beta-amylase (3.2.1.2)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2457—Pullulanase (3.2.1.41)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/246—Isoamylase (3.2.1.68)
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/22—Preparation of compounds containing saccharide radicals produced by the action of a beta-amylase, e.g. maltose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01002—Beta-amylase (3.2.1.2)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01041—Pullulanase (3.2.1.41)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01068—Isoamylase (3.2.1.68)

Definitions

  • the invention relates to the preparation of polysaccharide products with high resistance (anti-digestion) structure, in particular to a composite enzyme and a multi-physical field coupling and Synergistic directional enzymatic hydrolysis regulates the degree of ordered degradation of starch chains, thereby efficiently forming a processing method for resistant dextrins.
  • Resistant dextrin is a small-molecule water-soluble dietary fiber that undergoes structural reorganization after starch gelatinization or glycosylation transfer. Structures such as 3-glycosidic bond, glucan and ⁇ -1,6 glucoside have the functions of preventing colon cancer, lowering cholesterol levels, regulating blood sugar metabolism, and promoting the absorption of Ca 2+ and Fe 3+ minerals.
  • enzyme resistance generally originates from the highly dense crystalline lamellar structure, the modified products often have limitations such as high molecular weight, high hardness, and low palatability, which limit their market application. Therefore, it is of great significance to prepare edible-safe low-molecular-weight RD and its derived functional foods according to the microstructure regulation strategy.
  • the preparation methods of RD mainly include physical method, chemical method and biological enzyme method.
  • chemical methods tend to cause the starch structure to be inhomogeneous by chemical reagents, and the residues of chemical reagents in the obtained products and the discharge of production sewage are still problems. Therefore, the advantages of physical and biological enzymatic means in the preparation of modified starch are particularly prominent.
  • biological enzymes are safe and reliable for human health, they often require a high amount of enzyme or a long enzymatic hydrolysis time to improve the yield of modified products, resulting in expensive preparation and low efficiency, so they have obvious limitations in actual production. .
  • extrusion is a continuous physical processing technology that integrates transportation, mixing, heating, shearing and molding.
  • the extrusion chamber can provide a high-efficiency and short-term micro-mixing environment for the enzyme reaction.
  • enzymatic extrusion has been tried to be applied to the production process of rice wine, liquor, starch sugar and other products.
  • the enzymatic hydrolysis pretreatment time of the above-mentioned combined methods is still long, and the precise combination of complex enzymes has not been considered, and it is difficult to reasonably coordinate the efficient processing environment of its multi-physics coupling. Therefore, the biological enzyme-extrusion processing is still It is necessary to further develop new ideas on the existing basis, in order to obtain RDs with controllable chain length distribution (Chain length of distributions, CLDs), branching degree or molecular weight.
  • the present invention provides a composite enzyme.
  • the action site of the composite enzyme includes ⁇ -1,4 glycosidic bond or ⁇ -1,4 glycosidic bond and ⁇ -1,6 glycosidic bond.
  • the purpose of directional unzipping of starch chains is achieved, thereby forming a retrogradation matrix with low degree of polymerization (DP) CLDs.
  • the present invention additionally provides a method for efficiently preparing low molecular weight RD by using the above-mentioned composite enzyme, utilizing the temperature zone distribution of gradient heating, according to the restricted gelatinization enzymatic hydrolysis sequence of "branched chain unwinding, straight chain grading", in the composite enzyme
  • the base of retrograded CLDs was formed under the induction of , and then recrystallized to obtain RDs with the target structure.
  • the present invention provides a compound enzyme, comprising A-type amylase and B-type amylase, and the preparation method is as follows: the A-type amylase and B-type amylase are dissolved in an acetate buffer (pH 5.2 ), and pre-activated for 30 min in a water bath at 50 °C for later use.
  • the ratio of the total enzymatic activity of type A amylase to the total enzymatic activity of type B amylase is 1:1.5-6.
  • the A-type amylase is selected from one or more of ⁇ -amylase and ⁇ -amylase according to any proportion.
  • type B amylase is one or more of pullulanase and isoamylase.
  • the compound enzyme provided by the present invention is used in the preparation of RD.
  • the present invention provides a method for preparing RD.
