Method for preparing high-purity mannan oligosaccharide from konjac fine powder

Abstract

The invention discloses a method for preparing high-purity mannan oligosaccharide from konjac fine powder, which comprises the following steps: washing the konjac fine powder with alcohol, treating with alpha-amylase and glucoamylase, degrading konjac glucomannan by using a compound enzyme consisting of beta-glucanase and beta-mannase, and finally adjusting the pH value to an isoelectric point to prepare the high-purity konjac mannan oligosaccharide. According to the method, high-content impurities such as starch, protein and alkaloid in the konjak fine powder are removed, the problem of accumulation control of konjak processing byproducts is solved, the utilization rate and the economic value of konjak resources are improved, and the source of mannan oligosaccharide is enriched. The method realizes recycling of the konjac fine powder, has the advantages of being simple in process, low in equipment requirement and suitable for industrial production, and can be popularized and applied on a large scale.

Description

Technical Field

[0001] The invention belongs to the technical field of konjac by-product reprocessing, and particularly relates to a method for preparing high-purity manno-oligosaccharide from konjac fine powder. Background Art

[0002] Konjac, also known as "konjac", is a perennial herbaceous plant of the genus Amorphophallus in the family Araceae. It belongs to the yam crop in cultivation and is widely planted in Sichuan, Yunnan, Hubei and other places in my country. In recent years, konjac processing enterprises have developed rapidly, and the output and consumption of konjac flour have gradually increased worldwide, but a large number of processing by-products have not been well treated and utilized. At present, the industrial production process of konjac flour mainly adopts two methods: wet method and dry method, that is, konjac is first processed into taro slices, and then the taro slices are ground, crushed, air-selected, screened, and purified to obtain the finished product. After the taro slices are ground and crushed, the by-product collected when they are separated by air blast is called flying powder, and the mesh number greater than 120 after screening and classification is called fine powder, and the two are collectively referred to as konjac processing by-products. Among them, konjac fine powder contains about 50% starch and glucomannan, 10% protein, a small amount of vitamins and minerals, and has certain nutritional value, but because it also contains impurities such as alkaloids, its egg smell and special odor limit its research and application scope.

[0003] Manno-oligosaccharides are composed of mannose, glucose or galactose residues connected by α-1,2, α-1,3, β-1,3 or β-1,4 glycosidic bonds. They have special effects on the body's metabolism, mainly reflected in: ① promoting the growth and reproduction of beneficial bacteria in the body's intestines; ② directly adsorbing pathogens and eliminating intestinal toxins; ③ enhancing the body's immune function and disease resistance; ④ having a lipid-lowering effect, which can improve lipid metabolism, enhance the absorption of mineral elements, and reduce blood ammonia concentration. Due to its unique physiological functions, manno-oligosaccharides are recognized as an important functional additive and will be widely used in many fields such as food, health products and feed. At present, manno-oligosaccharides are mainly obtained by degrading konjac flour with glucanase, and obtaining glucomanno-oligosaccharides by removing impurities and refining. The preparation method generally adopted is the intermittent reaction method, but this method is energy-consuming, time-consuming, and the enzyme utilization efficiency is not high, and a large amount of konjac flour is required as a raw material.

[0004] Chinese patent CN101914597B discloses a method for preparing konjac glucomannan oligosaccharide, which uses konjac to prepare konjac flour, then performs enzymatic hydrolysis reaction between the konjac flour and endomannanase to obtain a generated solution of konjac glucomannan oligosaccharide, and continuously takes out the generated solution of konjac glucomannan oligosaccharide, replenishes the slurry of the konjac flour once every 3-6 hours of reaction, and after replenishing the feed 9-20 times, the reaction is stopped after another 3-6 hours of reaction, the generated solution of konjac glucomannan oligosaccharide is collected and combined, and dried to obtain the konjac glucomannan oligosaccharide, thereby improving the efficacy and utilization efficiency of the mannanase, and obtaining the target glucomannan oligosaccharide product to the greatest extent with a purity of more than 90%.

