A green and efficient method for extracting beta-glucan from highland barley

Abstract

The application discloses a kind of green efficient extraction of highland barley beta-glucan method, comprising the following steps: highland barley whole grain grinding powder;With highland barley whole grain powder is prepared powder;Ethanol enzyme removal impurity: highland barley powder is extracted with 80% ethanol, filtration and drying;Hot water extraction: distilled water extraction, centrifugal supernatant is combined;Enzymatic hydrolysis impurity: supernatant is sequentially added in high temperature resistant alpha-amylase, saccharifying enzyme, trypsin and is subjected to enzymatic hydrolysis, enzyme inactivation, centrifugal, and concentrates supernatant;Crude extraction: after alcohol precipitation, centrifugal washing, water freeze-drying is obtained beta-glucan extract.The application mainly uses clean energy such as ethanol, water, does not use organic chemical substance;The method is simple in operation, equipment cost is lower, and it is relatively energy saving and emission reduction;Extraction efficiency is higher, easy to factory implementation.

Description

Technical Field

[0001] This invention relates to the field of functional components of highland barley, specifically to a green and efficient method for extracting β-glucan from highland barley. Background Technology

[0002] Qinghai Province in my country has a vast area of ​​highland barley cultivation, high yield, and excellent quality. However, the highland barley industry suffers from a lack of sophisticated processing enterprises, which are small in scale, have low technological content, produce low-grade and low-quality products with outdated processing techniques, resulting in low added value and hindering the development of highland barley production bases. Highland barley is rich in nutrients, containing 18 essential amino acids, starch, and various vitamins. Its protein and lysine content are higher than that of wheat and rice.

[0003] β-glucan plays a crucial role in controlling blood sugar levels, lowering blood lipids, reducing cholesterol levels, and enhancing immunity. It can prevent or help improve type II diabetes, prevent colon cancer, improve serum short-chain fatty acid levels, and promote lipid metabolism in the liver. It can also be used as a raw material in the food industry. In addition, highland barley β-glucan exhibits high resistance to digestion, overcoming α-amylase hydrolysis and human stomach acid. It can reduce specific intestinal flora, decrease hydrogen sulfide production, thereby reducing the risk of colon cancer, and also has a significant anti-fatigue effect. Methods for extracting β-glucan mainly include three-phase partitioning, ultrasonic-microwave synergistic extraction, ultrasonic-assisted extraction, microwave-assisted (MAE) extraction, hot water extraction, ultrasonic method, microwave method, water extraction + alkali extraction, ultra-high pressure extraction, ultra-high pressure + ultrasonic method, acid hydrolysis, alkaline extraction, biotransformation or microbial fermentation, pressurized hot water extraction, and purification using ammonium sulfate precipitation, column separation, dialysis, etc. Despite the variety of methods, the extraction rate is low, the process is complex, the steps are cumbersome, and the energy consumption is high, making it difficult to implement in industrial applications. Summary of the Invention

[0004] This invention discloses a green and efficient method for extracting β-glucan from highland barley. Addressing potential problems encountered in literature and practice, this patent employs a more energy-efficient and industrially applicable hot water extraction method, optimizes the extraction process steps, refines the step parameters, and further improves extraction efficiency to meet the needs of industrial extraction.

[0005] The technical solution of the present invention is as follows:

[0006] A green and efficient method for extracting β-glucan from highland barley includes the following steps:

[0007] 1. Grind highland barley into flour

[0008] Barley flour was obtained by grinding whole barley grains. The flour was then sieved through a 100-mesh sieve in a laboratory mill. Studies showed that the size of the grinding particles affected the extraction efficiency. Therefore, in the energy-saving process, an optimal grinding particle size should be selected to maximize the dissolution of β-glucan during extraction.

[0009] 2. Ethanol reflux extraction

[0010] Barley powder and 80% ethanol were mixed at a ratio of 1:10 w / v and extracted by reflux at 80°C for 2 hours. After filtration, the precipitate was dried at 60°C to constant weight, pulverized by a mill, and sieved through a 100-mesh sieve.

