Compound enzyme composition and application thereof

Abstract

The invention provides a compound enzyme composition and application thereof. The compound enzyme composition contains low-temperature amylase and glucoamylase with the activity ratio of (0.8-1.2): (0.8-1.2), wherein the amino acid sequence of the low-temperature amylase is as shown in SEQ ID NO: 1. The compound enzyme composition can adapt to starch with complex crystal structures such as corn starch, rice starch and wheat starch, the prepared porous starch can serve as a slow-release carrier, an embedding material is efficiently loaded in a pore structure through the excellent adsorption characteristic and slow-release characteristic of the porous starch, the embedding material is continuously released in an acting part or an acting environment, and the slow-release effect is achieved. Efficient embedding and effective slow release of the embedding material are achieved, and the method is suitable for loading the embedding material such as active substances or flavor substances in products such as drugs, food and health-care products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food processing. More specifically, it relates to a composite enzyme composition and its application. BACKGROUND

[0002] Sustained-release carriers, which usually delay the release rate of the embedded substances (such as flavor substances, active substances, etc.) through porous structures and the like, are commonly used in the fields of food, medicine, health products, etc. For example: (1) in the field of food: for extending the release of flavors, protecting active ingredients, extending shelf life, etc.; (2) in the field of medicine: for controlling drug release, targeted delivery, protecting unstable ingredients, etc.; (3) in the field of health products: for ensuring sustained effects, reducing side effects, etc.

[0003] Porous starch stands out among many sustained-release carriers due to its good adsorption properties and sustained-release performance, and corn starch, rice starch, and wheat starch are the most easily obtained starch raw materials. However, the current method for preparing porous starch is to use alpha-amylase or glucoamylase for enzymatic hydrolysis of starch. This enzymatic hydrolysis scheme has poor adaptability to starches with complex crystal structures such as corn starch, rice starch, and wheat starch, and the porous starch obtained by hydrolysis has poor adsorption and sustained-release performance. SUMMARY

[0004] The present application aims to overcome the deficiencies of the prior art and provides a composite enzyme composition. The specific low-temperature amylase and glucoamylase are compounded, which can adapt to starches with complex crystal structures such as corn starch, rice starch, and wheat starch. The porous starch obtained by enzymatic hydrolysis of the starch has excellent adsorption and sustained-release performance.

[0005] The first object of the present application is to provide a composite enzyme composition.

[0006] The second object of the present application is to provide the use of the above-mentioned composite enzyme composition in the preparation of porous starch.

[0007] The third object of the present application is to provide a method for preparing porous starch.

[0008] The fourth object of the present application is to provide the porous starch prepared by the above-mentioned method.

[0009] The fifth object of the present application is to provide the use of the above-mentioned porous starch as and / or in the preparation of sustained-release carriers.

[0010] The sixth object of the present application is to provide a sustained-release carrier.

[0011] The seventh object of the present application is to provide the use of the above-mentioned sustained-release carrier in medicine, food, or health products.

[0012] The above objects of the present application are achieved by the following technical solutions: The application provides a complex enzyme composition, in particular: containing low-temperature amylase and saccharifying enzyme with an activity ratio of 0.8-1.2:0.8-1.2, the amino acid sequence of the low-temperature amylase is shown as SEQ ID NO:1.

[0013] SEQ ID NO:1: MKKFLNFLFLLVFLTIIGCDKTSNLTSNLNNSSISASLVSEWNNAYFRGTPNSWNTSPMTKVASNTWEITVTFNNGDSSGGPRFKIDRYGDWSENYPSQDYYVSPNKTYKITFYDSTHEIKVTEINNSSSSINGTMMQYFEWYLPNDGSLWNKVASESSTLSNMGITALWLPPAYKGQAGSADVGYGVYDMYDLGEFNQKGTIRTKYGTKDEYLNAINVAHQNGIQIYGDVVFNHRMGADGKENVSATRVDWNNRNVTYETKTISAWTDFHFPGRNGKYSTFNWKWYHFDGVDWDDNSKQKAIFRFTGKSWDWEVDTENNNYDYLMGADLDMDHPEVVQELKDWGKWYLDFTGVDGFRLDAVKHIKFTFFNDWLDYLRSSTGKELFTVGEYWSYDINKLNNYITKSNGKMSLFDAPLHMNFHNASNGNGYYDMKNIFNGTLVQSNPLKAVTIVENHDTQPGQALESPVKDWFKPLAYAMILLRQDGYPCVFYGDMYGAQNIKSQYNTIKKLVEARKLYAYGEQRDYLDNPDIIGWTRLGDANHPKAMATILTDASGGSKWMYVGKANAKFIDYLGNRSDVIYSNNDGWAEFKVNGGSVSVWIEQ.