  • the method includes: mixing compound enzyme and starch, and after each set temperature zone loaded by the extrusion equipment is stabilized, introducing the mixed material into the cavity . After being treated by screw shear co-extrusion, it is cooled and regenerated to form low molecular weight RD with high crystallinity.
  • the screw shear co-extrusion treatment adopts a temperature field with gradient temperature rise, that is, at least the low-temperature screw shear co-extrusion treatment of the front section and the high-temperature screw shear co-extrusion treatment of the rear section are included; the treatment temperature of the front section is 70 ° C. and below, the processing temperature of the latter stage is higher than that of the former stage, and is at least 60°C and above.
  • the low temperature field promotes the first hydrolysis of ⁇ -1,6 glycosidic bonds by B-amylase, so that the branched double helix outside the starch crystal cluster is unchained.
  • the enzyme can effectively hydrolyze ⁇ -1,4 glycosidic bonds or ⁇ -1,4 in the high temperature zone. Glycosidic bond, in the case of increasing the contact area between the enzyme and starch granules, the straight chain is more easily degraded.
  • the inside of the extruder cavity is essentially a "high substrate" environment for the enzyme.
  • extrusion is mostly a means of structural modification at the non-molecular level.
  • the activity reduction is mostly due to the application of extreme high temperature and high pressure conditions.
  • the gradient heating program adopted in the present invention promotes the pressure inside the extruder cavity to be lower than 0.8Mpa, the amylase can fully exert its enzyme activity in a short time, so that the extruded material can enter the die area for gelatinization.
  • the enzymatic activity of the A-type amylase is 5-20 U/g
  • the enzymatic activity of the B-type amylase is 7.5-120 U/g
  • the above-mentioned enzymatic activity is calculated on the dry basis (g) of the material.
  • the screw shear co-extrusion treatment includes five-stage warm zone extrusion, and the temperature is 20-50°C (zone I), 40-70°C (zone II), 60-90°C (zone III), and 80-90°C (zone III). 110°C (IV zone), 100 ⁇ 130°C (die head zone), and the temperature of the five temperature zones are increased in turn; the screw speed is 150 ⁇ 400r/min.
  • 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.
  • the moisture content of the mixed material of the composite enzyme and starch is adjusted to be 20-40 wt%.
  • the cooling retrogradation is as follows: the structurally reconstituted amyloid obtained after the screw shear co-extrusion treatment is placed at 0-10° C., and then cooled and recrystallized for 2-8 days.
  • the present invention Compared with the traditional enzymatic extrusion processing technology, the present invention reasonably utilizes the simultaneous action of screw extrusion ( ⁇ m-level non-directional shearing) and enzymes (nm-level specific shearing) to make starch ordered (single/ Double helix), disorder (amorphous region) and the degradation order and degree of molecular structure are regulated, and then differential CLDs are formed, and on this basis, crystallization forms resistant helical structures dominated by different DP chains, including branched Chain double helix, linear single helix and linear-branched heterohelix, etc.
  • the CLDs regulation strategy adopted in the present invention makes compound enzymatic extrusion an efficient means of degradation and recombination of starch microdomains, which shortens the pretreatment time of the enzyme solution and simplifies many of the enzymatic assisted extrusion.
  • Process on the other hand, can also establish the relationship between starch CLDs and their resistant fine structure, which can be used to guide production practice.
  • Fig. 1 is the process flow diagram of the RD prepared by the present invention
  • Fig. 2 is the X-ray diffraction (XRD) pattern of the RD prepared by the embodiment of the present invention and the comparative example, which is used to illustrate the crystal form change of the RD prepared by the present invention.
  • XRD X-ray diffraction
  • a preparation method of synchronous extrusion and restriction enzymolysis of RD the steps are as follows:
  • step (1) Temperature-controlled high-shear extrusion: the moisture content of common corn starch is adjusted to 40wt%, and the mixed enzyme liquid compounded in step (1) is added simultaneously during extrusion.
  • the system parameters of the twin-screw extruding barrel are: the temperature zone distribution is 40°C, 60°C, 80°C, 100°C, and 120°C (die zone); the screw speed is 250r/min, and the amyloid is extruded in one step;
  • Retrogradation control The extruded amyloid was placed at 0°C for cooling and retrogradation for 2 days. After being freeze-dried in a vacuum, it is ground through a 200-mesh sieve to obtain the RD;
  • the polysaccharide glucose was converted into the starch value of different digestive components using a conversion coefficient of 0.9, and TS was the total starch mass (g).
