Process for purifying a fermentation broth containing beta-1,3 / alpha-1,3-glucan

Abstract

This invention pertains to the purification of microbial fermentation broth, specifically a method for purifying fermentation broth containing β-1,3 / α-1,3-glucan. The method involves ethanol extraction and filtration to extract β-1,3 / α-1,3-glucan, including fermentation broth pretreatment, pH adjustment of the clarified broth after filtration, at least two ethanol extractions and filtrations, and drying and pulverizing the resulting fibrous material to obtain β-1,3 / α-1,3-glucan. This invention solves the problems of low extraction rate and low product purity in existing technologies, offering advantages such as high extraction rate and product yield, low protein and impurity content, and low production cost.

Description

Technical Field

[0001] This invention pertains to the purification of microbial fermentation broth, and specifically refers to a method for purifying fermentation broth containing β-1,3 / α-1,3-glucan. Background Technology

[0002] In April 2021, an announcement listed β-1,3 / α-1,3-glucan as a new food ingredient, specifying its molecular formula as (C6H2O). 10 O5) n , n≥615; its structural formula is as follows:

[0003] β-1,3 / α-1,3-glucan is a product made primarily from sucrose, produced by *Rhizobium pusa* (…). Rhizobium pusense It is made through processes such as fermentation, alcohol precipitation, filtration, separation, drying, and pulverization.

[0004] The announcement also specified quality requirements for β-1,3 / α-1,3-glucan, including its appearance (off-white to pale yellow powder), content (≥90g / 100g), protein content (≤3g / 100g), and ash content (≤5g / 100g), and clarified the methods for determining its content.

[0005] Existing research indicates that β-1,3 / α-1,3-glucan can promote the proliferation and differentiation of T cells and B cells, systematically enhancing the host's immune response through the synergistic effect of multiple immunomodulatory pathways. This substance can enhance the phagocytic capacity of macrophages, improving their efficiency in clearing harmful substances such as bacteria and viruses; simultaneously, it can promote the secretion of cytokines, including inflammatory factors such as TNF-α, IL-1α, IL-1β, and IL-6, as well as various chemokines. β-1,3 / α-1,3-glucan also possesses multiple physiological functions, including regulating blood sugar, lowering cholesterol, anti-tumor activity, anti-infection, and antioxidant activity. These functions give it broad application prospects in functional foods, health products, and pharmaceuticals.

[0006] The literature with publication number CN106434495A discloses a method for preparing β-1,3-glucan fermentation broth from *Rhizobium praecox*. Rhizobium pusenseCGMCC No. 12954 was inoculated into a sterilized culture medium containing carbon sources, nitrogen sources, and necessary nutrients. Fermentation was carried out with aeration and stirring. Fermentation ended when the viscosity of the culture medium stopped increasing or the fermentation period was ≤48 hours. Current purification methods involve multiple steps including alcohol precipitation, dissolution, Sevag protein removal, and further alcohol precipitation. The existing purification methods described above have the following problems: First, the process is cumbersome, the extraction rate is low, and it is not suitable for industrial production. Second, the extraction process easily causes degradation of β-1,3 / α-1,3-glucan molecules, affecting the bioactivity of the product. Third, the product quality is unstable. Due to the limitations of the extraction process, the content of β-1,3 / α-1,3-glucan in the product fluctuates greatly, making it difficult to consistently meet the standard requirement of over 90%. Fourth, the content of impurities such as protein (5-8g / 100g) and ash (8-10g / 100g) in the product is also high, affecting the purity (usually 60%-70%) and safety of the product. Fifth, the production cost is high. Currently, the market price of high-purity β-1,3-glucan is about 20,000-30,000 yuan / kg, which greatly limits the promotion and application of β-1,3-glucan. In addition to the low-yield fermentation process, the complex extraction and purification process and the strict quality control requirements are important factors contributing to the high cost. Summary of the Invention

[0007] The purpose of this invention is to provide a method for purifying fermentation broth containing β-1,3 / α-1,3-glucan. By pretreating the fermentation broth to effectively remove cells and proteins, the purified β-1,3 / α-1,3-glucan is significantly superior to existing technologies and meets the strict quality standards and purity requirements for food raw materials.

