Method for increasing fructo-oligosaccharide content and co-producing calcium gluconate

Abstract

The invention discloses a method for increasing the content of fructo-oligosaccharide and co-producing calcium gluconate, and belongs to the technical field of biological engineering. The invention discloses a method for increasing the content of fructo-oligosaccharide and co-producing calcium gluconate, which comprises the following steps: adding fructosyl transferase, glucose oxidase and catalase into a sucrose solution and regulating the pH value to obtain a reaction solution of fructo-oligosaccharide and calcium gluconate; and filtering, crystallizing, centrifuging and drying the reaction liquid to obtain calcium gluconate. And meanwhile, carrying out ion exchange on the centrifugate, and concentrating to obtain the fructo-oligosaccharide syrup. According to the method disclosed by the invention, the high-content fructooligosaccharide is directly prepared from the sucrose through the synergistic effect of the fructosyl transferase, the glucose oxidase and the catalase, the purification and impurity removal processes in the fructooligosaccharide production process are simplified, and efficient separation of the fructooligosaccharide and the calcium gluconate is realized through subsequent processes of filtration, crystallization and the like; the co-production of fructo-oligosaccharide and calcium gluconate is realized, so that the resource utilization efficiency is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically to a method for increasing the content of fructooligosaccharides and co-producing calcium gluconate. Background Technology

[0002] Fructooligosaccharides (FOS), as an important functional sugar, have attracted widespread attention in the food and pharmaceutical industries due to their unique physiological functions and broad application value. Studies have shown that FOS possesses various physiological activities, including anti-caries properties, low energy content, lowering serum cholesterol, reducing blood pressure, and protecting the intestines, making it a crucial raw material for the health industry. Meanwhile, calcium gluconate, as an important calcium supplement, is widely used in the pharmaceutical field for the prevention and treatment of calcium deficiency-related diseases such as osteoporosis and rickets, and also shows promising application prospects in the food additive field. In recent years, with increasing public awareness of health, the demand for functional sugars and calcium supplements has grown rapidly, driving the rapid development of related production technologies.

[0003] Although significant progress has been made in the production technology of fructooligosaccharides (FOS) and calcium gluconate, existing production methods still face numerous challenges and struggle to meet the growing market demand. Traditional FOS production methods mainly include enzymatic and microbial fermentation methods. Enzymatic synthesis is currently the most commonly used technology. This method uses fructosyltransferase as the core, catalyzing the transfer of fructosyl groups from sucrose molecules to acceptor molecules to generate FOS, offering advantages such as mild reaction conditions and high product specificity. However, the traditional enzymatic production process is relatively complex. During the conversion stage, the accumulation of glucose byproducts inhibits enzyme activity, resulting in an FOS content of approximately 55-60%. After steps such as filtration, ion exchange, and concentration, chromatographic separation is still required to produce a 90% FOS product. This results in limited FOS yield, a complex production process, and high production costs. To address this issue, researchers have attempted to use glucose oxidase and fructosyltransferase synergistically. By oxidizing glucose to gluconic acid, the glucose content in the system is reduced, thereby increasing FOS yield. Patent CN201710500905 describes a one-step enzymatic process for preparing high-purity fructooligosaccharides from mycelia by constructing an engineered strain expressing glucose oxidase. However, the fructooligosaccharide content is only 71.2%, and some byproducts are generated during fermentation. These byproducts require additional processing steps for removal, increasing the complexity and cost of large-scale production. Further research is needed to achieve efficient and economical large-scale production.

[0004] The main production methods for calcium gluconate include chemical synthesis and fermentation. While traditional chemical synthesis is a mature process, it suffers from high energy consumption and severe environmental pollution. Fermentation, on the other hand, is difficult to scale up due to low production efficiency and low product yield. In recent years, the application of multi-enzyme synergistic technology in calcium gluconate production has gradually attracted attention. For example, by hydrolyzing sucrose into glucose and fructose using invertase, then converting glucose into gluconic acid using glucose oxidase, and finally adding calcium carbonate for neutralization, efficient preparation of calcium gluconate has been achieved. This technology not only improves raw material utilization but also reduces byproduct generation, providing a new approach for the green production of calcium gluconate. However, it still faces many challenges in actual production. For instance, how to optimize reaction conditions to improve the yield of calcium gluconate and how to simplify the separation process to reduce production costs require further research. Furthermore, current publicly available technical reports focus on preparing composite products of fructooligosaccharides and calcium gluconate; no reports have yet demonstrated the efficient separation of high-content fructooligosaccharides from calcium gluconate, which limits the improvement of resource utilization efficiency and further reduction of production costs.

