Enzymatic preparation method of high-proportion long-chain isomaltooligosacharide

Abstract

The invention discloses an enzymatic preparation method of high-proportion long-chain isomaltooligosacharide. According to the method, starch / maltodextrin (with the DE value of 5-10) is taken as a raw material, and the high-proportion long-chain isomaltooligosacharide is prepared through yeast fermentation and purification by virtue of reconstruction of a bonding mode among glycosyl units under composite synergistic catalysis of alpha-amylase, pullulanase, maltotriose producing enzyme and alpha-glucosidase. The purity of the prepared isomaltooligosacharide reaches 95.93%, and the proportion of the long-chain isomaltooligosacharide is 34.12%, which is far higher than the proportion of the long-chain isomaltooligosacharide in the existing commercial products by 5-15%. The long-chain isomaltooligosacharide can remain in the intestinal tract for a long time, so that the long-chain isomaltooligosacharide can exert stronger life benefiting activity and is very concerned. According to the method, the high-purity and high-proportion long-chain isomaltooligosacharide can be prepared, the problems that the isomaltooligosacharide is poor in purity, the proportion of the long-chain isomaltooligosacharide is not high and the like are effectively solved, and the obtained product has wide application in the food industry.

Description

TECHNICAL FIELD

[0001] The present application relates to an enzymatic preparation method of long-chain oligomeric isomaltose, and belongs to the field of functional food. BACKGROUND

[0002] Oligomeric isomaltose is a kind of oligomeric glucose with a degree of polymerization of usually 2-10, which is mainly connected by α-1, 6 glycosidic bond between molecules, and has the characteristics of low sweetness, low heat and low glycemic index, and can be used as a healthy sugar substitute for diabetic patients. At the same time, oligomeric isomaltose can promote the growth of probiotic genera such as bifidobacterium and lactobacillus, adjust the balance of intestinal flora, promote intestinal peristalsis, prevent and relieve constipation and diarrhea, and inhibit the growth of harmful bacteria in the intestinal tract and the generation of putrefactive substances. Therefore, oligomeric isomaltose is widely used in food industry, feed industry and health care product industry as sweetener, flavoring agent, filling agent and feed additive, etc. The component with a degree of polymerization of 4-10 in oligomeric isomaltose is called long-chain oligomeric isomaltose, which has higher research value and application potential in probiotic function, food industry application, etc. due to its longer retention time in the intestinal tract and stronger probiotic activity.

[0003] Currently, the commercial production of isomalto-oligosaccharides at home and abroad mainly adopts the method of direct enzymatic synthesis. Specifically, starch is used as the substrate, and heat-resistant alpha-amylase is used for high-temperature jet liquefaction. Then, beta-amylase, pullulanase and other enzymes are added to obtain a saccharification liquid. Finally, alpha-glucosidase is added to synthesize isomalto-oligosaccharides. In this reaction, in order to improve the utilization rate of starch substrate, it is necessary to gelatinize the starch at high temperature, which requires a large amount of energy consumption. In addition, alpha-glucosidase cuts maltose to generate glucose, and then transfers the glucose group to maltose to generate panose under the action of alpha-glucosidase. The generation of a large amount of by-product glucose leads to a generally low yield of isomalto-oligosaccharides, which is generally less than 55% (Maurya, R. et al., Sci Rep 13, 12708 (2023); Wang Shuya et al., Tianjin University of Science and Technology, Vol. 30, No. 2, April 2015). In addition, the transglycosylation reaction of alpha-glucosidase is carried out in units of glucose groups, and maltose is mainly generated as the acceptor molecule. Therefore, the synthesis of long-chain isomalto-oligosaccharides requires higher polymerization degree of maltose oligosaccharide molecules, resulting in a low proportion of long-chain isomalto-oligosaccharides in the product. For example, commercial isomalto-oligosaccharides are divided into IMO-50 type (IG2+P+IG3+Gn≥50%), IMO-70 type and IMO-90 type, and most of the products are IMO-50 type, in which the proportion of long-chain isomalto-oligosaccharides is low. Du Guangpeng et al. prepared isomalto-oligosaccharides with a concentration of isomaltotriose greater than 35% by transglycosidase using maltose as the substrate (CN 103805654A). In Chinese patent CN10529657A, maltose was used as the substrate, and immobilized alpha-glucosidase was used to improve the IMO triose content of the product. The content of isomaltose, panose and isomaltotriose in the final product reached 75% of the dry matter, which improved the health performance to a certain extent, but the polymer of the product was not improved.

