Method for measuring content of glucomannan in konjak

Abstract

The invention belongs to the technical field of konjac glucomannan content determination, and particularly relates to a method for determining the content of glucomannan in konjac. According to the method, ethanol precipitation with the mass percent of 70%-75% at the temperature of 4-6 DEG C is combined with a directional enzymolysis technology, glucomannan is selectively precipitated through an ethanol solution, meanwhile, interference substances such as starch and protein are removed through enzymolysis reaction specificity, and efficient purification and accurate quantification of konjac glucomannan are achieved in cooperation with a low-temperature vacuum drying technology. The method can effectively simplify the sample pretreatment and detection process, improves the detection efficiency, is suitable for detecting the glucomannan content of the fresh konjak whole flour planted in the field and the konjak refined flour in the processing plant, has the advantages of low cost, high efficiency and environmental protection, and is suitable for popularization and application. The method provides an efficient and reliable solution for quality control and large-scale detection of konjak products.

Description

Technical Field

[0001] The present invention belongs to the technical field of determination of glucomannan content, and particularly relates to a method for determining the glucomannan content in konjac. Background Art

[0002] Konjac ( Amorphophallus konjac ) is a perennial herb of the genus Amorphophallus in the family Araceae. The konjac corm is rich in a high-molecular polysaccharide - glucomannan (KGM), which has broad application prospects in the fields of food, medicine, chemical engineering, and environmental protection. Konjac glucomannan is a water-soluble dietary fiber, formed by connecting D-glucose and D-mannose through β-1,4 glycosidic bonds to form a long-chain structure. Its excellent viscosity and biological activity make it favored in the fields of food thickeners, dietary supplements, etc.

[0003] At present, the detection of konjac glucomannan content mainly uses precipitation method, spectrophotometry, and high-performance liquid chromatography. These methods are mostly used for the determination of glucomannan in konjac refined powder. Konjac refined powder refers to the steps of drying, crushing, and screening konjac slices, and then subjecting the screened konjac powder to air separation, grinding, re-air separation, screening, and grading by dry method, wet method, or combined dry and wet method to finally obtain konjac refined powder. It can be seen that the processing steps for determining glucomannan using konjac refined powder are long and the process is complex. In order to reduce the pretreatment of konjac, there are also detections of glucomannan in fresh konjac, that is, konjac whole powder, in the prior art. Luo Yumei et al. reported a method for determining the main components of fresh konjac based on spectrophotometry. This method requires multiple pretreatment steps such as grinding, filtering, and suction filtration to extract glucomannan, and after extraction, complex operations such as swelling, acidolysis, neutralization, and colorimetry are also required. Finally, the glucomannan content is indirectly calculated through a glucose standard curve. However, this method not only has a long determination period but also is cumbersome to operate. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a method for determining the glucomannan content in konjac. This method optimizes the extraction process of glucomannan through low-temperature ethanol extraction combined with directional enzymolysis technology, simplifies the detection steps, shortens the determination period, and significantly improves the efficiency of the glucomannan content determination method.

[0005] To achieve the above object, the technical solution of the present invention is as follows.

[0006] A method for determining the glucomannan content in konjac, comprising the following steps: Placing konjac powder in an alcohol solvent, extracting at 4°C - 6°C for 2h - 3h to release glucomannan with a complete structure in the alcohol solvent, and then centrifuging to obtain a first precipitate; Enzymatically hydrolyzing the starch and protein in the first precipitate, and then performing alcohol precipitation and centrifuging. The obtained second precipitate is glucomannan; Weigh the mass of the glucomannan and calculate the content of the glucomannan in the konjac powder; The alcohol solvent is an ethanol solution with a mass percentage of 70% - 75%.

[0007] By extracting glucomannan in konjac powder at a temperature of 4°C - 6°C, the present invention can ensure the integrity of the glucomannan structure and the sufficient dissolution of glucomannan. Moreover, it can effectively avoid the problem of the destruction of the glucomannan structure caused by enzymatic degradation and thermal degradation during the subsequent enzymatic hydrolysis process, which helps to further protect the structural integrity of glucomannan. After enzymatic hydrolysis, the glucomannan in konjac powder can be effectively extracted, which effectively simplifies the pretreatment process and thus effectively improves the measurement efficiency.

