A method for preparing high-yield and high-purity beta-glucan from highland barley

Abstract

The application provides a high-yield and high-purity preparation method of highland barley beta-glucan, which comprises the following steps: first, alkali treatment; then, ultrasonic treatment by a pretreatment solution composed of betaine, 1,4-butanediol and ethylene glycol in a molar ratio of 1:1:2; then, water is added to make the weight-volume ratio of the material solution be 1g:10-15mL, and the solution is extracted at 85-95 DEG C for 1-1.5 hours, and the filtrate is taken; then, alcohol precipitation treatment is carried out, the precipitate is redissolved by adding water, and then activated carbon is added for treatment; after filtration, concentration and drying, the highland barley beta-glucan is obtained. When the highland barley beta-glucan is prepared, the yield is as high as 90% or more, and the purity of the obtained beta-glucan is as high as 95% or more. Meanwhile, the pretreatment step in the application has the advantages of short time consumption and low energy consumption. Therefore, the application has a good application prospect in the preparation of highland barley beta-glucan.

Description

Technical Field

[0001] This invention pertains to the extraction technology of natural components from highland barley, specifically to the preparation technology of β-glucan, and more specifically to a method for preparing high-yield, high-purity highland barley β-glucan. Background Technology

[0002] Barley (Hordeum vulgare var. coeleste Linnaeus) is an annual herbaceous plant belonging to the Poaceae family and the Hordeum genus. It has three erect, smooth stalks that can reach a height of 100 cm. The leaf sheaths are smooth, with two auricles on each side that clasp the stem. The ligule is membranous, and the leaf blades are slightly rough. Barley is a staple food, fuel, and livestock feed for residents of Tibetan areas in China, and it is also a raw material for beer, medicine, and health product production. The β-glucan content in barley ranges from 3.7% to 16.9%, higher than that in other grains such as wheat, barley, and oats.

[0003] Currently, traditional methods for preparing β-glucan from grains mainly include water extraction, acid extraction, alkali extraction, enzyme extraction, and microbial enrichment. Water extraction is a common method for extracting water-soluble β-glucan, with advantages such as mild extraction conditions, low cost, and minimal glucan degradation. However, its disadvantages include a long cycle time and generally low extraction yield and purity. [1] Adding acid to the water extraction method can increase the yield of β-glucan; however, the addition of acid usually increases the degradation of β-glucan. [2] Similarly, using a high-concentration alkaline solution can also increase the yield of β-glucan, but the presence of the alkaline solution will also lead to the degradation of β-glucan molecules. [3] Enzymatic extraction requires the selection of appropriate enzymes; different enzyme choices often result in significantly different extraction outcomes. For example, in Ahmed... [4] In the study, enzymatic extraction yielded a lower yield of barley β-glucan than hydrothermal extraction; while in Maheshwari G... [5] In the studies, enzymatic extraction yielded higher yields than hydrothermal and alkaline extraction. In addition, other studies have employed auxiliary methods to improve β-glucan extraction levels, such as ultrasound-assisted extraction. [6] Microwave-assisted [7] .

[0004] The aforementioned traditional preparation methods, whether used alone or in combination, struggle to achieve a balance between extraction rate and purity. They often result in either high yield but low purity, or high purity but low yield, rarely achieving both. To overcome this limitation, microbial fermentation has emerged in recent years. Researchers, by examining the enrichment effects of different fermentation bacteria on β-glucan, have found that suitable fermentation bacteria can significantly improve the yield. [8] Similar studies [9-10]This also indicates that microbial fermentation is a relatively good method for preparing β-glucan. However, microbial fermentation also fails to simultaneously achieve a balance between yield and purity, and requires stringent environmental protection measures, resulting in high production costs.

[0005] Therefore, developing a simple method for preparing high-yield and high-purity barley β-glucan is urgently needed in this field.

[0006] [1] Jia Ying. Extraction, separation, purification and properties of water-soluble β-glucan from highland barley bran [D]. East China University of Science and Technology, 2013.

[0007] [2]Ahmad A, Anjum FM, Zahoor T, et al. Extraction and characterization of beta-D-glucan from oat for industrial utilization. [J]. International journal of biological macromolecules, 2010, 46 (3): 304-309. DOI: 10.1016 / j.ijbiomac.2010.01.002.

[0008] [3]Babu LR,Joy D B.GREEN EXTRACTION TECHNIQUES,STRUCTURAL ANALYSISANDANTIOXIDANT ACTIVITES OF–GLUCAN PRESENT IN OATS[J].Current Advances inMechanical Design&Production VII,2016,7(1):511-519.DOI:DOI:10.1016 / B978-008043711-8 / 50052-0.