[0004] Therefore, in order to solve the problems of low purity and low proportion of long-chain isomalto-oligosaccharides in the current isomalto-oligosaccharide industry, an enzymatic preparation method of high-proportion long-chain isomalto-oligosaccharides is developed, which not only has important significance for the development of the isomalto-oligosaccharide industry, but also has positive significance for promoting the "Healthy China" strategic plan. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide an enzymatic preparation method of high-proportion long-chain oligoisomaltose. The specificity of the transglycosylation reaction of alpha-glucosidase is utilized, that is, maltotriose has higher transglycosylation efficiency and higher proportion of long-chain oligoisomaltose generation, linear starch chains are prepared by the synergistic hydrolysis of alpha-amylase and pullulanase on starch, then the linear starch chains are hydrolyzed by maltotriose generating enzyme to prepare high-concentration maltotriose syrup as a high-quality substrate for the transglycosylation reaction of alpha-glucosidase, and finally the purification is performed through yeast fermentation. When maltodextrin is used as the substrate, the degradation of starch by alpha-amylase can be omitted, and the preparation of high-proportion long-chain oligoisomaltose is directly performed from the pullulanase debranching reaction. The purity of the high-proportion long-chain oligoisomaltose of the present application is as high as 90% to 97%, and the proportion of long-chain oligoisomaltose is as high as 30% to 40%. The product has high purity, high solubility and high long-chain oligoisomaltose proportion, is a new type of prebiotic, and can be widely applied to food, medicine and other fields as a sugar substitute.

[0006] The purpose of the present application is achieved at least by one of the following technical solutions.

[0007] An enzymatic preparation method of high-proportion long-chain oligoisomaltose comprises the following steps:

[0008] (1) Adjusting the pH of a starch slurry reaction liquid or a maltodextrin aqueous solution with a DE value of 5 to 10 to 4.0 to 6.5, then adding pullulanase and incubating at 50 to 65 DEG C for 2 to 4 hours to perform a debranching reaction;

[0009] (2) Adjusting the pH of the reaction liquid obtained in step (1) to 6.0 to 7.5, then adding maltotriose generating enzyme after the reaction liquid is cooled to 45 to 55 DEG C and incubating for 4 to 8 hours;

[0010] (3) Adjusting the pH of the reaction liquid after step (2) to 6.0 to 7.0, then adding alpha-glucosidase and reacting at 60 to 65 DEG C for 9 to 15 hours, and the obtained reaction liquid is a crude oligoisomaltose solution;

[0011] (4) Adjusting the pH of the crude oligoisomaltose solution obtained in step (3) to 5.0 to 6.0, then adding yeast powder, and incubating at 25 to 35 DEG C for 24 to 36 hours to purify the oligoisomaltose through yeast fermentation; collecting the membrane filtrate, inactivating the enzyme activity, and centrifuging to obtain the supernatant, which is then spray-dried to obtain high-proportion long-chain oligoisomaltose.

[0012] Further, the mass concentration of the starch slurry reaction liquid or the maltodextrin aqueous solution with a DE value of 5 to 10 is 10% to 30%.

[0013] Further, the starch slurry reaction solution in step (1) is obtained by the following steps: mixing raw starch, CaCl2 and water to obtain a starch slurry, ultrasonic treatment for 5-10 min, 30-35℃ incubation, pH adjustment to 6.5-7.7, then adding alpha-amylase into the starch slurry and reacting for 6-12 h.