  • N de (X) was obtained by using the detector signal.
  • the Mw of the RD prepared in this example is 6.128 kDa, the content of RD in starch is 46.60%, and the content of low DP branches (DP ⁇ 6) is 58.24%.
  • a preparation method of synchronous extrusion and restriction enzymolysis of RD the steps are as follows:
  • Pre-activation of mixed enzyme liquid According to the moisture content of the pre-mixed material and the dry weight of starch, the concentration of the enzyme liquid for preparing A-type amylase (medium-temperature ⁇ -amylase) is: 10U/g; for preparing B-type amylase (Pululanase) enzyme solution concentration: 30U/g.
  • step (1) Temperature-controlled high-shear extrusion: the moisture content of common corn starch is adjusted to 30wt%, and the mixed enzyme solution prepared in step (1) is added simultaneously during extrusion.
  • the system parameters of the twin-screw extruder barrel are: the temperature zone distribution is 30°C, 50°C, 70°C, 90°C, and 110°C (die zone); the screw speed is 200 r/min, and the amyloid is extruded in one step;
  • Retrogradation control The extruded amyloid was placed at 5°C for cooling and retrogradation for 5 days. After being freeze-dried in a vacuum, it is ground through a 200-mesh sieve to obtain the RD;
  • the conversion coefficient of 0.9 was used to convert glucose into starch values of different digestive components, and TS was the total starch mass (g).
  • N de (X) was obtained by using the detector signal.
  • the Mw of the RD prepared in this example is 2.835kDa, the content of RD in starch is 58.27%, and the content of low DP branched chain (DP ⁇ 6) is 64.73%.
  • a preparation method of synchronous extrusion and restriction enzymolysis of RD the steps are as follows:
  • step (1) Temperature-controlled high-shear extrusion: the moisture content of common corn starch is adjusted to 20wt%, and the mixed enzyme solution prepared in step (1) is added simultaneously during extrusion.
  • the system parameters of the twin-screw extruding barrel are: the temperature zone distribution is 20°C, 40°C, 60°C, 80°C, and 100°C (die zone); the screw speed is 150r/min, and the amyloid is extruded in one step;
  • Retrogradation control The extruded amyloid was placed at 10°C for 8 days after cooling for retrogradation. After being freeze-dried in a vacuum, it is ground through a 200-mesh sieve to obtain the RD;
  • the conversion coefficient of 0.9 was used to convert glucose into starch values of different digestive components, and TS was the total starch mass (g).
  • N de (X) was obtained by using the detector signal.
  • the Mw of the RD prepared in this example is 0.908 kDa, the content of RD in starch is 65.43%, and the content of low DP branches (DP ⁇ 6) is 69.22%.
  • a preparation method of synchronous extrusion of RD the steps are as follows:
  • the present comparative example is set to extrusion without enzyme, that is, the composite enzyme is not added;
  • Temperature-controlled high-shear extrusion adjust the moisture content of common corn starch to 30wt%.
  • the system parameters of the twin-screw extruder barrel are: the temperature zone distribution is 30°C, 50°C, 70°C, 90°C, and 110°C (die head area); the screw speed is 150 r/min, and the amyloid is extruded in one step;
  • Retrogradation control The extruded amyloid was placed at 4°C for cooling and retrogradation for 4 days. After being freeze-dried in a vacuum, it is ground through a 200-mesh sieve to obtain the RD;
  • the conversion coefficient of 0.9 was used to convert glucose into starch values of different digestive components, and TS was the total starch mass (g).
  • N de (X) was obtained by using the detector signal.
  • the Mw of the sample prepared in this example is 37.23kDa, the RS content is 42.50%, and the content of low DP branches (DP ⁇ 6) is 19.52%, that is, a small molecular weight RD cannot be successfully prepared.
  • a preparation method of synchronous extrusion and single enzymolysis of RD the steps are as follows:
  • the present embodiment is set to a single amylase joint extrusion process, that is, the concentration of the enzyme liquid for preparing A-type amylase ( ⁇ -amylase) is: 20U/g. Before extrusion, it was pre-activated in a 50°C water bath for 30min;
  • Temperature-controlled high-shear extrusion adjust the moisture content of common corn starch to 30wt%.