[0008] The present invention selects to carry out the extraction under the low-temperature condition of 4°C - 6°C mainly because at room temperature of 23°C - 28°C, due to the high stability of the intermolecular hydrogen bonds of glucomannan, the dissolution and release are insufficient, resulting in low extraction efficiency, and microorganisms are likely to grow at room temperature, which may contaminate the extract; while at high temperature of 60°C - 80°C, although the solubility of glucomannan increases, it will cause partial molecular degradation and damage its structural integrity, affecting the measurement accuracy. In addition, an ethanol solution with a mass percentage of 50% cannot fully precipitate glucomannan, resulting in a low measurement result; an ethanol solution with a mass percentage of 90% will extract other impurities together, making the measurement result too high. Therefore, the present invention selects an ethanol solution with a mass percentage of 70% - 75%, which can not only achieve the complete precipitation of glucomannan under low-temperature conditions but also effectively avoid impurity interference and ensure the accuracy of the measurement result.

[0009] In another preferred embodiment, the konjac powder is konjac whole powder or konjac refined powder; the konjac whole powder refers to the product obtained by washing, crushing, and drying konjac.

[0010] In another preferred embodiment, the mass-volume ratio of the konjac whole powder or konjac refined powder to the alcohol solvent is 1 g: 3 mL - 4 mL.

[0011] In another preferred embodiment, the specific process of the enzymatic hydrolysis is as follows: Mix the first precipitate with α-amylase and carry out enzymatic hydrolysis for 40 min - 50 min at 45°C - 50°C and a pH value of 6 - 7, then add protease and continue enzymatic hydrolysis for 30 min - 40 min at 40°C - 45°C and a pH value of 7.5 - 8.

[0012] In another preferred embodiment, the mass ratio of the α-amylase to the first precipitate is 1: 50 - 100; the mass ratio of the protease to the first precipitate is 1: 100 - 200.

[0013] In another preferred embodiment, the protease is papain or trypsin.

[0014] In another preferred embodiment, the reagent used for alcohol precipitation is absolute ethanol.

[0015] In another preferred embodiment, before the konjac flour or konjac refined flour is mixed with the alcohol solvent, it also includes soaking in water. The specific process is as follows: Soak the konjac flour or konjac refined flour in water for 20 min to 30 min, and centrifuge to discard the supernatant.

[0016] In another preferred embodiment, the specific calculation formula for the content of glucomannan in konjac flour is as follows: A = B×ε / C×100%; In the formula, A represents the content of glucomannan, in %; B represents the mass of the extracted glucomannan, in g; C represents the mass of konjac flour, in g; ε represents the correction coefficient, and ε is 0.9.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for determining the content of glucomannan in konjac. By extracting at a low temperature of 4°C to 6°C and with an ethanol solution with a mass percentage of 70% to 75%, it not only ensures the structural integrity of glucomannan but also enables its full precipitation, improving the measurement accuracy, achieving the high-efficiency purification and rapid determination of glucomannan, effectively simplifying the pre-treatment steps and improving the detection efficiency. The method in the present invention can be used to determine the glucomannan in konjac flour and konjac refined flour, directly extract the glucomannan in the konjac dry matter, and calculate its content by mass ratio, significantly simplifying the determination process and improving the detection efficiency. Compared with the prior art, the present invention abandons the cumbersome steps such as wine washing, swelling, acidolysis, and neutralization in the traditional method, and only needs to purify the glucomannan in konjac flour to complete the determination, greatly shortening the detection cycle.

[0018] The present invention further improves the purity of glucomannan by specifically enzymatically removing interfering substances such as residual starch and protein. The present invention adopts the technology of low-temperature ethanol precipitation combined with directional enzymatic hydrolysis to effectively remove interfering substances such as starch and protein, and at the same time conducts quantitative analysis by the mass ratio of konjac glucomannan to konjac flour, simplifying the detection steps and shortening the detection cycle.