[0011] 3. Hot water extraction

[0012] The ground barley powder was extracted with distilled water at 52°C for 2 h (material-to-liquid ratio of 1:10 w / v), followed by centrifugation at 5000 rpm for 10 min. This step was repeated 3 times, and the supernatants were combined.

[0013] 4. Enzymatic decontamination

[0014] Add 10 U / g thermostable α-amylase to the supernatant, maintain pH at 6.5, heat in a 95°C water bath for 2 hours, then cool to 60°C. Adjust pH to 4.5 with dilute hydrochloric acid, add 200 U / g of saccharifying enzyme, and react at 60°C for 30 minutes. Adjust pH to 7.5 with 0.1 mol / L sodium hydroxide solution, add 200 U / mg of trypsin, and react at 50°C for 1 hour. After enzymatic hydrolysis, inactivate the enzyme in a 100°C water bath for 20 minutes, then cool to room temperature. Remove the precipitate by centrifugation at 5000 rpm for 15 minutes, and concentrate the supernatant.

[0015] 5. Obtain the crude extract.

[0016] Add anhydrous ethanol to 80%, precipitate the precipitate, centrifuge at 5000 rpm for 15 min, wash the precipitate twice with anhydrous ethanol, reconstitute with water, freeze dry under vacuum to remove ethanol, and freeze dry to obtain water-extracted β-glucan extract (HBBG).

[0017] Beneficial effects

[0018] This invention modifies the enzyme combination in the extraction method of β-glucan, thereby reducing the overall amount of enzyme used. Compared with other physical or chemical extraction methods, it mainly uses clean energy sources such as ethanol and water, without using organic chemicals; this method is simple to operate, has low equipment costs, and is energy-efficient and emission-reducing; it has high extraction efficiency and is easy to implement in industrial settings. Detailed Implementation

[0019] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0020] Example 1

[0021] A method for preparing β-glucan from barley includes the following steps:

[0022] (1) Grinding highland barley into flour

[0023] Grind 50g of whole barley grains using a grinder, sieve through a 100-mesh sieve, and store at 4℃ for later use.

[0024] (2) Ethanol extraction

[0025] 50g of highland barley powder was soaked in 500ml of 80% ethanol and extracted by reflux at 80℃ for 2h. After filtration, the precipitate was dried at 60℃ to constant weight, pulverized by a mill, and sieved through a 100-mesh sieve.

[0026] (3) Hot water extraction

[0027] The ground barley powder was extracted with distilled water at 52°C for 2 h, followed by centrifugation at 5000 rpm for 10 min. This step was repeated 3 times, and the supernatants were combined.

[0028] (4) Enzymatic decontamination

[0029] Add 10 U / g thermostable α-amylase to the supernatant, maintain pH at 6.5, heat in a 95℃ water bath for 2 h, then cool to 60℃. Adjust pH to 4.5 with 1 mol / L dilute hydrochloric acid, add 200 U / g saccharifying enzyme, and react at 60℃ for 30 min. Adjust pH to 7.5 with 2 mol / L sodium hydroxide solution, add 200 U / mg trypsin, and react at 50℃ for 1 h. After enzymatic hydrolysis, inactivate the enzyme in a 100℃ water bath for 20 min, and then cool to room temperature. Remove the precipitate by centrifugation at 5000 rpm for 15 min, and concentrate the supernatant.

[0030] (5) Obtain crude extract

[0031] After adding anhydrous ethanol to 80%, the mixture was precipitated by ethanol precipitation, centrifuged at 5000 rpm for 15 min, and the precipitate was washed twice with anhydrous ethanol. After redissolving in water, the ethanol was removed by vacuum freeze-drying, and the water-extracted β-glucan extract (HBBG) was obtained by freeze-drying.

[0032] Comparative Example 1

[0033] The difference from Example 1 is that step (2) is omitted, while the other steps are the same, and β-glucan is extracted.

[0034] Comparative Example 2

[0035] The difference from Example 1 is that in step (3), hot water extraction is performed for 2 hours, and the extraction is not repeated three times.