[0014] Further, the alpha-amylase is derived from deep-sea bacteria Pontibacillus sp. ZY, and the enzyme addition amount is 1-5 U / g raw starch.

[0015] Further, the pullulanase is derived from Bacillus licheniformis, and the enzyme addition amount is 100-150 U / g substrate.

[0016] Further, the maltotriose generating enzyme is derived from Microbacterium imperiale, and the enzyme addition amount is 150-200 U / g substrate; and the alpha-glucosidase is derived from Aspergillus niger, and the enzyme addition amount is 600-1200 U / g substrate.

[0017] Further, the yeast powder in step (4) is a high-sugar-tolerant yeast produced by Angel Yeast Co., Ltd., and the addition amount of the yeast powder is 5-10 g / L.

[0018] Further, in step (4), the reaction solution is centrifuged at 7000-8000 rpm for 20-30 min to obtain supernatant, filtered by a 0.45 μm filter membrane, and then spray-dried.

[0019] The application further provides a high-proportion long-chain oligoisomalto-oligosaccharide prepared by the preparation method, wherein the purity of oligoisomalto-oligosaccharide in the high-proportion long-chain oligoisomalto-oligosaccharide is 90-97%, and the proportion of long-chain oligoisomalto-oligosaccharide reaches 30-40%.

[0020] The application further provides a high-proportion long-chain oligoisomalto-oligosaccharide prepared by the preparation method, wherein the purity of oligoisomalto-oligosaccharide in the high-proportion long-chain oligoisomalto-oligosaccharide is 90-97%, and the proportion of long-chain oligoisomalto-oligosaccharide reaches 30-40%.

[0021] The application further provides a high-proportion long-chain oligoisomalto-oligosaccharide prepared by the preparation method, wherein the purity of oligoisomalto-oligosaccharide in the high-proportion long-chain oligoisomalto-oligosaccharide is 90-97%, and the proportion of long-chain oligoisomalto-oligosaccharide reaches 30-40%.

[0022] Compared with the prior art, the application has the following advantages:

[0023] (1) The present application provides an enzymatic preparation method of high proportion long-chain oligoisomaltose, which can realize efficient conversion of raw starch granules or malt dextrin. The method is to prepare oligoisomaltose by the synergistic effect of α-amylase, pullulanase, maltotriose generating enzyme and α-glucosidase on starch or malt dextrin, and the conversion rate of the substrate is 45.05%, which is significantly better than the yield of traditional commercial oligoisomaltose (30-45%). In the starch hydrolysis process, α-amylase (AmyZ1) first acts on raw starch, effectively destroys the crystal structure of starch, significantly reduces the crystallinity, thereby generating a large amount of soluble starch sugar, and providing more reaction sites for the subsequent action of pullulanase. In addition, a variety of enzymes act on the starch chain structure in turn, and the enzymatic product of the previous step provides a substrate with higher affinity for the subsequent enzyme reaction, thereby realizing an efficient starch conversion process.

[0024] The present application provides an enzymatic preparation method of high proportion long-chain oligoisomaltose, which can realize the enrichment of long-chain oligoisomaltose. The method first effectively hydrolyzes the crystal structure of starch granules by α-amylase; then pullulanase is used to debranch the starch chain to provide linear starch chains as substrates for maltotriose generating enzyme; maltotriose generating enzyme efficiently hydrolyzes linear starch chains to generate a large amount of maltotriose, which provides donor / acceptor molecules for transglycosylation reaction of α-glucosidase; and α-glucosidase uses maltotriose as the acceptor molecule for transglycosylation reaction to prepare high proportion oligoisomaltose.