  • the system parameters of the twin-screw extruding barrel are: the temperature zone distribution is 20°C, 40°C, 60°C, 80°C, and 100°C (die zone); the screw speed is 150r/min, and the amyloid is extruded in one step;
  • Retrogradation control The extruded amyloid was placed at 4°C for cooling and retrogradation for 4 days. After being freeze-dried in a vacuum, it is ground through a 200-mesh sieve to obtain the RD;
  • the conversion coefficient of 0.9 was used to convert glucose into starch values of different digestive components, and TS was the total starch mass (g).
  • N de (X) was obtained by using the detector signal.
  • the Mw of the sample prepared in this example is 24.86kDa, the RS content is 46.54%, and the content of low DP branches (DP ⁇ 6) is 27.13%, that is, a small molecular weight RD cannot be successfully prepared.

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Abstract

提供了一种复合酶以及配合同步挤压制备抗性糊精的方法。该方法基于不同的糖苷键作用位点以选定复配淀粉酶的比例，将复合酶与淀粉进行高剪切共挤压加工，通过挤压腔内的热机械多物理场耦合(μm级非定向剪切)并协同酶解定点解聚作用(nm级专一性剪切)，精细调控淀粉直链和支链的断裂顺序及程度，以形成差异性链长分布。该方法将内置双螺杆腔体作为高效酶解淀粉的反应器，在增大其轴向扩散速率、滞留时间和作用面积的条件下降低酶液用量，形成了高结晶度的抗性糊精(M w：0.5～8kDa)。

Description

一种复合酶及抗性糊精的制备方法 技术领域
本发明涉及制备具有高抗性(抗消化)结构的多糖类产品，尤其是涉及一种复合酶及一种在高剪切及热能、机械能输入的挤压环境下，通过多物理场耦合及协同定向酶解作用调控淀粉链的有序降解程度，从而高效形成抗性糊精的加工方法。
背景技术
抗性糊精(Resistant dextrin，RD)是淀粉糊化解链或发生糖基化转移后结构重组的一种小分子水溶性膳食纤维，其分子中含有α-1,2糖苷键、α-1,3糖苷键、缩葡聚糖和β-1,6葡萄糖苷等结构，具有预防结肠癌、降低胆固醇水平、调节血糖代谢、促进Ca 2+、Fe 3+矿物质吸收等功能。然而，由于酶抗性一般来源于高度致密结晶的片层排布结构，导致其改性产物常具有高分子量、高硬度、低适口性等局限性，限制了其市场应用。因此，根据微结构调控策略制备食用安全的低分子量RD及其衍生功能食品的意义重大。