[0008] The overall technical concept of this invention is: A purification method for fermentation broth containing β-1,3 / α-1,3-glucan, employing ethanol extraction and filtration to extract β-1,3 / α-1,3-glucan, includes the following process steps: A. Fermentation broth pretreatment Add acid to the fermentation broth containing β-1,3 / α-1,3-glucan to adjust the pH to 2-3, raise the temperature to 110℃-120℃, and maintain it for 15-30 minutes. The acid added can be citric acid and / or hydrochloric acid and / or sulfuric acid and / or phosphoric acid. B. Adjust the pH of the filtered solution. After filtering the fermentation broth in step A, take the clear liquid and add alkali to adjust the pH of the clear liquid to 6-8; the alkali added is sodium hydroxide solution and / or potassium hydroxide solution. C. After at least two ethanol extractions and filtrations, the fibrous material obtained from the filtration is dried and pulverized to obtain β-1,3 / α-1,3-glucan.

[0009] The main functions of this invention in pretreating fermentation broth are twofold: First, adding acid rapidly dissolves the bacterial cells in the fermentation broth. Heating not only promotes the denaturation and precipitation of bacterial proteins, but more importantly, it cleverly utilizes the poor temperature resistance of β-1,3 / α-1,3-glucan. Its viscosity decreases significantly after heating and recovers upon cooling, facilitating the filtration of bacterial cells and proteins. Subsequent purification requires only ethanol extraction, eliminating multiple repetitive steps, reducing reagent, equipment, and manpower investment, and lowering overall costs. Second, the acid-adding and heating process of this invention fully denatures and precipitates proteins, filtering out bacterial cells and most impurities. Ethanol extraction specifically enriches glucan, reducing impurity residue and ensuring stable purity. This effectively solves the problems of incomplete protein removal, easy residue of protein impurities, and the introduction of impurities, solvent residue, low product purity, and large fluctuations in content during multi-step ethanol precipitation in existing technologies. Third, the purification process involves fewer steps, eliminating repeated dissolution and alcohol precipitation. The acid addition and temperature control are gentle, minimizing damage to the polysaccharide molecular structure and maximizing the preservation of its biological activity. This effectively solves the problem in existing purification processes where mechanical stirring and repeated solvent action during multi-step alcohol precipitation and dissolution easily damage the glycosidic bonds of dextran, leading to molecular degradation and decreased biological activity. Fourth, the purification process is simple, can be operated continuously, and requires no complex equipment. This improves production efficiency while reducing energy consumption and costs, making it more practical. It also addresses the problems of time-consuming and complex multi-step purification processes in existing technologies, which are difficult to adapt to continuous industrial production.

[0010] The main functions of adjusting the pH of the clarified liquid by adding alkali before ethanol extraction are as follows: First, after acidification and heating, excessive acidic substances remain in the fermentation broth. An acidic environment can damage the glycosidic bonds of β-1,3 / α-1,3-glucan, leading to molecular degradation and decreased biological activity. Adding alkali can quickly neutralize the residual acid, adjusting the pH of the fermentation broth to neutral or weakly alkaline, preventing damage to the β-1,3 / α-1,3-glucan structure and ensuring its biological activity and molecular weight uniformity. Second, β-1,3 / α-1,3-glucan has more stable solubility in a neutral or weakly alkaline environment. Alkali adjustment allows for sufficient molecular dispersion, resulting in efficient enrichment during subsequent ethanol extraction, reducing loss during the extraction process and improving product recovery. Third, the neutralization process promotes the denaturation and precipitation of residual trace proteins, sugars, and other impurities. This, combined with the previously filtered bacterial cells and protein impurities, further reduces the impurity content, helping to ensure that the purity of β-1,3 / α-1,3-glucan consistently reaches above 90%.

[0011] The specific technical concept of this invention also includes: To ensure a gentle reaction, avoid damage to the molecular structure of β-1,3 / α-1,3-glucan, effectively preserve its original functional properties, prevent excessive alkali residue from increasing product impurities, and precisely control the pH to avoid affecting subsequent ethanol extraction and product quality, the preferred technical solution is to use a low-concentration sodium hydroxide solution and / or potassium hydroxide solution in step B.

[0012] Furthermore, in step B, the mass percentage concentration of the low-concentration sodium hydroxide solution and / or potassium hydroxide solution is 5% to 10%.