[0019] The method in the present invention has the advantages of simple operation, low cost, high accuracy, environmental friendliness, etc., is suitable for large-scale popularization and application, and provides an efficient and reliable solution for the rapid detection of the content of konjac glucomannan. Specific Embodiments

[0020] Next, in combination with specific embodiments of the present invention, the technical solutions in the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0021] In each embodiment of the present invention, the methods described, unless otherwise specified, are all conventional methods. The materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0022] Currently, the spectrophotometry is a commonly used method for the determination of glucomannan content. However, its determination process is complex and requires steps such as grinding, filtering, and suction filtration to extract glucomannan. Subsequently, operations such as swelling, acid hydrolysis, neutralization, and colorimetry are also required. Finally, the glucomannan content is indirectly determined through a glucose standard curve. This method not only takes a long time and has cumbersome steps, but also has high requirements for the experimental environment and is difficult to meet the needs of rapid detection.

[0023] In view of the above problems, the present invention proposes a method for the determination of glucomannan content in konjac. High-purity glucomannan is extracted through low-temperature ethanol precipitation and directed enzymolysis techniques, and the content is directly determined by mass ratio, significantly simplifying the detection process and improving the detection efficiency and accuracy. This method is applicable to the rapid detection of fresh konjac whole powder in the field planting front line and can also be used for the determination of glucomannan content in konjac refined powder in processing plants. It has the advantages of low cost, high efficiency, and environmental friendliness, providing a new solution for the quality control and large-scale detection of konjac products.

[0024] Compared with the prior art, the present invention has a wide range of applications. It can detect both fresh konjac whole powder and the glucomannan content in konjac refined powder, meeting the detection needs of different scenarios. And it has high purification efficiency. Through low-temperature ethanol precipitation at 4°C to 6°C and directed enzymolysis techniques, interfering substances such as starch and protein are effectively removed, significantly improving the purification efficiency of glucomannan; the entire detection process is simplified. A determination system for the mass ratio of glucomannan to dry matter is established, eliminating cumbersome steps such as alcohol washing, swelling, acid hydrolysis, neutralization, and colorimetry in the traditional method, greatly shortening the detection time; ethanol is used to replace toxic organic solvents, reducing the detection cost and at the same time reducing the difficulty of waste liquid treatment, meeting the requirements of environmental friendliness. The present invention is applicable to the rapid detection at the konjac planting site and the raw material quality control of processing enterprises, providing reliable technical support for the quality monitoring of the konjac industrial chain and having important popularization and application value.

[0025] Next, a method for the determination of glucomannan content in konjac will be specifically described.

[0026] Example 1 A method for the determination of glucomannan content in konjac includes the following steps: S1. Select fresh konjac corms with smooth epidermis, no pests and diseases, no rot, and good appearance, rinse them with clean water to remove the soil and impurities on the surface; cut the cleaned konjac corms into thin slices with a thickness of 3 mm to increase the surface area and improve the drying speed.

[0027] S2. Place the konjac slices in a low-temperature drying oven, control the temperature at 50 °C, take them out after drying until completely dehydrated, and calculate the water content of the fresh konjac; grind the dried konjac slices to 100 meshes, soak them in distilled water for 20 min, then centrifuge at a low speed of 3000 rpm / min for 10 minutes, and discard the supernatant to remove water-soluble impurities to obtain konjac flour.

[0028] S3. Mix the konjac flour with a 70% ethanol solution by mass-volume ratio of 1 g: 3 mL, let it stand at 4 °C for 2 hours to precipitate glucomannan into the ethanol solution, centrifuge at a low speed of 3000 rpm / min for 8 minutes, and discard the supernatant to obtain the first precipitate.

[0029] S4. Mix the first precipitate with α-amylase at a mass ratio of 1: 50, enzymatically hydrolyze it at 45 °C and pH 6 for 40 min, then add papain and continue to enzymatically hydrolyze it at 40 °C and pH 7.5 for 30 min, where the mass ratio of papain to the first precipitate is 1: 100; perform alcohol precipitation with absolute ethanol at 4 °C, centrifuge at 3500 rpm / min for 5 min, and discard the supernatant to obtain the second precipitate.