目前RD的制备方法主要有物理法、化学法和生物酶法等。然而，化学法易使淀粉结构受化学试剂诱导呈现非均态，且所得产物的化学试剂残留及生产污水排放等仍是问题。因此，物理和生物酶手段在制备变性淀粉中的优势尤为突显。然而，生物酶虽然对人体健康安全可靠，但往往需要高酶量或较长的酶解时间以提高改性产物的产率，导致制备成本昂贵、效率低下，故在实际生产中的局限性明显。挤压作为一种常用的淀粉基物料加工方式，是集运输、混合、加热、剪切和成型等单元为一体的连续式物理加工技术。挤压腔作为酶反应器，可提供高效、短时的酶反应微混合环境。近年来，酶促挤压已被尝试应用于黄酒、白酒、淀粉糖等产品的生产工艺中。然而在实际应用中，上述联合手段的酶解预处理时间仍然较长，复合酶的精准搭配未及考虑，难以合理地协同其多物理场耦合的高效加工环境，因此生物酶-挤压加工仍需在现有基础上进一步开创新思路，以期得到链长分布(Chain length of distributions,CLDs)、分支度或分子量可控的RD。
发明内容
本发明提供了一种复合酶，该复合酶的作用位点包括α-1,4糖苷键或β-1,4糖苷键、α-1,6糖苷键，通过对其复配比例的调控，达到对淀粉链的定向解链目的，从而形成具有低聚合度(Degree of polymerization,DP)CLDs的回生基质。
本发明另外提供了一种采用上述复合酶高效制备低分子量RD的方法，利用梯度升温的温区分布，按照“支链解旋、直链分级”的限制性糊化酶解顺序，在复合酶的诱导下形成回生的CLDs基础，进而重结晶得到具备目标结构的RD。
作为本发明的一方面，本发明提供一种复合酶，包括A类淀粉酶和B类淀粉酶，其配制方法如下：将A类淀粉酶和B类淀粉酶于醋酸盐缓冲液(pH 5.2)中混合配制，并于50℃水浴条件下预活化30min备用。A类淀粉酶的总酶活与B类淀粉酶的总酶活的比例为1:1.5～6。
进一步地，A类淀粉酶选自α-淀粉酶、β-淀粉酶中的一种或多种按照任意配比组成。
进一步地，B类淀粉酶为普鲁兰酶、异淀粉酶中的一种或多种。
作为本发明的另一方面，本发明提供的复合酶在制备RD中应用。
作为本发明的另一方面，本发明提供一种RD的制备方法，该方法为：将复合酶与淀粉混合，待挤压设备加载的各设定温区稳定后，在腔体中引入混合物料。经螺杆剪切共挤压处理后冷却回生，形成高结晶度的低分子量RD。且所述螺杆剪切共挤压处理采用梯度升温的温度场，即至少包含前段的低温螺杆剪切共挤压处理和后段的高温螺杆剪切共挤压处理；前段的处理温度在70℃及其以下，后段的处理温度高于前段，且至少在60℃及其以上。
挤压时，低温场促使B类淀粉酶首先发挥α-1,6糖苷键位点的水解作用，以致淀粉结晶簇外部的支链双螺旋解链。经过低温区对A类淀粉酶的再次预活化，配合螺杆捏合区和反向阻滞区的轴向混合输送，该酶可在高温区有效水解α-1,4糖苷键或β-1,4糖苷键，在增大酶与淀粉颗粒接触面积的情况下，直链更易降解。挤压机腔内实质是酶的“高底物”环境，在底物包酶“中心向周围辐射”的酶解形式下，酶中心与底物的反应配位与替换速率加快，从而极大提高了酶的作用效率。此外，挤压多是非分子水平的结构改性手段，对于分子水平 的酶，其活性降低多是由于极端高温高压条件的施加，然而本发明采用的梯度升温程序促使挤压机腔内部压力低于0.8Mpa，可使淀粉酶在短期内充分发挥酶活力，以便挤压物料进入模头区进行糊化。
进一步地，A类淀粉酶的酶活为5～20U/g、B类淀粉酶的酶活为7.5～120U/g，上述酶活以物料干基(g)计。
进一步地，螺杆剪切共挤压处理包含五段温区挤压，温度依次为20～50℃(I区)、40～70℃(Ⅱ区)、60～90℃(Ⅲ区)、80～110℃(Ⅳ区)，100～130℃(模头区)，五段温区的温度依次提高；螺杆转速为150～400r/min。
进一步地，挤压物料为玉米淀粉、高直链玉米淀粉、蜡质玉米淀粉、马铃薯淀粉、小麦淀粉、木薯淀粉、甘薯淀粉、大米淀粉中的一种或多种。
作为优选的方案，所述挤压前，调节复合酶与淀粉的混合物料的水分含量为20～40wt％。
作为优选的方案，所述冷却回生为：将经螺杆剪切共挤压处理后得到的结构重组淀粉样置于0～10℃条件下，待冷却重结晶2～8天。
发明的有益技术效果如下：
1.本发明与传统酶法挤压加工工艺相比，合理利用了螺杆挤压(μm级非定向剪切)与酶(nm级专一性剪切)的同步作用对淀粉有序(单/双螺旋)、无序(无定形区)和分子结构的降解顺序和程度进行调控，进而形成了差异性CLDs，并在此基础上结晶形成以不同DP链为主导的抗性螺旋结构，包括支链双螺旋、直链单螺旋和直链-支链杂螺旋等。