[0009] [4]Ahmad A, Anjum FM, Zahoor T, et al. Extraction and characterization of beta-D-glucan from oat for industrial utilization. [J]. International journal of biological macromolecules, 2010, 46 (3): 304-309. DOI: 10.1016 / j.ijbiomac.2010.01.002.

[0010] [5]Maheshwari G,Sowriraian S,Joseph B.Extraction and Isolation ofβ-Glucan from Grain Sources—A Review[J].Journal of Food Science,2017,82(7-9):1535-1545.DOI:10.1111 / 1750-3841.13765.

[0011] [6] Deng Aihua, Yang Pinhong, Liu Yejia, et al. Optimization of ultrasonic-assisted extraction process of β-glucan from highland barley [J]. Grain Science and Technology and Economy, 2022(003):047.

[0012] [7] Luo Yanping, Li Jialin, Zhang Xuefei. Optimization of microwave-assisted extraction process of β-glucan from highland barley [J]. Agricultural Products Processing, 2016, (14):

[0013] [8] Chen Yu, Wang Wei, Jiao Yingchun, et al. Effects of different starter cultures on the enrichment of β-glucan in highland barley [J]. Food Research and Development, 2023, 44(04):

[0014] [9] Liu Xinqi, He Xianzhe, Liu Jiechun, et al. Optimization of the extraction process of β-glucan from barley bran by fermentation and study on its physicochemical properties [J]. Food Industry Technology, 2020, 41(07):

[0015]

[10] Zhang Fenbo, Chen Qiong, Jiang Tao, et al. Optimization of fermentation process for enriching β-glucan and γ-aminobutyric acid in barley from *Ganoderma lucidum* [J]. Modern Food Science and Technology, 2022, 38(11): Summary of the Invention

[0016] To address the shortcomings of existing technologies, the present invention aims to provide a simple preparation method that yields high efficiency and purity of β-glucan from highland barley. Specifically, the β-glucan yield from highland barley needs to reach over 90%, and the purity needs to reach over 95%.

[0017] To achieve this goal, the present invention provides the following technical solution:

[0018] A method for preparing high-yield, high-purity β-glucan from highland barley, the method comprising the following steps:

[0019] (1) The barley is soaked in an alkaline solution at a temperature of 20-40°C for 3-5 minutes; the alkaline solution includes an aqueous solution of potassium hydroxide or sodium hydroxide.

[0020] (2) Remove the alkaline solution from the product obtained in step (1), add a pretreatment solution, and ultrasonically treat it at 20-40°C for 5-10 minutes; the pretreatment solution is an aqueous solution composed of betaine, 1,4-butanediol and ethylene glycol in a molar ratio of 1:1:2.

[0021] (3) Remove the pretreatment liquid from the product obtained in step (2), add water to make the weight-to-volume ratio of material to liquid 1g:10-15mL, extract at 85-95℃ for 1-1.5 hours, and take the filtrate.

[0022] (4) The product obtained in step (3) is subjected to alcohol precipitation, the precipitate is redissolved in water, activated carbon is added for treatment, and after filtration, concentration and drying, the product is obtained.

[0023] Preferably, the pH of the alkaline solution is 8 to 8.5.

[0024] Preferably, in step (1), the pH of the alkaline solution is 8.2, the soaking time is 4 minutes, and the temperature is 30°C; in step (2), the pretreatment temperature is 30°C and the treatment time is 8 minutes.

[0025] Preferably, in step (2), the ultrasonic power is 300W.

[0026] Preferably, the method for removing the alkaline solution in step (2) or the method for removing the pretreatment solution in step (3) is to filter and then wash with water.

[0027] Preferably, in step (3), the weight-to-volume ratio of the liquid to the material is 1g:12mL, the extraction temperature is 90℃, and the extraction time is 75 minutes.

[0028] Preferably, in step (4), anhydrous ethanol is used for alcohol precipitation, and the precipitation time is 12 hours.

[0029] Preferably, in step (4), when adding activated carbon to the reconstituted solution, the weight-to-volume ratio of activated carbon to the reconstituted solution is 1 g / 100 mL, and after addition, the solution is treated at 50°C for 20 minutes.

[0030] Preferably, in step (4), the drying is freeze drying.

[0031] The present invention also provides β-glucan prepared by the above preparation method.

[0032] In traditional preparation methods, hydrothermal extraction is a typical method, but the purity of the extracted β-glucan is generally low, for example, Zhang Feng...