[0005] Chinese patent CN105803016A discloses a method for preparing konjac mannan oligosaccharide, wherein sweet potato and barley are cooked and fermented to prepare maltose solution containing mold, and then the obtained maltose solution is gradually added to the konjac that has been ground and filtered, and then the mixture is mixed and cooked until the mixture becomes viscous, and finally konjac mannan oligosaccharide is obtained by drying and crushing. The method has the advantages of simple operation, easy availability of raw materials, no pollutants, and a yield of mannan oligosaccharide of more than 90%, and a purity of more than 92%.

[0006] The existing preparation of konjac mannooligosaccharide generally directly uses konjac or konjac refined flour as raw materials. Although high-quality konjac mannooligosaccharide can be prepared, the consumption of konjac raw materials is large, and the problem of accumulation of konjac processing byproducts cannot be solved. Therefore, it is necessary to develop a method for preparing mannooligosaccharide by konjac processing byproducts, thereby solving the problems of konjac processing byproducts storage and low konjac resource utilization. Summary of the invention

[0007] In view of the above technical problems, the present invention provides a method for preparing high-purity manno-oligosaccharides from konjac flour. The konjac flour is subjected to the processes of removing alkaloids, removing starch, saccharifying, degrading konjac glucomannan and removing protein, so as to prepare high-purity manno-oligosaccharides, solve the problem of stockpiling of konjac processing by-products, and improve the utilization rate of konjac resources.

[0008] In order to achieve the above object, the present invention provides a method for preparing high-purity manno-oligosaccharides from konjac flour, comprising the following steps: (1) Removing alkaloids: washing konjac powder with an ethanol solution, filtering and drying to obtain konjac flour; (2) Destarchization: placing konjac flour in an α-amylase solution and mixing to obtain a mixed solution, steaming and cooling to obtain a gelatinized solution; (3) Saccharification: Add saccharifying enzyme to the gelatinized liquid, filter after the reaction to obtain saccharification residue; (4) Degradation of konjac glucomannan: placing the saccharification residue in a complex enzyme solution to obtain a mixed solution, enzymatically hydrolyzing and centrifuging to obtain a supernatant; (5) Deproteinization: Adjust the pH of the supernatant to the isoelectric point of the protein, let it stand, centrifuge, and dry to obtain high-purity manno-oligosaccharides.

[0009] Preferably, the mass concentration of the ethanol solution in step (1) is 30%-50%, the number of washing times is 3-5 times, the ethanol solution has a gradient concentration during washing, and the concentration of the ethanol solution gradually increases with the increase in the number of washing times; the mass ratio of konjac powder to ethanol solution is 1:20-30.

[0010] Preferably, the amount of konjac flour in step (2) is 15%-20% of the total mass of the mixed solution; the amount of α-amylase added is 10-20 U / g, where g is the mass of konjac flour.

[0011] Preferably, the cooking temperature in step (2) is 90-100° C. and the cooking time is 1-2 h.

[0012] Preferably, the amount of saccharifying enzyme added in step (3) is 100-200 U / g, the reaction temperature is 50-60° C., and the reaction time is 1-2 h; wherein g is the mass of konjac flour.

[0013] Preferably, the amount of the complex enzyme added in step (4) is 0.1%-1% of the mass of the mixed solution; and the amount of the saccharification residue added is 20%-25% of the mass of the mixed solution.

[0014] Further preferably, the complex enzyme consists of β-glucanase and β-mannanase, and the mass ratio of the β-glucanase to the β-mannanase is 1:1-1.5.

[0015] Preferably, the enzymatic hydrolysis temperature in step (4) is 40-60°C, and the enzymatic hydrolysis time is 2-12h.

[0016] Preferably, the pH value in step (5) is 3-4, and the standing time is 0.5-1 h.

[0017] Preferably, the drying air inlet temperature in step (5) is 170-180°C, and the air outlet temperature is 80-90°C.

[0018] Preferably, the solution for adjusting the pH value in step (5) is an acid solution, more preferably an HCl solution, with a concentration of 1 mol / L.

[0019] The beneficial effects of the present invention are: 1. The by-product of konjac processing, konjac powder, is used to prepare konjac mannan oligosaccharide, which solves the problem of large output and serious stockpiling of by-products in the konjac processing process, improves the utilization rate and economic value of konjac resources, reduces the processing cost of konjac processing by-products by enterprises, and enriches the source of mannan oligosaccharide.