2.本发明采用的CLDs调控策略使得复合酶法挤压成为一种高效的淀粉微结构域降解及重组手段，这一方面缩短了酶液预处理时间，合并简化了酶法辅助挤压的诸多工艺，另一方面也可建立起淀粉CLDs与其抗性精细结构的关系，用于指导生产实践。
附图说明
图1为本发明制备的RD的工艺流程图；
图2为本发明实施例和对比例制备的RD的X-射线衍射(XRD)图谱，用于说明本发明制备的RD的晶型变化。
具体实施方式
以下通过实施例来进一步阐释本发明，下列实施例用于说明目的而非用于限制本发明范围。
实施例1
一种RD的同步挤压及限制性酶解的制备方法，步骤如下：
(1)混合酶液预活化：根据预调混合物料的水分含量和淀粉干基质量，配制A类淀粉酶(耐高温α-淀粉酶)的酶液浓度为：5U/g；配制B类淀粉酶(普鲁兰酶)的酶液浓度为：30U/g。将酶液混合后，即复配成耐高温α-淀粉酶:普鲁兰酶＝1:6的混合酶液。在挤压前，将其置于50℃水浴条件下预活化30min；
(2)控温高剪切挤压：调节普通玉米淀粉水分含量至40wt％，挤压时同步添加经步骤(1)复配好的混合酶液。双螺杆挤压机筒的***参数为：温区分布依次为40℃、60℃、80℃、100℃、120℃(模头区)；螺杆转速为250r/min，一步挤出淀粉样；
(3)回生控制：挤出淀粉样置于0℃条件下待冷却回生2天。经真空冷冻干燥后，研磨过200目筛，即制得所述RD；
(4)分子量检测：采用高效液相分子排阻色谱与多角度光散射仪及折光检测器连用***(HPSEC-MALLS-RI)，并利用Mark-Houwink参数计算和校准得出样品的重均分子量(Mw)；
(5)体外模拟消化：现配混合酶液于37℃下对样品进行计时消化。混合酶液的酶活配比为：胰酶(500U/ml):葡萄糖苷酶(700U/ml):转化酶(400U/ml)。利用葡萄糖氧化酶检测试剂盒(GOPOD-FORMAT)检测游离糖(Free-sugar glucose,FSG)、消化20min后葡萄糖(Glucose of 20,G20)、消化120min后葡萄糖(Glucose of 120,G120)和总糖(Total glucose,TG)处的吸光度，并计算抗性淀粉(Resistant starch,RS)的得率(％)，公式如下：
RS(％)＝(TG-G120)×0.9/TS×100
其中，利用转化系数0.9将多糖葡萄糖转化为不同消化组分的淀粉值，TS为总淀粉质量(g)。
(6)CLDs检测：采用凝胶辅助糖电泳技术，即PA-800Plus Face***在N-CHO涂层的毛细管中测定支链淀粉链的数量分布，即利用探测器信号得到N de(X)。
本实施例中制得的RD，其Mw为6.128kDa，淀粉中RD含量为46.60％， 且低DP支链(DP<6)的含量为58.24％。
实施例2
一种RD的同步挤压及限制性酶解的制备方法，步骤如下：
(1)混合酶液预活化：根据预调混合物料的水分含量和淀粉干基质量，配制A类淀粉酶(中温α-淀粉酶)的酶液浓度为：10U/g；配制B类淀粉酶(普鲁兰酶)的酶液浓度为：30U/g。将酶液混合后，即复配成中温α-淀粉酶:普鲁兰酶＝1:3的混合酶液。在挤压前，将其置于50℃水浴条件下预活化30min；
(2)控温高剪切挤压：调节普通玉米淀粉水分含量至30wt％，挤压时同步添加经步骤(1)复配好的混合酶液。双螺杆挤压机筒的***参数为：温区分布依次为30℃、50℃、70℃、90℃、110℃(模头区)；螺杆转速为200r/min，一步挤出淀粉样；
(3)回生控制：挤出淀粉样置于5℃条件下待冷却回生5天。经真空冷冻干燥后，研磨过200目筛，即制得所述RD；
(4)分子量检测：采用高效液相分子排阻色谱与多角度光散射仪及折光检测器连用***(HPSEC-MALLS-RI)，并利用Mark-Houwink参数计算和校准得出样品的重均分子量(Mw)；