[0025] The present application provides an enzymatic preparation method of high proportion long-chain oligoisomaltose, which can realize the preparation of high-purity oligoisomaltose. The purity of the oligoisomaltose prepared by the method is more than 90%, which is much higher than the purity of 50% and 70% of commercial products. The products of pullulanase and β-amylase used in the saccharification stage of the existing process are mainly maltose and glucose, and the chain length is relatively short. In the present method, maltotriose generating enzyme is used, which acts on the debranched malt dextrin, and the product is mainly maltotriose, which has a relatively long degree of polymerization. After transglycosylation reaction by α-glucosidase, less glucose byproduct is generated in the product. In addition, after the enzymatic reaction, yeast fermentation and purification are carried out, which greatly improves the purity of oligoisomaltose. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The chromatogram of separating each standard by anion exchange chromatography in the examples. DETAILED DESCRIPTION

[0027] In order to better understand the present application, the present application will be further described in detail in conjunction with the examples, but the embodiments of the present application are not limited thereto.

[0028] An enzymatic preparation method of high proportion long-chain oligoisomaltose, comprising the following steps and process conditions:

[0029] Slurry mixing and reaction: malt dextrin (DE value 5-10) is mixed with water to form a slurry with a dry basis mass fraction of 10%-30%, CaCl2 is added, the pH is adjusted to 6.5-7.7, and then ultrasonic treatment is performed at a power of 500-700 W for 5-10 min.

[0030] Reaction: the slurry or raw starch slurry reaction solution in step (1) is kept in a constant temperature water bath at 30-35°C and continuously stirred, 1-5 U / g of substrate of α-amylase is added, and reaction is performed for 6-12 h; then the temperature is adjusted to 50-65°C, the pH is adjusted to 4.0-6.5, 100-150 U / g of substrate of pullulanase is added, and reaction is performed for 2-4 h; then 150-200 U / g of maltotriose generating enzyme is added, and reaction is performed for 4-8 h; finally, 600-1200 U / g of α-glucosidase is added, and reaction is performed for 9-15 h to obtain a crude product. Yeast powder is added to the crude product reaction solution, and fermentation is performed at 30°C for 24-36 h.

[0031] Reaction termination and enzyme inactivation: after reaction is completed, the supernatant is obtained by centrifugal filtration, and enzyme inactivation is performed in a water bath.

[0032] To better achieve the object of the application, preferably, the selected raw starch is corn starch or rice starch.

[0033] Preferably, the concentration of CaCl2 in the reaction system is 0.5-1.5 mM.

[0034] Preferably, the stirring rate is 250-450 rpm.

[0035] In the examples, the determination method of each component in oligoisomaltose: after dilution of the supernatant after reaction or purification by a certain multiple, centrifugation with a 0.22 μm microporous filter, and then qualitative and quantitative analysis by high performance anion exchange chromatography, the chromatographic conditions are as follows: the chromatographic column is CarboPA200 chromatographic column (3x250mm), CarbopacTM PA200 (3x150mm) and PA200 pre-column (4x50mm). The system uses PEEK tube (0.24mm i.d.), gradient mixer (2mm), ED amperometric cell with 0.25 μL channel volume, pH reference electrode of Ag / AgCl, 0.002 gasket and gold electrode. Mobile phase: 100 mM NaOH (phase A) and 150 mM sodium acetate + 100 mM NaOH mixture (phase B); elution program: 0-10 min, keep 100% A phase; 40 min, 100% B phase; 50 min, 100% A phase. The column temperature is 40℃, the injection amount is 10 μL, and the flow rate is 0.5 mL / min. Qualitative and quantitative analysis of each component according to the standard curve, and calculation of the content of each component and the total yield of the reaction;

[0036] The α-amylase used in the examples was prepared by the laboratory of Anhui University (AmyZ1, CN109679937A "A raw starch hydrolyzing enzyme with high specific activity, its coding gene and application"); the pullulanase (A30278G190) was purchased from Genencor (China) Bioengineering Co., Ltd.; the maltotriose generating enzyme (AMT 1.2L) and α-glucosidase were purchased from Japan Takeda Enzyme Products Co., Ltd.