[0036] Comparative Example 3

[0037] The difference from Example 1 is that in step (4), the thermostable α-amylase is replaced with α-amylase, while the other steps are the same, and β-glucan is extracted.

[0038] Comparative Example 4

[0039] The difference from Example 1 is that in step (4), 10 U / g of thermostable α-amylase was added to the supernatant, the pH was maintained at 6.5, and the mixture was heated in a 95°C water bath for 2 hours, then cooled to 60°C. The pH was adjusted to 7.5 with 2 mol / L sodium hydroxide solution, and 200 U / mg of trypsin was added and reacted at 50°C for 1 hour. The pH was adjusted to 4.5 with 1 mol / L dilute hydrochloric acid, and 200 U / g of saccharifying enzyme was added and reacted at 60°C for 30 minutes. After enzymatic hydrolysis, the mixture was placed in a 100°C water bath for 20 minutes to inactivate the enzyme, and then cooled to room temperature. The precipitate was removed by centrifugation at 5000 rpm for 15 minutes, and the supernatant was concentrated.

[0040] The other steps are the same.

[0041] Comparative Example 5

[0042] Combine the conditions of Comparative Examples 1-3, keeping other conditions unchanged, and extract β-glucan.

[0043] The β-glucan extracted from Example 1 and Comparative Examples 1-5 was tested to obtain the purity and extraction rate.

[0044] Purity testing: The purity of β-glucan was measured using a commercial assay kit (Megazyme International Ireland, Bray, Ireland) according to the method of AOAC (995.16).

[0045] Extraction rate detection:

[0046] Molecular weight determination: The polydispersity of the β-glucan product was determined by gel permeation chromatography combined with multi-angle laser scattering and a refractive index detector (Wyatt Technologies, Santa Barbara, California, USA). A mobile phase of sodium nitrate (0.1 M) and sodium azide (0.02% w / v) was used at a flow rate of 0.4 mL / min. The column (Shodex Ohpak SB-805 HQ) and refractive index detector were maintained at 60°C. Measurement data were analyzed using Astra 6.0 software.

[0047]

Claims

1. A green and efficient method for extracting β-glucan from highland barley, characterized in that, Includes the following steps: (1) Grinding whole barley grains into powder; powder is obtained by grinding whole barley grains. (2) Ethanol extraction: Barley powder was extracted with 80% ethanol, and the precipitate was dried; (3) Hot water extraction: Extract with distilled water, centrifuge and combine the supernatants; (4) Enzymatic decontamination: Thermoresistant α-amylase, saccharifying enzyme and trypsin were added to the supernatant in sequence for enzymatic hydrolysis, enzyme activity was inactivated, centrifuged and the supernatant was concentrated; (5) Crude extraction: After alcohol precipitation, centrifugation and washing, lyophilization was performed to obtain β-glucan extract; In step (4), the amount of heat-resistant α-amylase added is 10 U / g, the pH value is 6.5, and the enzymatic hydrolysis is carried out at 95℃ for 2 hours; In step (4), the amount of saccharifying enzyme added is 200 U / g, the pH value is 4.5, and the enzymatic hydrolysis is carried out at 60℃ for 30 min; In step (4), the amount of trypsin added is 200 U / g, the pH value is 7.5, and the enzyme is hydrolyzed at 50℃ for 1 h; In step (3), the centrifugation conditions are 5000 rpm for 10 min, and in steps (4) and (5), the centrifugation conditions are 5000 rpm for 15 min.

2. The method for green and efficient extraction of β-glucan from highland barley according to claim 1, characterized in that, In step (2), barley powder and 80% ethanol are mixed at a ratio of 1:10 w / v, and the reflux extraction temperature is 80℃ for 2 hours.

3. The method for green and efficient extraction of β-glucan from highland barley according to claim 1, characterized in that, In step (3), the sample was extracted with distilled water at 52°C for 2 h, and the extraction was repeated three times. The material-to-liquid ratio was 1:10 w / v.

4. The method for green and efficient extraction of β-glucan from highland barley according to claim 1, characterized in that, In step (5), the final solution required for alcohol precipitation has an ethanol concentration of 80%.

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