[0013] To ensure extraction efficiency and fully extract β-1,3 / α-1,3-glucan, the preferred technical means is to effectively control production costs. The preferred technical means is that the ethanol extraction in step C includes primary extraction and secondary extraction, with the volume percentage concentration of ethanol being 85% to 95%, and the primary extraction is carried out at a volume ratio of fermentation broth to ethanol of 1:4 to 6.

[0014] Furthermore, the secondary extraction involves adding ethanol at a ratio of 2 to 3 times the mass of the fibrous material after the primary extraction and filtration, allowing the fibrous material to be fully immersed in the ethanol for 30 to 50 minutes.

[0015] To avoid damaging the molecular structure of β-1,3 / α-1,3-glucan and effectively preserve its original functional properties, the preferred technical means is to dry the extracted fibrous material at a temperature of 60℃~70℃.

[0016] To verify the technical effects of the present invention, the applicant conducted the following experiments: I. Determination of protein content in the product.

[0017] Refer to the first method of GB 5009.5-2016 II. Determination of ash content in the product.

[0018] Refer to the first method of GB 5009.4-2016 III. Determination of β-1,3 / α-1,3-glucan content in the product Refer to the appendix of the "Announcement on Six New Foods Including β-1,3 / α-1,3-glucan and dihydroquercetin" (No. 5 of 2021).

[0019] The essential features and significant technical advancements of this invention are as follows: 1. This invention employs acid dissolution of bacterial cells followed by temperature filtration to remove bacterial cells and proteins. This approach cleverly utilizes the characteristics of β-1,3 / α-1,3-glucan, simplifying the process and reducing operational difficulty and costs. Secondly, it enhances purification efficiency, ensuring stable and compliant product purity. Thirdly, it avoids polysaccharide molecule degradation, preserving the product's biological activity. Fourthly, it is suitable for industrial production, improving production efficiency.

[0020] 2. The main functions of adding acid and heating and adding alkali to adjust the pH before ethanol extraction in this invention are: first, to neutralize residual acid and stabilize the dextran structure; second, to optimize the ethanol extraction effect and improve the dextran recovery rate; and third, to further remove impurities and improve product purity.

[0021] 3. The main functions of adjusting pH with low-concentration alkali in this invention are: first, to avoid degradation of β-1,3 / α-1,3-glucan; second, to prevent the introduction of new impurities; and third, to precisely control pH and ensure extraction stability.

[0022] 4. This invention employs a series of technical measures, including acid-addition and heating to remove bacterial cells and proteins, alkali-addition to improve the recovery rate of β-1,3 / α-1,3-glucan, low-concentration alkali to stabilize the β-1,3 / α-1,3-glucan structure and achieve precise control, and auxiliary ethanol extraction, to ensure that the protein content in the purified product is stable at 2%–3%, the ash content at 4%–5%, and the β-1,3 / α-1,3-glucan content at 90%–93%. This not only meets the quality requirements for β-1,3 / α-1,3-glucan in the "Announcement on Six 'New Foods' including β-1,3 / α-1,3-glucan and dihydroquercetin" (No. 5 of 2021), but also controls the production cost to 4,000–6,000 yuan / kg. This greatly expands the application range of β-1,3 / α-1,3-glucan and enhances its market competitiveness. Detailed Implementation

[0023] The present invention will be further described below with reference to embodiments, but this is not intended to limit the present invention. The scope of protection of the present invention shall be determined by the contents of the claims. Any equivalent technical means substitution made in accordance with the specification shall not depart from the scope of protection of the present invention. Example 1

[0024] A purification method for fermentation broth containing β-1,3 / α-1,3-glucan, employing ethanol extraction and filtration to extract β-1,3 / α-1,3-glucan, includes the following process steps: A. Fermentation broth pretreatment Add acid to the fermentation broth containing β-1,3 / α-1,3-glucan to adjust the pH to 2-3, raise the temperature to 120℃ and maintain it for 30 minutes. Citric acid is used as the acid added. B. Adjust the pH of the filtered solution. After filtering the fermentation broth in step A, take the clear liquid and add alkali to adjust the pH of the clear liquid to 6-8; the alkali added should be a sodium hydroxide solution with a mass percentage concentration of 5%-10%. C. After at least two ethanol extractions and filtrations, the fibrous material obtained from the filtration is dried and pulverized to obtain β-1,3 / α-1,3-glucan; the ethanol extraction in step C includes a first extraction and a second extraction, with the volume percentage concentration of ethanol being 85% to 95%, and the first extraction is carried out at a volume ratio of fermentation broth: ethanol = 1: 6.