[0014] The complex enzyme composition can be suitable for corn starch, rice starch, wheat starch and other starches with complex crystal structure, and the prepared porous starch can be used as a sustained-release carrier, and the embedded objects can be efficiently loaded in the pore structure through the excellent adsorption and sustained-release properties, and can be continuously released in the action site or environment, so that the embedded objects can be efficiently embedded and effectively released, and the active substances or flavor substances in the products such as medicines, foods and health products can be embedded. Therefore, the application of the complex enzyme composition in the preparation of porous starch should also be within the protection scope of the application.

[0015] Based on the above, the application further provides a preparation method of the porous starch, i.e. using the above-mentioned complex enzyme composition to perform enzymolysis on the starch.

[0016] Preferably, the starch is first prepared into a starch suspension before the enzymolysis, and heated at 45-55 ℃ for 8-12 min, most preferably heated at 50 ℃ for 10 min.

[0017] Further preferably, the preparation method of the starch suspension is to add the starch into the phosphate buffer so that the final concentration of the starch in the phosphate buffer is 18wt%-22wt%, most preferably 20wt%.

[0018] More preferably, the pH of the phosphate buffer is 6-7, most preferably 6.5.

[0019] Preferably, the starch is one or more of corn starch, rice starch and wheat starch, most preferably corn starch.

[0020] Preferably, the use amount ratio of the complex enzyme composition to the starch is 190-210 U:1 g, most preferably 200 U:1 g.

[0021] Preferably, the enzymolysis time is 7.5-8.5 h, most preferably 8 h.

[0022] Preferably, the enzymolysis temperature is 45-55 ℃, most preferably 50 ℃.

[0023] Preferably, after the enzymolysis, enzyme inactivation is performed, such as adjusting the pH of the system to 9.5-10.5 and incubating for 4-6 min, most preferably adjusting the pH of the system to 10 by using sodium hydroxide and incubating for 5 min.

[0024] Further preferably, after the enzyme inactivation, the pH of the system is adjusted to neutral, such as by using hydrochloric acid.

[0025] Preferably, after the enzymolysis, solid-liquid separation is performed on the enzymolysis solution, such as centrifugation at 4500-5500 rpm for 13-17 min, most preferably centrifugation at 5000 rpm for 15 min.

[0026] Further preferably, after the solid-liquid separation, the obtained solid is post-treated, such as sequentially washed, dried and crushed.

[0027] More preferably, the washing is washing with water.

[0028] More preferably, the drying is drying at 40-50 ℃ for 20-28 h, most preferably drying at 45 ℃ for 24 h.

[0029] More preferably, the pulverization is to 50-150 mesh, most preferably 100 mesh.

[0030] The porous starch prepared by the above method can be used as a sustained-release carrier, and the embedding material is efficiently loaded in the pore structure by the excellent adsorption and sustained-release properties, and is continuously released in the action site or environment, realizing efficient embedding and effective sustained release of the embedding material, and is suitable for loading active substances or flavor substances in products such as medicines, foods, and health products. Therefore, the porous starch prepared by the above method, the application of the above porous starch in the preparation of a sustained-release carrier, a sustained-release carrier containing the above porous starch, and the application of the sustained-release carrier in medicines, foods, or health products should also be within the protection scope of the present application.

[0031] The present application has the following beneficial effects: The composite enzyme composition of the present application can be suitable for corn starch, rice starch, wheat starch and other starches with complex crystal structure. The prepared porous starch can be used as a sustained-release carrier, and the embedding material is efficiently loaded in the pore structure by the excellent adsorption and sustained-release properties, and is continuously released in the action site or environment, realizing efficient embedding and effective sustained release of the embedding material, and is suitable for loading active substances or flavor substances in products such as medicines, foods, and health products. DETAILED DESCRIPTION

[0032] The present application will be further described in conjunction with specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0033] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0034] Corn starch was purchased from Henan Midaner Trading Co., Ltd.

[0035] Glucoamylase was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd.

[0036] Low-temperature amylase, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0037] Low-temperature alpha-amylase was purchased from Beijing Keyeibo Biotechnology Co., Ltd.

[0038] Glucoamylase, LKE-zyme GAM, was purchased from Shandong Longkete Enzyme Preparation Co., Ltd.

[0039] Example 1 Preparation of a composite enzyme composition The low-temperature amylase and the glucoamylase with an activity ratio of 1:1 were mixed uniformly.

[0040] Example 2 Preparation of a composite enzyme composition Mix the cold-stable amylase and the glucoamylase at an activity ratio of 0.8:1.2.

[0041] Example 3 Preparation of a composite enzyme composition Mix the cold-stable amylase and the glucoamylase at an activity ratio of 1.2:0.8.