[11] The provided method yielded β-glucan with a purity of only 64.72%. Further purification is needed to improve the purity, but even then, the purity of the purified β-glucan is only 88.7%. Furthermore, the yield of β-glucan obtained by hydrothermal extraction is also relatively low. Even with the assistance of ultrasound and microwave, the increase in yield is limited, making it difficult to maximize the extraction of β-glucan from barley.

[12] Other methods, such as acid and alkaline methods, often sacrifice the purity of the obtained β-glucan in order to increase the yield (or in terms of yield). As mentioned earlier, although enzymatic hydrolysis can achieve high yields or purity—for example, the method for extracting and purifying β-glucan from barley grains provided in CN106117389B can achieve a purity of 98.86%—its process is complex, difficult to control, and the extraction rate is only 31.1%. Furthermore, as mentioned earlier, microbial fermentation also presents environmental biosafety issues, and the cost of building a β-glucan production plant is relatively high.

[0033] Based on the mature hydrothermal extraction method, this invention makes a bold innovation in the pretreatment of extraction by adopting a two-stage pretreatment method. The first step is to soak the sample in an alkaline solution at low temperature for a short time, and then introduce a pretreatment solution composed of betaine, 1,4-butanediol and ethylene glycol for low-temperature ultrasonic pretreatment. After that, hydrothermal extraction is carried out, which can significantly improve the yield and purity.

[0034] In the method of this invention, the temperature, duration, and pretreatment solution in the pretreatment step are crucial. The alkaline solution can be sodium hydroxide or potassium hydroxide, etc., with a temperature not exceeding 40°C and a duration not exceeding 5 minutes. The temperature in the second pretreatment step should not exceed 40°C, and the duration should not exceed 10 minutes. As shown in the comparative scheme of this invention, differences in temperature, duration, and pretreatment solution can lead to a significant reduction in the final yield and purity. Furthermore, the order of the pretreatment steps and the presence or absence of each step also have a significant impact on the results.

[0035] To the inventor's knowledge, although there are some reports in the field of pretreatment before water extraction, for example, Cao Jing et al.

[13] used ethanol reflux pretreatment and ethanol combined with enzyme pretreatment when extracting β-glucan from Ganoderma lucidum fruiting bodies, and then carried out water extraction, which improved the yield. However, the pretreatment time of this method is long, ranging from 45 minutes to 2 hours, and the extraction cycle is too long. In addition, in terms of the extraction of β-glucan from barley, there are very few reports on improving the yield and purity through pretreatment. Those skilled in the art mainly improve the parameters of existing extraction methods or combine different extraction methods to achieve the corresponding improvement in yield and purity. Therefore, before this invention, those skilled in the art were still constrained by the aforementioned defects in the prior art in the preparation of β-glucan from barley. No effective path has been found to improve the yield and purity of β-glucan from barley to more than 90% and 95% respectively through a simple method.

[0036] Therefore, the technical contribution of this invention to the field is that it provides a pretreatment method with short time consumption and low energy consumption, which prepares high-yield and high-purity barley β-glucan by hydrothermal extraction.

[0037] Furthermore, this invention unexpectedly discovered that the β-glucan obtained by this invention possesses high antioxidant properties. Numerous reports in the art have found that the functions of β-glucan obtained by different extraction methods often vary significantly. The high antioxidant properties of the β-glucan obtained by this invention may be due to the mild extraction method, minimal degradation and destruction of β-glucan in the preparation route, and the homogeneity of its molecular composition.

[0038] The beneficial effects of this invention are:

[0039] This invention achieves a yield of over 90% and a purity of over 95% for the preparation of highland barley β-glucan. Furthermore, the pretreatment steps in this invention are time-efficient and energy-saving. Therefore, this invention has promising applications in the preparation of highland barley β-glucan. The highland barley β-glucan obtained by this invention also exhibits a high level of antioxidant activity.

[0040]

[11] Zhang Feng. Isolation, purification and physicochemical properties of β-glucan from highland barley [D]. Southwest Agricultural University, 2005.

[0041]

[12] You Maolan, Qin Xiaoli, Duan Jiaojiao, et al. Ultrasonic-microwave synergistic extraction of β-glucan from highland barley [J]. Food and Fermentation Industries, 2019, 45(08): Detailed Implementation

[0042] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are still within the scope of protection of the present invention.

[0043] Example 1

[0044] (1) Soak barley bran powder (particle size 80-110 μm) in sodium hydroxide aqueous solution at a volume-to-weight ratio of 100 mL: 1, adjust the pH to 8.2, and treat in a water bath (30°C) for 4 minutes. During the treatment, stir slowly (200 rpm) to ensure that the barley is evenly dispersed in the solution.