[0025] The secondary extraction involves adding ethanol at a ratio of 3 times the mass of the fibrous material after the primary extraction and filtration, allowing the fibrous material to be fully immersed in the ethanol for 50 minutes.

[0026] The extracted fibrous material is dried at a temperature of 60℃~70℃. Example 2

[0027] A purification method for fermentation broth containing β-1,3 / α-1,3-glucan, employing ethanol extraction and filtration to extract β-1,3 / α-1,3-glucan, includes the following process steps: A. Fermentation broth pretreatment Add acid to the fermentation broth containing β-1,3 / α-1,3-glucan to adjust the pH to 2-3, raise the temperature to 110℃ and maintain it for 15 minutes. The acid added is hydrochloric acid. B. Adjust the pH of the filtered solution. After filtering the fermentation broth in step A, take the clear liquid and add alkali to adjust the pH of the clear liquid to 6-8; the alkali added should be a potassium hydroxide solution with a mass percentage concentration of 5%-10%. C. After at least two ethanol extractions and filtrations, the fibrous material obtained from the filtration is dried and pulverized to obtain β-1,3 / α-1,3-glucan. The ethanol extraction in step C includes a first extraction and a second extraction. The volume percentage concentration of ethanol is 85% to 95%. The first extraction is carried out at a volume ratio of fermentation broth to ethanol = 1:4.

[0028] The secondary extraction involves adding ethanol at a ratio of 2 to 3 times the mass of the fibrous material after the primary extraction and filtration, allowing the fibrous material to be fully immersed in the ethanol for 30 minutes.

[0029] The extracted fibrous material is dried at a temperature of 60℃~70℃. Example 3

[0030] A purification method for fermentation broth containing β-1,3 / α-1,3-glucan, employing ethanol extraction and filtration to extract β-1,3 / α-1,3-glucan, includes the following process steps: A. Fermentation broth pretreatment Add acid to the fermentation broth containing β-1,3 / α-1,3-glucan to adjust the pH to 2-3, raise the temperature to 115℃ and maintain it for 23 minutes. The acid added is sulfuric acid. B. Adjust the pH of the filtered solution. After filtering the fermentation broth in step A, take the clear liquid and add alkali to adjust the pH of the clear liquid to 6-8; the alkali added is sodium hydroxide solution and / or potassium hydroxide solution. C. After at least two ethanol extractions and filtrations, the fibrous material obtained from the filtration is dried and pulverized to obtain β-1,3 / α-1,3-glucan.

[0031] In step B, a sodium hydroxide solution and a potassium hydroxide solution with a mass percentage concentration of 7.5% are selected.

[0032] Step C involves ethanol extraction, which includes a primary extraction and a secondary extraction. The volume percentage concentration of ethanol is 85%–95%, and the primary extraction is performed at a volume ratio of fermentation broth to ethanol of 1:5.

[0033] The secondary extraction involves adding ethanol at a ratio of 2.5 times the mass of the fibrous material after the primary extraction and filtration, allowing the fibrous material to be fully immersed in the ethanol for 40 minutes.

[0034] The extracted fibrous material is dried at a temperature of 60℃~70℃. Example 4

[0035] A purification method for fermentation broth containing β-1,3 / α-1,3-glucan, employing ethanol extraction and filtration to extract β-1,3 / α-1,3-glucan, includes the following process steps: A. Fermentation broth pretreatment Add acid to the fermentation broth containing β-1,3 / α-1,3-glucan to adjust the pH to 2-3, raise the temperature to 118℃ and maintain it for 25 minutes. The acid added should be sulfuric acid or phosphoric acid. B. Adjust the pH of the filtered solution. After filtering the fermentation broth in step A, take the clear liquid and add alkali to adjust the pH of the clear liquid to 6-8; the alkali added is a 9% sodium hydroxide solution by mass percentage. C. After at least two ethanol extractions and filtrations, the fibrous material obtained from the filtration is dried and pulverized to obtain β-1,3 / α-1,3-glucan. Step C involves both primary and secondary ethanol extractions. The volume percentage concentration of ethanol is 85%–95%, and the primary extraction is performed at a volume ratio of fermentation broth to ethanol of 1:4.2.

[0036] The secondary extraction involves adding ethanol at a ratio of 2.3 times the mass of the fibrous material after the primary extraction and filtration, allowing the fibrous material to be fully immersed in the ethanol for 35 minutes.