[0020] 2. By alcohol washing and enzyme treatment, the alkaloids, total phenols and other materials in the konjac powder are removed, and the sulfur dioxide residue brought in during processing for the purpose of color protection can also be helped to be removed simultaneously, and no special smell, high-purity manno-oligosaccharides are obtained, and the difficult problem of special smell in animal feed application is overcome, and the physiological activity benefit is enhanced; Compared with single enzyme treatment, the manno-oligosaccharides with higher quality can be obtained. In addition, the alcohol washing and enzyme treatment adopted by the present invention are applicable to multiple konjac flour products with certain peculiar smell, such as konjac fly powder, konjac powder etc.

[0021] 3. Use hydrochloric acid solution to adjust the pH value of the supernatant after the composite enzyme degradation treatment to reach the isoelectric point of the protein, so that the protein in the supernatant can be precipitated. The protein can be separated by simple centrifugation to further improve the purity of manno-oligosaccharides. The operation is simple and the removal effect is good.

[0022] 4. The present invention has simple process and low equipment requirements, is suitable for industrial-scale production, and can be promoted and applied on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a process flow chart of the present invention.

[0024] Figure 2 LogMp-RT (number average molecular weight) calibration curve for molecular weight detection of mannooligosaccharides prepared for standards.

[0025] Figure 3 LogMw-T (weight average molecular weight) calibration curve for molecular weight detection of mannooligosaccharides prepared for standards.

[0026] Figure 4 LogMn-RT (number average molecular weight) calibration curve for molecular weight detection of mannooligosaccharides prepared for standards. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present invention and should not be understood as limiting the present invention. The protection scope of the present invention shall be based on the contents recorded in the claims. Any modification or replacement made by those skilled in the art to the technical solution of the present invention without creative work shall fall within the protection scope of the present invention.

[0028] In the following examples, the konjac flour has a viscosity of 10000 mPa·s, a glucomannan content of 30%, a sulfur dioxide content of 860 ppm, a khaki color, and a particle size of more than 120 meshes; The activity of the thermostable α-amylase is 2000U; the activity of the saccharifying enzyme is 50000U; the activity of the β-glucanase is 65000U; and the activity of the β-mannanase is 20000U.

[0029] Example 1 (1) Removal of alkaloids: taking konjac powder, washing it with 30%, 30% and 50% ethanol solutions at a solid-liquid ratio of 1:30 for 10 min, 10 min and 30 min respectively to remove alkaloids and other impurities, and then filtering and drying to obtain konjac flour; (2) Destarchization: adding konjac flour to a solution containing thermostable α-amylase, mixing to obtain a mixed solution, and then cooking at 98°C for 90 min, cooling to 60°C at room temperature, and obtaining a gelatinized solution; wherein the mass fraction of konjac flour in the mixed solution is 18%, and the amount of thermostable α-amylase added is 20 U / g (g is the mass of konjac flour); (3) Saccharification: add 200 U / g saccharifying enzyme (g is the mass of konjac powder) to the gelatinized liquid while stirring, perform enzymatic hydrolysis at 60°C for 1 h, filter press, and wash with clean water three times to obtain saccharification residue; (4) Degradation of konjac glucomannan: Place the saccharification residue in water, then add the composite enzyme, mix well to obtain a composite enzymatic solution, react at 50°C for 10 hours, then boil for 10 minutes to inactivate the enzyme, and centrifuge to retain the supernatant; the mass ratio of the saccharification residue, water and composite enzyme is 300:1000:10 (the amount of the composite enzyme is 0.7%), and the composite enzyme is composed of β-glucanase and β-mannanase added at a mass ratio of 1:1.5; (5) Deproteinization: The pH value of the supernatant was adjusted to 3.8 with 1 mol / L HCl solution. After standing for 30 min, the supernatant was centrifuged at 4000 r / min for 15 min. The supernatant was retained and spray-dried to obtain light yellow manno-oligosaccharides. The inlet air temperature of the spray-drying was 170 °C and the outlet air temperature was 80 °C.