[0037] Comparative Example 1

[0038] The rice starch granules were mixed with water and stirred to obtain a slurry with a concentration of 30 wt%, CaCl2 was added to make the concentration 1 mM, and after ultrasonic treatment for 5 min, the pH was adjusted to 7.0, and the slurry was incubated at 35℃ with stirring. The α-amylase was added to the slurry at an addition amount of 5 U / g for hydrolysis reaction for 8 h. The pH was adjusted to 5.5, and the slurry was incubated at 60℃ with stirring. The pullulanase was added to the slurry at an addition amount of 150 U / g of substrate for debranching reaction for 3 h. The temperature was adjusted to 50℃, and the maltotriose generating enzyme was added at an addition amount of 150 U / g of malt dextrin for reaction for 6 h to prepare a high-concentration maltotriose reaction solution. The temperature was adjusted to 60℃, and the α-glucosidase was added at an addition amount of 1000 U / g of malt dextrin for reaction for 12 h. After adding yeast powder to the reaction solution, the slurry was incubated at 30℃ with stirring at 500 rpm for 24 h. After centrifugal filtration, a high-concentration long-chain oligoisomaltose product was obtained.

[0039] Test results show that the total yield of oligoisomaltose is 46.73%, the purity is 92.08%, and the proportion of long-chain oligoisomaltose is 22.29%.

[0040] Comparative Example 2

[0041] The corn starch particles were mixed with water and stirred to obtain a slurry with a concentration of 30 wt%, CaCl2 was added to make the concentration 1 mM, and after ultrasonic treatment for 5 min, the pH was adjusted to 6.8, and the slurry was incubated at 35°C with stirring. Alpha-amylase was added to the slurry at an addition amount of 3 U / g for hydrolysis reaction for 8 h. The pH was adjusted to 5.5, and the slurry was incubated in a water bath at 65°C with stirring. Pullulanase was added to the slurry at an addition amount of 125 U / g of substrate for debranching reaction for 3 h. The temperature was adjusted to 50°C, and 150 U / g of beta-amylase was added for reaction for 6 h to prepare a high-concentration maltose reaction solution. The temperature was adjusted to 60°C, and 900 U / g of maltodextrin alpha-glucosidase was added for reaction for 12 h. After adding yeast powder to the reaction solution, the solution was stirred at 30°C at 500 rpm, and after fermentation for 24 h, a high-concentration long-chain oligoisomaltose product was obtained after centrifugal filtration.

[0042] Testing showed that the total yield of oligoisomaltose was 35.84%, the purity was 93.28%, and the proportion of long-chain oligoisomaltose was 17.14%.

[0043] Comparative Example 3

[0044] Maltodextrin with a DE value of 12 was mixed with water and stirred to obtain a slurry with a concentration of 30 wt%, CaCl2 was added to make the concentration 1 mM, and after ultrasonic treatment for 5 min, the pH was adjusted to 5.5, and the slurry was incubated in a water bath at 65°C with stirring. Pullulanase was added to the slurry at an addition amount of 125 U / g of maltodextrin for debranching reaction for 3 h. The temperature was adjusted to 50°C, and 150 U / g of maltodextrin maltotriose-generating enzyme was added for reaction for 6 h to prepare a high-concentration maltotriose reaction solution. The temperature was adjusted to 60°C, and 900 U / g of maltodextrin alpha-glucosidase was added for reaction for 12 h. After adding yeast powder to the reaction solution, the solution was stirred at 30°C at 500 rpm, and after fermentation for 24 h, a high-concentration long-chain oligoisomaltose product was obtained after centrifugal filtration.

[0045] Testing showed that the total yield of oligoisomaltose was 29.73%, the purity was 92.62%, and the proportion of long-chain oligoisomaltose was 17.04%.