[0037] The extracted fibrous material is dried at a temperature of 60℃~70℃. Example 5

[0038] A purification method for fermentation broth containing β-1,3 / α-1,3-glucan, employing ethanol extraction and filtration to extract β-1,3 / α-1,3-glucan, includes the following process steps: A. Fermentation broth pretreatment The pH of the fermentation broth containing β-1,3 / α-1,3-glucan was adjusted to 2-3 by adding acid, and the temperature was raised to 118℃ and maintained for 26 minutes. Citric acid and hydrochloric acid were used as the acids added. B. Adjust the pH of the filtered solution. After filtering the fermentation broth in step A, take the clear liquid and add alkali to adjust the pH of the clear liquid to 6-8; the alkali added is a 6% potassium hydroxide solution by mass percentage. C. After at least two ethanol extractions and filtrations, the fibrous material obtained from the filtration is dried and pulverized to obtain β-1,3 / α-1,3-glucan.

[0039] Step C involves ethanol extraction, which includes a primary extraction and a secondary extraction. The volume percentage concentration of ethanol is 85%–95%, and the primary extraction is performed at a volume ratio of fermentation broth to ethanol of 1:5.6.

[0040] The secondary extraction involves adding ethanol at a ratio of 2.8 times the mass of the fibrous material after the primary extraction and filtration, allowing the fibrous material to be fully immersed in the ethanol for 45 minutes.

[0041] The extracted fibrous material is dried at a temperature of 60℃~70℃.

[0042] The applicant tested the protein, ash, and β-1,3 / α-1,3-glucan content in the products prepared in Examples 1-5, and the relevant results are as follows: protein(%) Ash content (%) β-1,3 / α-1,3-glucan (%) Product purified by the method in Example 1 2.1 4.1 90.5 Product purified by the method in Example 2 2.5 4.0 91.0 Product purified by the method in Example 3 2.3 4.0 92.2 Product purified by the method in Example 4 2.1 4.8 90.8 Product purified by the method in Example 5 2.6 4.5 90.2

Claims

1. A method for purifying fermentation broth containing β-1,3 / α-1,3-glucan, comprising ethanol extraction and filtration to extract β-1,3 / α-1,3-glucan, characterized in that... The process includes the following steps: A. Fermentation broth pretreatment Add acid to the fermentation broth containing β-1,3 / α-1,3-glucan to adjust the pH to 2-3, raise the temperature to 110℃-120℃, and maintain it for 15-30 minutes. The acid added can be citric acid and / or hydrochloric acid and / or sulfuric acid and / or phosphoric acid. B. Adjust the pH of the filtered solution. After filtering the fermentation broth in step A, take the clear liquid and add alkali to adjust the pH of the clear liquid to 6-8; the alkali added is sodium hydroxide solution and / or potassium hydroxide solution. C. After at least two ethanol extractions and filtrations, the fibrous material obtained from the filtration is dried and pulverized to obtain β-1,3 / α-1,3-glucan.

2. The purification method for fermentation broth containing β-1,3 / α-1,3-glucan according to claim 1, characterized in that... In step B, a low concentration of sodium hydroxide solution and / or potassium hydroxide solution is selected.

3. The purification method for fermentation broth containing β-1,3 / α-1,3-glucan according to claim 2, characterized in that... In step B, the mass percentage concentration of the low-concentration sodium hydroxide solution and / or potassium hydroxide solution is 5% to 10%.

4. The purification method for fermentation broth containing β-1,3 / α-1,3-glucan according to claim 1, characterized in that... Step C involves ethanol extraction, which includes primary and secondary extraction. The volume percentage concentration of ethanol is 85%–95%, and primary extraction is performed at a volume ratio of fermentation broth to ethanol of 1:4–6.

5. The purification method for fermentation broth containing β-1,3 / α-1,3-glucan according to claim 4, characterized in that... The secondary extraction involves adding ethanol at a ratio of 2 to 3 times the mass of the fibrous material after the primary extraction and filtration, allowing the fibrous material to be fully immersed in the ethanol for 30 to 50 minutes.

6. The purification method for fermentation broth containing β-1,3 / α-1,3-glucan according to claim 1, 4 or 5, characterized in that... The extracted fibrous material is dried at a temperature of 60℃~70℃.

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