[0030] Example 2 The method and steps are the same as those of Example 1, wherein in step (1), the konjac powder is washed 5 times with 30% ethanol by mass, each time for 15 minutes; The mass fraction of konjac flour in the mixed solution in step (2) is changed to 20%; In step (4), the mass ratio of saccharification residue, water and compound enzyme was changed to 300:1000:12 (the dosage of compound enzyme was 0.9%); In step (5), the pH is adjusted to 4.0 to prepare manno-oligosaccharides.

[0031] Example 3 The method and steps are the same as those of Example 1, wherein the mass fraction of konjac flour in the mixed solution in step (2) is changed to 15%, and the amount of thermostable α-amylase is changed to 10 U / g; In step (3), the amount of saccharifying enzyme in the mixed solution was changed to 100 U / g; In step (4), the mass ratio of the complex enzyme β-glucanase to β-mannanase was changed to 1:1.2, and the enzymatic reaction time was shortened to 6 h; and manno-oligosaccharides were prepared.

[0032] Example 4 The method and steps are the same as those of Example 1, wherein the mass fraction of konjac flour in the mixed solution in step (2) is changed to 15%; the amount of thermostable α-amylase added is changed to 15 U / g; In step (3), the amount of saccharifying enzyme in the mixed solution is changed to 150 U / g; In step (4), the mass ratio of saccharification residue, water and complex enzyme was changed to 300:1000:7 (the dosage of complex enzyme was 0.5%) to prepare manno-oligosaccharides.

[0033] Comparative Example 1 The method and steps were the same as those in Example 1, except that step (1) was omitted to prepare manno-oligosaccharides.

[0034] Comparative Example 2 The method and steps were the same as those in Example 1, except that (2) and (3) were omitted to prepare manno-oligosaccharides.

[0035] Comparative Example 3 The method and steps are the same as those in Example 1, except that the complex enzyme in step (4) is replaced by an equal amount of β-glucanase to prepare manno-oligosaccharides.

[0036] Comparative Example 4 The method and steps are the same as those in Example 1, except that the complex enzyme in step (4) is replaced by an equal amount of β-mannanase to prepare manno-oligosaccharides.

[0037] Comparative Example 5 The method and steps are the same as those in Example 1, except that the mass ratio of β-glucanase to β-mannanase in step (4) is changed to 1:2 to prepare manno-oligosaccharides.

[0038] Comparative Example 6 The method and steps were the same as those in Example 1, except that the mass ratio of saccharification residue, water and complex enzyme in step (4) was changed to 300:1000:1 (the amount of complex enzyme was 0.07%) to prepare manno-oligosaccharides.

[0039] Comparative Example 7 The method and steps are the same as those in Example 1, except that steps (1), (2), (3) and (5) are deleted and enzymatic hydrolysis is directly performed according to step (4) to prepare manno-oligosaccharides.

[0040] Comparative Example 8 The method and steps are the same as those of Example 1, except that the mass fraction of konjac flour in the mixed solution in step (2) is changed to 30%.

[0041] Comparative Example 9 The method and steps are the same as those in Example 1, except that the konjac powder in step (1) is replaced by konjac powder.

[0042] Results test: The manno-oligosaccharides prepared in the above examples and comparative examples were taken, and the appearance was counted and the molecular weight of the finished product was indicated by the high performance gel permeation chromatography results. The detection method was referred to "A method for testing the molecular weight and molecular weight distribution of alginate by high performance gel permeation chromatography" (Zhang Ruixin, Wang Zhihui, Cong Wei, etc., Journal of Process Engineering, 2019), in which dextran with different molecular weights was used as the standard, and the LogMp-RT, LogMw-T and LogMn-RT calibration curves were shown in Figure 2-4 , the molecular weight of the prepared manno-oligosaccharide was detected after calibration with the standard; the yield was calculated at the same time, and the content of manno-oligosaccharide was detected with reference to the enterprise standard Q / YZ 0015-2024, where the yield was calculated as follows: yield = mass of final product ÷ mass of added konjac powder × 100%, the results are shown in the following table:

[0043] The results show that when only β-glucanase (Comparative Example 3) or only β-mannanase (Comparative Example 4) is used to degrade konjac glucomannan, the content and yield of manno-oligosaccharides are significantly affected, and the molecular weight of manno-oligosaccharides is concentrated at 1000Da, which increases the difficulty of manno-oligosaccharides. When the amount of β-mannanase in the complex enzyme is increased (Comparative Example 5), although the appearance and smell of manno-oligosaccharides are not affected, the molecular weight is further increased, which is not conducive to the subsequent application of manno-oligosaccharides.