[0046] Comparative Example 4

[0047] The malt dextrin with DE value of 20 was mixed with water and stirred to obtain a slurry with concentration of 30 wt%, CaCl2 was added to make the concentration 1 mM, after ultrasonic treatment for 5 min, the pH was adjusted to 5.5, and the slurry was kept in water bath at 65°C and stirred, pullulanase was added to the slurry at an addition amount of 125 U / g of malt dextrin for debranching, after reaction for 3 h, the temperature was adjusted to 50°C, maltotriose generating enzyme was added at an amount of 150 U / g of malt dextrin, and the reaction was carried out for 6 h to prepare a high-concentration maltotriose reaction solution; then the temperature was adjusted to 60°C, and α-glucosidase was added at an amount of 900 U / g of malt dextrin, and the reaction was carried out for 12 h. After adding yeast powder to the reaction solution, stirring was carried out at 30°C and 500 rpm, and after fermentation for 24 h, high-concentration long-chain oligoisomaltose product was obtained after centrifugal filtration.

[0048] Test results showed that the total yield of oligoisomaltose was 22.81%, the purity was 87.40%, and the long-chain oligoisomaltose accounted for 19.66%.

[0049] Example 1

[0050] The malt dextrin with DE value of 6 was mixed with water and stirred to obtain a slurry with concentration of 30 wt%, CaCl2 was added to make the concentration 1 mM, after ultrasonic treatment for 5 min, the pH was adjusted to 5.5, and the slurry was kept in water bath at 65°C and stirred, pullulanase was added to the slurry at an addition amount of 125 U / g for debranching, after reaction for 3 h, the temperature was adjusted to 50°C, maltotriose generating enzyme was added at an amount of 150 U / g, and the reaction was carried out for 6 h to prepare a high-concentration maltotriose reaction solution; then the temperature was adjusted to 60°C, and α-glucosidase was added at an amount of 900 U / g, and the reaction was carried out for 12 h. After adding yeast powder to the reaction solution, stirring was carried out at 30°C and 500 rpm, and after fermentation for 24 h, high-concentration long-chain oligoisomaltose product was obtained after centrifugal filtration.

[0051] Test results showed that the yield of oligoisomaltose was 45.03%, the purity was 95.93%, and the long-chain oligoisomaltose accounted for 34.12%.

[0052] Example 2

[0053] The corn starch particles were mixed with water and stirred to obtain a slurry with a concentration of 30 wt%, CaCl2 was added to make the concentration 1 mM, and after ultrasonic treatment for 5 min, the pH was adjusted to 6.8, and the slurry was incubated at 35°C with stirring. Alpha-amylase was added to the slurry at an addition amount of 3 U / g for hydrolysis reaction for 8 h. The pH was adjusted to 6.0, and the slurry was incubated at 55°C with stirring. Pullulanase was added to the slurry at an addition amount of 125 U / g for debranching reaction for 3 h. The temperature was adjusted to 50°C, and maltotriose-generating enzyme was added at an addition amount of 150 U / g for reaction for 6 h to prepare a high-concentration maltotriose reaction solution. The temperature was adjusted to 60°C, and alpha-glucosidase was added at an addition amount of 900 U / g for reaction for 12 h. Then, yeast powder was added to the reaction solution, and the solution was stirred at 500 rpm at 30°C for fermentation for 24 h. After centrifugal filtration, a high-concentration long-chain oligoisomaltooligosaccharide product was obtained.

[0054] Test results showed that the yield of oligoisomaltooligosaccharide was 43.52%, the purity was 93.41%, and the proportion of long-chain oligoisomaltooligosaccharide was 31.37%.

[0055] Example 3

[0056] The corn starch particles were mixed with water and stirred to obtain a slurry with a concentration of 30 wt%, CaCl2 was added to make the concentration 1 mM, and after ultrasonic treatment for 5 min, the pH was adjusted to 6.8, and the slurry was incubated at 35°C with stirring. Alpha-amylase was added to the slurry at an addition amount of 3 U / g for hydrolysis reaction for 8 h. The pH was adjusted to 6.0, and the slurry was incubated at 55°C with stirring. Pullulanase was added to the slurry at an addition amount of 125 U / g for debranching reaction for 3 h. The temperature was adjusted to 50°C, and maltotriose-generating enzyme was added at an addition amount of 150 U / g for reaction for 6 h to prepare a high-concentration maltotriose reaction solution. The temperature was adjusted to 60°C, and alpha-glucosidase was added at an addition amount of 900 U / g for reaction for 12 h. Then, yeast powder was added to the reaction solution, and the solution was stirred at 500 rpm at 30°C for fermentation for 24 h. After centrifugal filtration, a high-concentration long-chain oligoisomaltooligosaccharide product was obtained.