[0030] S5. Wash the second precipitate with 95% ethanol by mass twice, centrifuge at 3500 rpm / min for 5 min after each washing, collect the precipitate to obtain glucomannan, weigh the mass of the glucomannan, and calculate the content of the glucomannan in the konjac flour. The results are shown in Table 1.

[0031] Example 2 A method for determining the content of glucomannan in konjac includes the following steps: S1. Select fresh konjac corms with smooth epidermis, no pests and diseases, no rot, and good appearance, rinse them with clean water to remove the soil and impurities on the surface; cut the cleaned konjac corms into thin slices with a thickness of 3 mm to increase the surface area and improve the drying speed.

[0032] S2. Place the konjac slices in a low-temperature drying oven, control the temperature at 50 °C, take them out after drying until completely dehydrated, and calculate the water content of the fresh konjac; grind the dried konjac slices to 100 meshes, soak them in distilled water for 30 min, then centrifuge at a low speed of 3500 rpm / min for 15 minutes, and discard the supernatant to remove water-soluble impurities to obtain konjac flour.

[0033] S3. Mix konjac flour with 75% ethanol solution by a mass-to-volume ratio of 1 g: 4 mL, let it stand at 4 °C for 3 hours to precipitate glucomannan into the ethanol solution, centrifuge at a low speed of 3500 rpm / min for 15 minutes, discard the supernatant, and obtain the first precipitate.

[0034] S4. Mix the first precipitate with α-amylase at a mass ratio of 1:100, enzymatically hydrolyze it at 50 °C and pH 7 for 50 min, then add trypsin and continue to enzymatically hydrolyze it at 45 °C and pH 8 for 40 min, where the mass ratio of papain to the first precipitate is 1:200; perform alcohol precipitation with absolute ethanol at 5 °C, centrifuge at 3500 rpm / min for 5 min, discard the supernatant, and obtain the second precipitate.

[0035] S5. Wash the second precipitate with ethanol of mass percentage for 2 times, centrifuge at 3500 rpm / min for 5 min after each washing, collect the precipitate to obtain glucomannan, weigh the mass of the glucomannan, and calculate the content of the glucomannan in konjac powder. The results are shown in Table 1.

[0036] Example 3 A method for determining the content of glucomannan in konjac, comprising the following steps: S1. Select fresh konjac corms with smooth epidermis, no pests and diseases, no rot, and good appearance, rinse them with clean water to remove the soil and impurities on the surface; cut the cleaned konjac corms into slices with a thickness of 4 mm to increase the surface area and improve the drying speed.

[0037] S2. Place the konjac slices in a low-temperature drying oven, control the temperature at 55 °C, take them out after drying to complete water loss, and calculate the water content of fresh konjac; grind the dried konjac slices to 110 meshes, soak them in distilled water for 25 min, then centrifuge at a low speed of 3000 rpm / min for 10 minutes, discard the supernatant to remove water-soluble impurities, and obtain konjac flour.

[0038] S3. Mix konjac flour with 70% ethanol solution by a mass-to-volume ratio of 1 g: 3 mL, let it stand at 6 °C for 3 hours to precipitate glucomannan into the ethanol solution, centrifuge at a low speed of 3500 rpm / min for 15 minutes, discard the supernatant, and obtain the first precipitate.

[0039] S4. Mix the first precipitate and α-amylase at a mass ratio of 1:100, enzymatically hydrolyze for 50 min at 50 °C and a pH value of 7, then add trypsin and continue to enzymatically hydrolyze for 40 min at 45 °C and a pH value of 8, where the mass ratio of papain to the first precipitate is 1:200; perform alcohol precipitation with absolute ethanol at 5 °C, centrifuge at 3500 rpm / min for 5 min, discard the supernatant to obtain the second precipitate.