[0044] When the amount of the complex enzyme added is too small (Comparative Example 6), the content and yield of manno-oligosaccharides are further reduced, the molecular weight is further increased, and high-value manno-oligosaccharides cannot be obtained.

[0045] When one or more of the steps of alkaloid removal, starch removal, saccharification, and protein removal are not performed, the effective content of manno-oligosaccharides in the product will decrease due to the interference of sulfur dioxide with enzyme activity, partial starch degradation, insufficient enzyme chain degradation, etc., resulting in insufficient product purity (Comparative Examples 1, 2, and 7). When konjac powder is replaced with fly powder, it is affected by the composition of the fly powder, and the yield of manno-oligosaccharides prepared therefrom is very low (Comparative Example 9).

[0046] In summary, referring to the steps of Example 1, the manno-oligosaccharides prepared by combining the properties of the enzyme have a higher content of active ingredients, better quality, more cost-effective production costs, are more suitable for large-scale production, consume by-products produced during konjac processing, and reduce resource waste.

Claims

1. A method for preparing high-purity manno-oligosaccharides from konjac flour, characterized in that: The steps include: (1) Removing alkaloids: washing konjac powder with an ethanol solution, filtering and drying to obtain konjac flour; (2) Destarchization: placing konjac flour in an α-amylase solution and mixing to obtain a mixed solution, steaming and cooling to obtain a gelatinized solution; (3) Saccharification: Add saccharifying enzyme to the gelatinized liquid, filter after the reaction to obtain saccharification residue; (4) Degradation of konjac glucomannan: placing the saccharification residue in a complex enzyme solution to obtain a mixed solution, enzymatically hydrolyzing and centrifuging to obtain a supernatant; (5) Deproteinization: Adjust the pH of the supernatant to the isoelectric point of the protein, let it stand, centrifuge, and dry to obtain high-purity manno-oligosaccharides.

2. The method for preparing high-purity manno-oligosaccharides from konjac powder according to claim 1, wherein: The mass concentration of the ethanol solution in step (1) is 30%-50%, and the washing times are 3-5 times; the mass ratio of konjac powder to the ethanol solution is 1:20-30.

3. The method for preparing high-purity manno-oligosaccharides from konjac powder according to claim 1, wherein: The amount of konjac flour in step (2) is 15%-20% of the total mass of the mixed solution; the amount of α-amylase added is 10-20 U / g.

4. The method for preparing high-purity manno-oligosaccharides from konjac powder according to claim 1, wherein: The cooking temperature in step (2) is 90-100° C. and the cooking time is 1-2 h.

5. The method for preparing high-purity manno-oligosaccharides from konjac powder according to claim 1, characterized in that: The amount of saccharifying enzyme added in step (3) is 100-200 U / g, the reaction temperature is 50-60°C, and the reaction time is 1-2h.

6. The method for preparing high-purity manno-oligosaccharides from konjac powder according to claim 1, characterized in that: The amount of the complex enzyme added in step (4) is 0.1%-1% of the mass of the mixed solution; the amount of the saccharification residue added is 20%-25% of the mass of the mixed solution.

7. The method for preparing high-purity manno-oligosaccharides from konjac powder according to claim 6, characterized in that: The complex enzyme consists of beta-glucanase and beta-mannanase, and the mass ratio of the beta-glucanase to the beta-mannanase is 1:1-1.

5.

8. The method for preparing high-purity manno-oligosaccharides from konjac powder according to claim 1, characterized in that: The enzymatic hydrolysis temperature in step (4) is 40-60°C, and the enzymatic hydrolysis time is 2-12h.

9. The method for preparing high-purity manno-oligosaccharides from konjac powder according to claim 1, characterized in that: The pH value of step (5) is 3-4, and the standing time is 0.5-1h.

10. The method for preparing high-purity manno-oligosaccharides from konjac powder according to claim 1, characterized in that: The drying air inlet temperature in step (5) is 170-180°C, and the air outlet temperature is 80-90°C.

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