[0057] Test results showed that the yield of oligoisomaltooligosaccharide was 43.52%, the purity was 93.41%, and the proportion of long-chain oligoisomaltooligosaccharide was 31.37%.

[0058] The starch hydrolysis rate results of the examples and the comparative examples are shown in Table 1. The yield of oligoisomaltooligosaccharide and the proportion of long-chain oligoisomaltooligosaccharide of the comparative examples were both higher than those of the examples.

[0059] In Comparative Examples 1-4, the oligoisomaltose yield was 46.73%, 35.84%, 29.73%, and 22.81%, respectively. Except for Comparative Example 1, the oligoisomaltose yield was low, which might be related to the fact that the rice starch in Comparative Example 1 was a substrate with stronger affinity to AnyZ1 of the a-amylase. Meanwhile, in Comparative Examples 1-4, the long-chain oligoisomaltose yield was 16.29%, 17.14%, 17.04%, and 19.66%, respectively, which was generally lower than the long-chain oligoisomaltose content in Examples 1-3, indicating that the proportion of long-chain oligoisomaltose in the product was affected by the type of the substrate starch and the degree of polymerization of the substrate. Comparative Example 1 and Examples 2 and 3 were compared, and it was found that there was no significant difference in the yield and purity of the product in the two cases, but the proportion of long-chain oligoisomaltose was quite different, indicating that the soluble sugar obtained by using rice starch as the substrate was not conducive to the generation of maltotriose, and therefore was not conducive to the generation of long-chain oligoisomaltose in the transglycosylation stage; and the transglycosylation substrate with high maltotriose content prepared by using corn starch as the substrate and subjecting it to the action of maltotriose-generating enzyme was more conducive to the preparation of long-chain oligoisomaltose through transglycosylation. Comparative Example 2 and Examples 2 and 3 were compared, and it was found that there was no significant difference in the yield and purity of the product in the two cases, but the proportion of long-chain oligoisomaltose was quite different, indicating that the maltotriose generated by the maltotriose-generating enzyme was more suitable for the preparation of long-chain oligoisomaltose through transglycosylation than the maltose generated by the β-amylase. Comparative Example 2 and Comparative Examples 3 and 4 were compared, and it was found that the generation of high proportion of long-chain oligoisomaltose in the first three cases was also related to the generation of maltotriose, and the generation of maltotriose was affected by the degree of polymerization of the substrate. After the starch or low-DE maltodextrin was subjected to the debranching of the pullulanase, a linear starch chain with high degree of polymerization was generated, which was more suitable for the preparation of high-concentration maltotriose liquefaction liquid by the action of the maltotriose-generating enzyme; therefore, more long-chain oligoisomaltose could be generated through the transglycosylation of the a-glucosidase, and the utilization rate of the whole starch / maltodextrin was also improved.

[0060] Table 1 Proportion of each component in the product and yield

[0061]

[0062] The soluble sugar and oligoisomaltose after starch enzymolysis were analyzed by high-performance anion exchange chromatography (HPAEC) for accurate qualitative and quantitative analysis. The separation effect of each component was as shown in FIG. 1. It was found through comparative analysis that the retention time of each component in the mixed standard solution was consistent with the peak time of the corresponding single standard, which confirmed that the method had good separation effect. Figure 1 Figure 1 ​In the formula, G1, G2, G3, G4 respectively represent glucose, maltose, maltotriose, maltotetraose; IG2, P, IG3, IG4, IG5 are isomaltose, panose, isomaltotriose, isomaltotetraose, isomaltopentaose respectively.