[0042] Example 4 Preparation of porous starch Put 1 g of corn starch into a conical flask containing phosphate buffer (pH = 6.5) to obtain a 20wt% starch suspension, then place the conical flask in a water bath shaker, incubate at 50°C for 10 min, then add 200 U of the composite enzyme composition obtained in Example 1, and enzymatically hydrolyze at 50°C for 8 h, then use 2 M NaOH solution to adjust the pH to 10, incubate for 5 min to terminate the reaction, then add 2 M HCl solution to adjust the pH to neutral, then centrifuge at 5000 r / min for 15 min, wash the precipitate obtained by centrifugation with deionized water for 3 times, and dry in an oven at 45°C for 24 h, then crush through a 100 mesh sieve, and place in a dry vessel for standby.

[0043] Example 5 Preparation of porous starch Put 1 g of rice starch into a conical flask containing phosphate buffer (pH = 6) to obtain an 18wt% starch suspension, then place the conical flask in a water bath shaker, incubate at 55°C for 8 min, then add 210 U of the composite enzyme composition obtained in Example 2, and enzymatically hydrolyze at 55°C for 7.5 h, then use 2 M NaOH solution to adjust the pH to 9.5, incubate for 6 min to terminate the reaction, then add 2 M HCl solution to adjust the pH to neutral, then centrifuge at 5500 r / min for 13 min, wash the precipitate obtained by centrifugation with deionized water for 3 times, and dry in an oven at 40°C for 28 h, then crush through a 100 mesh sieve, and place in a dry vessel for standby.

[0044] Example 6 Preparation of porous starch Put 1 g of wheat starch into a conical flask containing phosphate buffer (pH = 7) to obtain a 22wt% starch suspension, then place the conical flask in a water bath shaker, incubate at 45°C for 12 min, then add 190 U of the composite enzyme composition obtained in Example 3, and enzymatically hydrolyze at 45°C for 8.5 h, then use 2 M NaOH solution to adjust the pH to 10.5, incubate for 4 min to terminate the reaction, then add 2 M HCl solution to adjust the pH to neutral, then centrifuge at 4500 r / min for 17 min, wash the precipitate obtained by centrifugation with deionized water for 3 times, and dry in an oven at 50°C for 20 h, then crush through a 100 mesh sieve, and place in a dry vessel for standby.

[0045] Comparative Example 1 The same as Example 4, except that 200 U of the complex enzyme composition obtained in Example 1 was replaced by 200 U of a low-temperature amylase.

[0046] Comparative Example 2 The same as Example 4, except that 200 U of the complex enzyme composition obtained in Example 1 was replaced by 200 U of a saccharifying enzyme.

[0047] Comparative Example 3 The same as Example 4, except that 100 U of the low-temperature amylase in the complex enzyme composition obtained in Example 1 was replaced by 100 U of a low-temperature alpha-amylase.

[0048] Comparative Example 4 The same as Example 4, except that 100 U of the saccharifying enzyme in the complex enzyme composition obtained in Example 1 was replaced by 100 U of a glucoamylase.

[0049] Comparative Example 5 The same as Example 4, except that the complex enzyme composition obtained in Example 1 was not added, i.e., after incubation at 50 °C for 10 min, the product was directly left to stand at 50 °C for 8 h.

[0050] Test Example 1: Adsorption properties of porous starch Two groups of 0.100 ± 0.005 g of the products obtained in Examples 4-6 and Comparative Examples 1-5 (the initial mass is denoted as M0) were weighed, and Group A was added to 1 mL of deionized water and mixed, and Group B was added to 1 mL of soybean oil and mixed, and then centrifuged at 3000 x g for 15 min, and the supernatant was discarded, and the mass of the precipitate was weighed as M, and the water / oil absorption rate was calculated according to “water / oil absorption rate (%) = (M-M0) / M0 x 100%”, and the results are shown in Table 1.

[0051] Table 1

[0052] It can be seen that: (1) The water / oil absorption rates of the products obtained in Examples 4-6 were significantly higher than those of Comparative Examples 1-5, indicating that the porous starch obtained by enzymatic hydrolysis using the complex enzyme composition of the present application has more excellent adsorption properties.

[0053] (2) The water / oil absorption rates of the products obtained in Examples 4-6 were significantly higher than those of Comparative Examples 3-4, indicating that it is the selection of the specific two enzymes in the present application that enables the excellent adsorption properties.

[0054] (3) Compared with the water absorption rate of Comparative Example 5 without enzyme hydrolysis, the water absorption rates of Comparative Examples 1-2 using single enzyme were increased by 34.0% and 19.9%, respectively, and the oil absorption rates were increased by 34.6% and 32.1%, respectively. The water absorption rate of Example 4 using a composite enzyme composition prepared by compounding two enzymes was increased by 64.9% (> 34.0% + 19.9% = 53.9%), and the oil absorption rate was increased by 72.3% (> 34.6% + 32.1% = 66.7%), indicating that in the composite enzyme composition of the present application, the low-temperature amylase and the saccharifying enzyme showed synergistic effects in improving the water absorption rate and oil absorption rate of porous starch.