[0045] (2) Filter the product obtained in step (1) and retain the filter residue; wash the filter residue with water three times, filter again, and retain the washed filter residue. Add pretreatment solution (enough to cover the filter residue) to the washed filter residue, and treat it for 8 minutes in a water bath (30°C) under 300W ultrasonication. During the treatment, stir slowly (200 rpm) to ensure that the washed filter residue is evenly dispersed in the solution. The pretreatment solution used is an aqueous solution composed of betaine, 1,4-butanediol and ethylene glycol in a molar ratio of 1:1:2 (water content is 50% w / w).

[0046] (3) Filter the product obtained in step (2) and retain the filter residue; wash the filter residue with water three times, filter again, and retain the water-washed filter residue. Add water to the water-washed filter residue obtained in this step so that the weight-to-volume ratio of the material to the liquid is 1g:12mL, extract at 90℃ for 75 minutes, and collect the filtrate.

[0047] (4) The product obtained in step (3) was subjected to alcohol precipitation with anhydrous ethanol for 12 hours. After the alcohol precipitation was completed, the precipitate was redissolved with water. Then, activated carbon was added to the redissolved solution at a weight-volume ratio of 1 g / 100 mL and treated at 50 °C for 20 minutes. After that, the product was filtered, heated and concentrated, and then freeze-dried to obtain barley β-glucan.

[0048] Example 2

[0049] (1) Soak barley bran powder (particle size 80-110 μm) in sodium hydroxide aqueous solution at a volume-to-weight ratio of 100 mL: 1, adjust the pH to 8.5, and treat in a water bath (20°C) for 5 minutes. During the treatment, stir slowly (200 rpm) to ensure that the barley is evenly dispersed in the solution.

[0050] (2) Filter the product obtained in step (1) and retain the filter residue; wash the filter residue with water three times, filter again, and retain the washed filter residue. Add pretreatment solution (enough to cover the filter residue) to the washed filter residue, and treat it for 10 minutes in a water bath (20°C) under 300W ultrasonication. During the treatment, stir slowly (200 rpm) to ensure that the washed filter residue is evenly dispersed in the solution. The pretreatment solution used is an aqueous solution composed of betaine, 1,4-butanediol and ethylene glycol in a molar ratio of 1:1:2 (water content is 50% w / w).

[0051] (3) Filter the product obtained in step (2) and retain the filter residue; wash the filter residue with water three times, filter again, and retain the water-washed filter residue. Add water to the water-washed filter residue obtained in this step so that the weight-to-volume ratio of the material to the liquid is 1g:15mL, extract at 95℃ for 60 minutes, and collect the filtrate.

[0052] (4) The product obtained in step (3) was subjected to alcohol precipitation with anhydrous ethanol for 12 hours. After the alcohol precipitation was completed, the precipitate was redissolved with water. Then, activated carbon was added to the redissolved solution at a weight-volume ratio of 1 g / 100 mL and treated at 50 °C for 20 minutes. After that, the product was filtered, heated and concentrated, and then freeze-dried to obtain barley β-glucan.

[0053] Example 3

[0054] (1) Soak barley bran powder (particle size 80-110 μm) in sodium hydroxide aqueous solution at a volume-to-weight ratio of 100 mL: 1, adjust the pH to 8.0, and treat in a water bath (40 °C) for 3 minutes. During the treatment, stir slowly (200 rpm) to ensure that the barley is evenly dispersed in the solution.

[0055] (2) Filter the product obtained in step (1) and retain the filter residue; wash the filter residue with water three times, filter again, and retain the washed filter residue. Add pretreatment solution (enough to cover the filter residue) to the washed filter residue, and treat it for 5 minutes in a water bath (40°C) under 300W ultrasonication. During the treatment, stir slowly (200 rpm) to ensure that the washed filter residue is evenly dispersed in the solution. The pretreatment solution used is an aqueous solution composed of betaine, 1,4-butanediol and ethylene glycol in a molar ratio of 1:1:2 (water content is 50% w / w).

[0056] (3) Filter the product obtained in step (2) and retain the filter residue; wash the filter residue with water three times, filter again, and retain the water-washed filter residue. Add water to the water-washed filter residue obtained in this step so that the weight-to-volume ratio of the material to the liquid is 1g:10mL, extract at 85°C for 90 minutes, and collect the filtrate.

[0057] (4) The product obtained in step (3) was subjected to alcohol precipitation with anhydrous ethanol for 12 hours. After the alcohol precipitation was completed, the precipitate was redissolved with water. Then, activated carbon was added to the redissolved solution at a weight-volume ratio of 1 g / 100 mL and treated at 50 °C for 20 minutes. After that, the product was filtered, heated and concentrated, and then freeze-dried to obtain barley β-glucan.