[0063] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A method for enzymatically preparing high-proportion long-chain isomaltooligosaccharides, characterized in that: The steps include: (1) The starch slurry reaction solution or the maltodextrin aqueous solution with a DE value of 5 to 10 is adjusted to a pH of 4.0 to 6.5, and then pullulanase is added and kept at 50 to 65° C. for 2 to 4 hours to perform a debranching reaction; (2) adjusting the pH of the reaction solution obtained in step (1) to 6.0-7.5, adding maltotriose generating enzyme after the reaction solution is cooled to 45-55° C., and keeping the reaction warm for 4-8 hours; (3) adjusting the pH of the liquid after the reaction in step (2) to 6.0-7.0, then adding α-glucosidase and reacting at 60-65° C. for 9-15 hours, and the resulting reaction liquid is a crude isomaltooligosaccharide solution; (4) The pH of the crude isomaltooligosaccharide solution obtained in step (3) is adjusted to 5.0-6.0, and then yeast powder is added, and the mixture is kept at 25-35° C. for 24-36 hours to purify the isomaltooligosaccharide by yeast fermentation; the membrane filtrate is collected, the enzyme activity is inactivated, and the mixture is centrifuged, and the obtained supernatant is spray-dried to obtain a high-proportion long-chain isomaltooligosaccharide.

2. The enzymatic preparation method of a high-proportion long-chain isomaltooligosaccharide according to claim 1, characterized in that: The mass concentration of the starch slurry reaction solution or the maltodextrin aqueous solution with a DE value of 5 to 10 is 10 to 30%.

3. The enzymatic preparation method of a high-proportion long-chain isomaltooligosaccharide according to claim 1, characterized in that: The starch slurry reaction liquid in step (1) is obtained by the following steps: raw starch, CaCl2 and water are mixed uniformly to obtain starch slurry, ultrasonically treated for 5 to 10 minutes, kept warm at 30 to 35°C, adjusted to a pH of 6.5 to 7.7, and then α-amylase is added to the starch slurry to react for 6 to 12 hours.

4. The enzymatic preparation method of a high-proportion long-chain isomaltooligosaccharide according to claim 1, characterized in that: The α-amylase is derived from the deep-sea bacterium Pontibacillus sp. ZY, and the added enzyme amount is 1 to 5 U / g of raw starch.

5. The enzymatic preparation method of a high-proportion long-chain isomaltooligosaccharide according to claim 1, characterized in that: The pullulanase is derived from Bacillus licheniformis, and the added enzyme amount is 100-150 U / g substrate.

6. The enzymatic preparation method of a high-proportion long-chain isomaltooligosaccharide according to claim 1, characterized in that: The maltotriose-generating enzyme is derived from Microbacterium imperiale, and the enzyme addition amount is 150-200 U / g substrate; the α-glucosidase is derived from Aspergillus niger, and the enzyme addition amount is 600-1200 U / g substrate.

7. The enzymatic preparation method of a high-proportion long-chain isomaltooligosaccharide according to claim 1, characterized in that: The yeast powder in step (4) is a high-sugar-tolerant yeast produced by Angel Yeast Co., Ltd., and the added amount of yeast powder is 5-10 g / L.

8. The enzymatic preparation method of a high-proportion long-chain isomaltooligosaccharide according to claim 1, characterized in that: In step (4), the reaction solution is centrifuged at 7000-8000 rpm for 20-30 min to obtain the supernatant, which is filtered through a 0.45 μm filter membrane and then spray-dried.

9. A high-ratio long-chain isomaltooligosaccharide prepared by the preparation method according to any one of claims 1 to 8, wherein the purity of the isomaltooligosaccharide in the high-ratio long-chain isomaltooligosaccharide is 90-97%, and the proportion of the long-chain isomaltooligosaccharide is 30-40%.

10. Use of a high-ratio long-chain isomaltooligosaccharide according to any one of claims 1 to 6 in the preparation of food and medicine.

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