[0005] Therefore, providing a method for increasing the content of fructooligosaccharides and co-producing calcium gluconate is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a method for increasing the content of fructooligosaccharides and co-producing calcium gluconate, thereby providing a new solution for the production of functional sugars and calcium supplements.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for increasing the content of fructooligosaccharides and co-producing calcium gluconate includes the following steps: (1) Dissolve sucrose in deionized water to prepare a sucrose solution with a concentration of 30-45% (w / v); (2) Adjust the pH of the sucrose solution obtained in step (1) to 5.0~7.0, add fructosyltransferase, and react for 4~6 hours at a temperature of 34.5~35.5℃, a rotation speed of 150~250r / min, and an aeration rate of 1.0~2.0L / min to obtain an oligofructose solution; The amount of fructosyltransferase added is 40-60 U of fructosyltransferase per 100g of sucrose; (3) Add glucose oxidase and catalase to the oligofructose solution obtained in step (2), and continue the reaction for 6 to 8 hours at a temperature of 30 to 40°C, a rotation speed of 150 to 250 r / min and an aeration rate of 1.0 to 2.0 L / min. During the reaction, add calcium carbonate to adjust the pH value to the range of 5.0 to 7.0. The amount of glucose oxidase added is 35-55 U of glucose oxidase per 100g of sucrose; the amount of catalase added is 65-85 U of catalase per 100g of sucrose. (4) Filter the reaction solution obtained in step (3) to remove impurities and obtain a filtrate containing fructooligosaccharides and calcium gluconate; (5) Crystallize, centrifuge and dry the filtrate obtained in step (4) to obtain calcium gluconate; at the same time, concentrate the centrifuged liquid to obtain fructooligosaccharide syrup.

[0008] Further, in step (3), a calcium carbonate solution with a concentration of 5-10% is added to adjust the pH value to the range of 5.0-7.0.

[0009] Furthermore, the specific steps of crystallization, centrifugation, and drying described in step (5) are as follows: The filtrate obtained in step (4) is placed in a reaction vessel and cooled to 15-25℃ at a gradient of 1.0-1.5℃ / h for 2-4 hours to crystallize. The crystallized liquid is centrifuged to obtain the centrifuged liquid and solid. The obtained solid is dried at 65-75℃ to obtain calcium gluconate. The obtained centrifuged liquid was subjected to ion exchange treatment with D001 (H type) resin and D354 (OH type) resin. The ion exchange solution was collected at room temperature at 2.0~3.0 BV / h. The ion exchange solution was concentrated at 65~75℃, 40~50 r / min, and -0.08~-0.1 MPa until the soluble solids content was 75%, to obtain fructooligosaccharide syrup with a product content of over 90%.

[0010] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method for increasing the content of fructooligosaccharides and co-producing calcium gluconate, which has the following beneficial effects: (1) The present invention uses a one-step conversion process with the synergistic action of fructosyltransferase, glucose oxidase and catalase to obtain a product with an oligofructose content of more than 90%, which reduces subsequent chromatographic separation and other processes, reduces production costs, and achieves a yield of more than 83%, which is 27% higher than the yield of the traditional process of 55%.

[0011] (2) The conversion stage of this invention is a complex of high content fructooligosaccharides and calcium gluconate. The proportion of calcium gluconate in the product is low, and it is difficult to achieve efficient separation of the two products. This invention achieves efficient separation of fructooligosaccharides and calcium gluconate through processes such as filtration and gradient cooling crystallization. The content of the crystalline calcium gluconate product can reach more than 99%, which reduces the production cost and makes the method more suitable for large-scale industrial production.