[0040] S5. Wash the second precipitate twice with ethanol with a mass percentage of 95%, centrifuge at 3500 rpm / min for 5 min after each wash, collect the precipitate to obtain glucomannan, weigh the mass of the glucomannan, and calculate the content of the glucomannan in konjac flour. The results are shown in Table 1.

[0041] Perform spectrophotometer detection on the glucomannan obtained in Examples 1 to 3 respectively. The specific process is as follows: Hydrolyze glucomannan in a boiling water bath with 3 mol / L H 2 SO 4 Then neutralize it to neutral with 6 mol / l NaOH solution, react with 3,5-dinitrosalicylic acid to form a brownish-red amino compound, and then perform colorimetry with a spectrophotometer to determine the purity of glucomannan. The results are shown in Table 1.

[0042] Table 1 Glucomannan purity Vacuum dry the glucomannan in Examples 1 to 3 at 50 °C and 100 Pa for 6 hours respectively, weigh the mass of the dried glucomannan, record the measurement data, substitute it into the formula for calculation, and the results are shown in Table 2 below. The calculation formula is as follows:

[0043] A = B×ε / C×100%; In the formula, A represents the glucomannan content, with the unit of %; B represents the mass of the extracted glucomannan, with the unit of g; C represents the mass of konjac flour, with the unit of g; ε represents the correction coefficient, and ε is 0.9.

[0044] Table 2 Glucomannan content extracted in Examples 1 to 3 Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concepts. Obviously, those skilled in the art can make various changes and deformations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and deformations of the present invention fall within the scope of the equivalent technology of the present invention, the present invention also intends to include these changes and deformations.

Claims

1. A method for determining the content of glucomannan in konjac, characterized in that: The following steps are involved: The konjac flour is placed in an alcohol solvent and extracted at 4°C to 6°C for 2h to 3h to release the glucomannan with a complete structure in the alcohol solvent, followed by centrifugation to obtain a first precipitate; The starch and protein in the first precipitate are enzymatically hydrolyzed, then alcohol precipitated, and centrifuged to obtain the second precipitate, i.e., glucomannan; the mass of the glucomannan is weighed, and the content of the glucomannan in the konjac flour is calculated; The alcohol solvent is an ethanol solution with a mass percentage of 70% to 75%.

2. the assay method of glucomannan content in the konjac according to claim 1, is characterized in that, The konjac flour is whole konjac flour or refined konjac flour; The whole konjac flour is obtained by washing, crushing and drying the konjac.

3. the assay method of glucomannan content in the konjac according to claim 2, is characterized in that, The mass volume ratio of konjac whole flour or konjac refined flour to alcohol solvent is 1g:3mL~4mL.

4. the assay method of glucomannan content in the konjac according to claim 1, is characterized in that, The specific process of the enzymolysis is as follows: The first precipitate is mixed with α-amylase, and enzymatically hydrolyzed at 45°C~50°C and pH 6~7 for 40min~50min, and then protease is added, and enzymatic hydrolysis is continued at 40°C~45°C and pH 7.5~8 for 30min~40min.

5. the assay method of glucomannan content in the konjac according to claim 4, is characterized in that, The mass ratio of the α-amylase to the first precipitate is 1:50-100; the mass ratio of the protease to the first precipitate is 1:100-200.

6. the assay method of glucomannan content in the konjac according to claim 5, is characterized in that, The protease is papain or trypsin.

7. The assay method for glucomannan content in the konjac according to claim 1, wherein The alcohol precipitation adopts anhydrous ethanol.

8. The method for determining the glucomannan content in the konjac according to claim 1, wherein The whole konjac flour or refined konjac flour is further soaked in water before being mixed with the alcohol solvent.

9. The method for determining the glucomannan content in the konjac according to claim 1, wherein The specific calculation formula of the content of the glucomannan in the konjac flour is as follows: A=B×ε / C×100%; Wherein, A represents the content of glucomannan in konjac flour, in units of %; B represents the mass of glucomannan extracted, in units of g; C represents the mass of konjac flour, in units of g; ε represents the correction coefficient, which is 0.9.

Images