(5)体外模拟消化：现配混合酶液于37℃下对样品进行计时消化。混合酶液的酶活配比为：胰酶(500U/ml):葡萄糖苷酶(700U/ml):转化酶(400U/ml)。利用葡萄糖氧化酶检测试剂盒(GOPOD-FORMAT)检测FSG、G20、G120和TG处的吸光度，并计算RS的得率(％)，公式如下：
RS(％)＝(TG-G120)×0.9/TS×100
其中，利用转化系数0.9将葡萄糖转化为不同消化组分的淀粉值，TS为总淀粉质量(g)。
(6)CLDs检测：采用凝胶辅助糖电泳技术，即PA-800Plus Face***在N-CHO涂层的毛细管中测定支链淀粉链的数量分布，即利用探测器信号得到N de(X)。
本实施例中制得的RD，其Mw为2.835kDa，淀粉中RD含量为58.27％，且低DP支链(DP<6)的含量为64.73％。
实施例3
一种RD的同步挤压及限制性酶解的制备方法，步骤如下：
(1)混合酶液预活化：根据预调混合物料的水分含量和淀粉干基质量， 配制A类淀粉酶(β-淀粉酶)的酶液浓度为：20U/g；配制B类淀粉酶(异淀粉酶)的酶液浓度为：30U/g。将酶液混合后，即复配成β-淀粉酶:异淀粉酶＝1:1.5的混合酶液。在挤压前，将其置于50℃水浴条件下预活化30min；
(2)控温高剪切挤压：调节普通玉米淀粉水分含量至20wt％，挤压时同步添加经步骤(1)复配好的混合酶液。双螺杆挤压机筒的***参数为：温区分布依次为20℃、40℃、60℃、80℃、100℃(模头区)；螺杆转速为150r/min，一步挤出淀粉样；
(3)回生控制：挤出淀粉样置于10℃条件下待冷却回生8天。经真空冷冻干燥后，研磨过200目筛，即制得所述RD；
(4)分子量检测：采用高效液相分子排阻色谱与多角度光散射仪及折光检测器连用***(HPSEC-MALLS-RI)，并利用Mark-Houwink参数计算和校准得出样品的重均分子量(Mw)；
(5)体外模拟消化：现配混合酶液于37℃下对样品进行计时消化。混合酶液的酶活配比为：胰酶(500U/ml):葡萄糖苷酶(700U/ml):转化酶(400U/ml)。利用葡萄糖氧化酶检测试剂盒(GOPOD-FORMAT)检测FSG、G20、G120和TG处的吸光度，并计算RS的得率(％)，公式如下：
RS(％)＝(TG-G120)×0.9/TS×100
其中，利用转化系数0.9将葡萄糖转化为不同消化组分的淀粉值，TS为总淀粉质量(g)。
(6)CLDs检测：采用凝胶辅助糖电泳技术，即PA-800Plus Face***在N-CHO涂层的毛细管中测定支链淀粉链的数量分布，即利用探测器信号得到N de(X)。
本实施例中制得的RD，其Mw为0.908kDa，淀粉中RD含量为65.43％，且低DP支链(DP<6)的含量为69.22％。
对比实施例1
一种RD的同步挤压的制备方法，步骤如下：
(1)为证明本发明中复合酶对淀粉微结构域的调控效果，本对比实施例设置为无酶挤压，即不添加所述复合酶；
(2)控温高剪切挤压：调节普通玉米淀粉水分含量至30wt％。双螺杆挤压机筒的***参数为：温区分布依次为30℃、50℃、70℃、90℃、110℃(模 头区)；螺杆转速为150r/min，一步挤出淀粉样；
(3)回生控制：挤出淀粉样置于4℃条件下待冷却回生4天。经真空冷冻干燥后，研磨过200目筛，即制得所述RD；
(4)分子量检测：采用高效液相分子排阻色谱与多角度光散射仪及折光检测器连用***(HPSEC-MALLS-RI)，并利用Mark-Houwink参数计算和校准得出样品的重均分子量(Mw)；
(5)体外模拟消化：现配混合酶液于37℃下对样品进行计时消化。混合酶液的酶活配比为：胰酶(500U/ml):葡萄糖苷酶(700U/ml):转化酶(400U/ml)。利用葡萄糖氧化酶检测试剂盒(GOPOD-FORMAT)检测FSG、G20、G120和TG处的吸光度，并计算RS的得率(％)，公式如下：
RS(％)＝(TG-G120)×0.9/TS×100
其中，利用转化系数0.9将葡萄糖转化为不同消化组分的淀粉值，TS为总淀粉质量(g)。