[0058] Comparative Example 1

[0059] Except for omitting the alkaline pretreatment in step (1), the rest is the same as in Example 1.

[0060] Comparative Example 2

[0061] Except for omitting the pretreatment step (2), the rest is the same as in Example 1.

[0062] Comparative Example 3

[0063] Except for the temperature of 50°C and the duration of 8 minutes in step (1), the rest is the same as in Example 1.

[0064] Comparative Example 4

[0065] Except for the temperature of 50 degrees Celsius and the duration of 12 minutes in step (2), the rest is the same as in Example 1.

[0066] Comparative Example 5

[0067] Except that the pretreatment solution is composed of betaine and 1,4-butanediol in a molar ratio of 1:1, the rest is the same as in Example 1.

[0068] Experimental Example 1

[0069] The yield, recovery rate, and purity of β-glucan from highland barley were determined for Examples 1-3 and Comparative Examples 1-5 using the following methods:

[0070] The method for detecting the β-glucan content in the barley β-glucan sample was based on the literature.

[13] The reporting method is as follows.

[0071] The formula for calculating the purity of β-barley glucan is: (weight of β-glucan / weight of β-barley glucan extract) × 100%.

[0072] The formula for calculating the yield of β-barley glucan is: (weight of β-barley glucan extract / weight of β-barley bran extract) × 100%.

[0073] The formula for calculating the yield of β-barley glucan is: (weight of β-barley glucan extract / weight of barley bran powder) × 100%.

[0074] The test results are shown in Table 1.

[0075] Table 1

[0076]

[0077]

[13] Zhang Juan, Du Xianfeng, Rao Yanqin. Study on determination of β-glucan content in oats by Congo red method [J]. Journal of Anhui Agricultural University, 2007(01):23-26.

[0078] Experimental Example 2

[0079] The antioxidant properties of the barley β-glucan obtained in Example 1 were tested. The detection methods are described in the references.

[14] The DPPH free radical scavenging activity was investigated. The DPPH free radical scavenging rate of 100% highland barley β-glucan stock solution was measured to be 89.4%.

[0080]

[14] Gu Feiyan. Extraction of β-glucan from highland barley and its application in cosmetics [D]. Shanghai University of Applied Technology, 2018.

Claims

1. A method for preparing high-yield, high-purity β-glucan from highland barley, characterized in that, The preparation method includes the following steps: (1) Soaking barley in an alkaline solution at a temperature of 20-40°C for 3-5 minutes; the alkaline solution includes an aqueous solution of potassium hydroxide or sodium hydroxide; the pH of the alkaline solution is 8-8.

5. (2) Remove the alkaline solution from the product obtained in step (1), add the pretreatment solution, and sonicate at 20~40℃ for 5~10 minutes with an ultrasonic power of 300W; the pretreatment solution is an aqueous solution composed of betaine, 1,4-butanediol and ethylene glycol in a molar ratio of 1:1:

2. (3) Remove the pretreatment liquid from the product obtained in step (2), add water to make the weight-to-volume ratio of material to liquid 1g:10~15mL, extract at 85~95℃ for 1~1.5 hours, and take the filtrate; (4) The product obtained in step (3) is subjected to alcohol precipitation, the precipitate is redissolved in water, and then activated carbon is added for treatment. After filtration, concentration and drying, the product is obtained.

2. The preparation method according to claim 1, characterized in that, In step (1), the pH of the alkaline solution is 8.2, the soaking time is 4 minutes, and the temperature is 30℃; in step (2), the pretreatment temperature is 30℃ and the treatment time is 8 minutes.

3. The preparation method according to claim 1, characterized in that, The method for removing the alkaline solution in step (2) or the method for removing the pretreatment solution in step (3) is to filter and then wash with water.

4. The preparation method according to claim 1, characterized in that, In step (3), the weight-to-volume ratio of the material and liquid is 1g:12mL, the extraction temperature is 90℃, and the extraction time is 75 minutes.

5. The preparation method according to claim 1, characterized in that, In step (4), anhydrous ethanol is used for alcohol precipitation, and the precipitation time is 12 hours.

6. The preparation method according to claim 1, characterized in that, In step (4), when adding activated carbon to the reconstituted solution, the weight-to-volume ratio of activated carbon to the reconstituted solution is 1 g / 100 mL. After adding, the solution is treated at 50 °C for 20 minutes.

7. The preparation method according to claim 1, characterized in that, In step (4), the drying is freeze drying.

8. Barley β-glucan prepared by the preparation method according to any one of claims 1 to 7.

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