[0055] Test Example 2: Slow-release properties of porous starch I. Method for determining the content of 2,6-dimethylpyrazine The content of volatile substances (2,6-dimethylpyrazine) in the sample was detected by gas chromatography-mass spectrometry.

[0056] Among them, the chromatographic conditions: the chromatographic column is Agilent 19091S-431UI (HP-5ms Ultra Inert), 1 m x 250 μm x 0.25 μm, the injection port temperature is 250 ℃, the detection port temperature is 280 ℃, the pressure is 45.6 kPa, the total flow rate is 50 mL / min, the split ratio is 3:1, the PAL injector, the carrier gas is helium (99.99%), the flow rate is 2.25 mL / min. The temperature program: keep at 60 ℃ for 1 min, then rise to 250 ℃ at 10 ℃ / min, keep for 5 min.

[0057] Mass spectrometry conditions: EI ionization mode, interface temperature 300 ℃, ion source temperature 250 ℃, collision gas nitrogen, flow rate 1.5 mL / min, full scan mode data collection, scan range m / z 40-450.

[0058] II. Standard curve of 2,6-dimethylpyrazine Dissolve 2,6-dimethylpyrazine in water to obtain 2,6-dimethylpyrazine solutions with concentrations of 20, 40, 60, 80, and 100 μg / mL. Detect the peak area of 2,6-dimethylpyrazine using gas chromatography-mass spectrometry, and plot the standard curve of 2,6-dimethylpyrazine with concentration as the abscissa and peak area as the ordinate.

[0059] III. Test method for slow-release properties of porous starch on volatile substances Take 2 g of the product obtained in Examples 4-6 and Comparative Examples 1-5, place in a 20 mL screw thread headspace bottle, add 5 mL of 400 μg / mL 2,6-dimethylpyrazine solution (the initial content of 2,6-dimethylpyrazine is denoted as A), shake at 200 rpm for 30 min, then take the supernatant, determine the content B of 2,6-dimethylpyrazine in the supernatant using gas chromatography-mass spectrometry and a standard curve of 2,6-dimethylpyrazine, and then stand for 8 h, and at the 4th and 8th hour, take the supernatant, determine the content C of 2,6-dimethylpyrazine in the supernatant using gas chromatography-mass spectrometry and a standard curve of 2,6-dimethylpyrazine, and then calculate the retention rate of 2,6-dimethylpyrazine in the porous starch at the time of testing according to "flavor retention rate (%) = (A-C) / (A-B) x 100%", and then characterize the slow-release effect of the product obtained in Examples 4-6 and Comparative Examples 1-5 on volatile substances.

[0060] IV. Test results of the slow-release characteristics of the porous starch on volatile substances The results are shown in Table 2.

[0061] Table 2

[0062] It can be seen that: (1) The flavor retention rates of the product obtained in Examples 4-6 at the 4th and 8th hour are significantly higher than those of Comparative Examples 1-5, indicating that the porous starch obtained by enzymatic hydrolysis using the composite enzyme composition of the present application has more excellent slow-release characteristics.

[0063] (2) The flavor retention rates of the product obtained in Examples 4-6 at the 4th and 8th hour are significantly higher than those of Comparative Examples 3-4, indicating that it is the selection of the specific two enzymes in the present application that enables the slow-release characteristics to be so excellent.

[0064] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A complex enzyme composition, characterized in that, The product contains a low-temperature amylase and a saccharifying enzyme with an activity ratio of 0.8–1.2:0.8–1.2, and the amino acid sequence of the low-temperature amylase is shown in SEQ ID NO:

1.

2. The application of the complex enzyme composition according to claim 1 in the preparation of porous starch.

3. A method for preparing porous starch, characterized in that, Starch is enzymatically hydrolyzed using the complex enzyme composition of claim 1.

4. The preparation method according to claim 3, characterized in that, The starch is one or more of corn starch, rice starch, and wheat starch.

5. The preparation method according to claim 3, characterized in that, The ratio of the compound enzyme composition to starch is 190–210 U: 1 g.

6. The preparation method according to claim 3, characterized in that, The enzymatic hydrolysis time is 7.5–8.5 h.

7. Porous starch prepared by any one of claims 3 to 6.

8. The use of the porous starch of claim 7 in the preparation of a sustained-release carrier.

9. A sustained-release carrier, characterized in that, It includes the porous starch of claim 7.

10. The use of the sustained-release carrier according to claim 9 in pharmaceuticals, food or health products.

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