(6)CLDs检测：采用凝胶辅助糖电泳技术，即PA-800Plus Face***在N-CHO涂层的毛细管中测定支链淀粉链的数量分布，即利用探测器信号得到N de(X)。
本实施例制得的样品，其Mw为37.23kDa，RS含量为42.50％，且低DP支链(DP<6)的含量为19.52％，即未能成功制备出小分子量的RD。
对比实施例2
一种RD的同步挤压及单一酶解的制备方法，步骤如下：
(1)为证明本发明中复合酶对淀粉微结构域的调控效果，本实施例设置为单一淀粉酶联合挤压加工，即配制A类淀粉酶(β-淀粉酶)的酶液浓度为：20U/g。在挤压前，将其置于50℃水浴条件下预活化30min；
(2)控温高剪切挤压：调节普通玉米淀粉水分含量至30wt％。双螺杆挤压机筒的***参数为：温区分布依次为20℃、40℃、60℃、80℃、100℃(模头区)；螺杆转速为150r/min，一步挤出淀粉样；
(3)回生控制：挤出淀粉样置于4℃条件下待冷却回生4天。经真空冷冻干燥后，研磨过200目筛，即制得所述RD；
(4)分子量检测：采用高效液相分子排阻色谱与多角度光散射仪及折光检测器连用***(HPSEC-MALLS-RI)，并利用Mark-Houwink参数计算和校 准得出样品的重均分子量(Mw)；
(5)体外模拟消化：现配混合酶液于37℃下对样品进行计时消化。混合酶液的酶活配比为：胰酶(500U/ml):葡萄糖苷酶(700U/ml):转化酶(400U/ml)。利用葡萄糖氧化酶检测试剂盒(GOPOD-FORMAT)检测FSG、G20、G120和TG处的吸光度，并计算RS的得率(％)，公式如下：
RS(％)＝(TG-G120)×0.9/TS×100
其中，利用转化系数0.9将葡萄糖转化为不同消化组分的淀粉值，TS为总淀粉质量(g)。
(6)CLDs检测：采用凝胶辅助糖电泳技术，即PA-800Plus Face***在N-CHO涂层的毛细管中测定支链淀粉链的数量分布，即利用探测器信号得到N de(X)。
本实施例制得的样品，其Mw为24.86kDa，RS含量为46.54％，且低DP支链(DP<6)的含量为27.13％，即未能成功制备出小分子量的RD。

Claims (9)

  1. 一种复合酶，其特征在于，包括A类淀粉酶和B类淀粉酶，所述A类淀粉酶的作用位点为α-1,4糖苷键或β-1,4糖苷键，所述B类淀粉酶的作用位点为α-1,6糖苷键；A类淀粉酶的总酶活与B类淀粉酶的总酶活的比例为1:1.5～6。
  2. 根据权利要求1所述的复合酶，其特征在于，所述A类淀粉酶选自α-淀粉酶、β-淀粉酶中的一种或多种按照任意配比组成。
  3. 根据权利要求1所述的复合酶，其特征在于，所述B类淀粉酶为普鲁兰酶、异淀粉酶中的一种或多种。
  4. 一种权利要求1所述的复合酶在制备抗性糊精中应用。
  5. 一种抗性糊精的制备方法，其特征在于：将权利要求1所述的复合酶与淀粉的混合物料经螺杆剪切共挤压处理，冷却回生后形成高结晶度的低分子量抗性糊精。所述混合物料中，A类淀粉酶的酶活为5～20U/g、B类淀粉酶的酶活为7.5～120U/g，上述酶活以物料干基(g)计。
  6. 根据权利要求5所述的制备方法，其特征在于，所述螺杆剪切共挤压处理包含五段温区挤压，温度依次为20～50℃、40～70℃、60～90℃、80～110℃，100～130℃，五段温区的温度依次提高；螺杆转速为150～400r/min。
  7. 根据权利要求5所述的制备方法，其特征在于，所述淀粉为玉米淀粉、高直链玉米淀粉、蜡质玉米淀粉、马铃薯淀粉、小麦淀粉、木薯淀粉、甘薯淀粉、大米淀粉中的一种或多种。
  8. 根据权利要求5所述的制备方法，其特征在于，复合酶与淀粉的混合物料的含水量为20～40wt％。
  9. 根据权利要求5所述的制备方法，其特征在于，所述冷却回生为：将经螺杆剪切共挤压处理后得到的结构重组淀粉样置于0～10℃条件下，待冷却重结晶2～8天。
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