[0012] (3) The present invention achieves the co-production of fructooligosaccharides and calcium gluconate in the same reaction system, thereby greatly improving resource utilization efficiency and reducing production costs. Detailed Implementation

[0013] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0014] Example 1 A method for increasing the content of fructooligosaccharides and co-producing calcium gluconate includes the following steps: (1) Add deionized water to sucrose to prepare 3 kg of sucrose solution with a concentration of 45% (w / v).

[0015] (2) Adjust the pH of the sucrose solution obtained in step (1) to 6.0~7.0 with 0.1M hydrochloric acid solution, transfer the solution to the reactor, add fructosyltransferase at a ratio of 40U fructosyltransferase per 100g sucrose, and react for 4 hours at a temperature of 34.5℃, a rotation speed of 150r / min and an aeration rate of 1.0L / min to obtain the oligofructose solution.

[0016] (3) Add glucose oxidase and catalase to the oligofructose solution obtained in step (2). The amount of glucose oxidase added is 35U of glucose oxidase per 100g of sucrose, and the amount of catalase added is 65U of catalase per 100g of sucrose. Continue the reaction for 8 hours at a temperature of 30℃, a rotation speed of 150r / min, and an aeration rate of 1.0L / min. During the reaction, use a 5% calcium carbonate solution to adjust the pH value within the range of 6.0~7.0.

[0017] (4) The reaction solution obtained in step (3) is filtered and impurities are removed by using perlite as a filter aid. The filter aid is made into a paste with the reaction solution and evenly coated on the surface of the filter medium. The filter is then filtered to form a sediment layer of 1-5 mm thickness. The reaction solution is then filtered to obtain a filtrate containing fructooligosaccharides and calcium gluconate.

[0018] (5) The filtrate obtained in step (4) is cooled to 20°C at a gradient of 1.0°C / h and crystallized for 3 hours. The crystallized liquid is centrifuged for 10 min (8000 r / min, 20°C), and the resulting solid is dried at 65°C to obtain calcium gluconate.

[0019] (6) At the same time, the centrifuged liquid obtained in step (4) is subjected to ion exchange treatment using D001 (H type) resin and D354 (OH type) resin (the filling ratio of cation and anion resin is 2:1). The ion exchange liquid is collected at room temperature at 2.0 BV / h. The ion exchange liquid is concentrated at 65℃, 40r / min, and -0.1MPa until the soluble solids content is 75%, thus obtaining fructooligosaccharide syrup.

[0020] Example 2 A method for increasing the content of fructooligosaccharides and co-producing calcium gluconate specifically includes the following steps: (1) Add deionized water to sucrose to prepare 3 kg of sucrose solution with a concentration of 30% (w / v).

[0021] (2) Adjust the pH of the sucrose solution obtained in step (1) to 6.0~7.0 using 0.1M hydrochloric acid solution. Transfer the solution to the reactor and add fructosyltransferase at a ratio of 50U per 100g of sucrose. React for 5 hours at a temperature of 35℃, a rotation speed of 200r / min, and an aeration rate of 1.5L / min to obtain the oligofructose solution.

[0022] (3) Add glucose oxidase and catalase to the oligofructose solution obtained in step (2). The amount of glucose oxidase added is 45U of glucose oxidase per 100g of sucrose, and the amount of catalase added is 75U of catalase per 100g of sucrose. Continue the reaction for 7 hours at a temperature of 35℃, a rotation speed of 200r / min, and an aeration rate of 1.5L / min. During the reaction, use a 7.5% calcium carbonate solution to adjust the pH value to the range of 6.0~7.0.

[0023] (4) The reaction solution obtained in step (3) is filtered and impurities are removed by using perlite as a filter aid. The filter aid is made into a paste with the reaction solution and evenly coated on the surface of the filter medium. The filter is then filtered to form a sediment layer of 1-5 mm thickness. The reaction solution is then filtered to obtain a filtrate containing fructooligosaccharides and calcium gluconate.

[0024] (5) The filtrate obtained in step (4) is cooled to 20°C at a gradient of 1.5°C / h and crystallized for 3 hours. The crystallized liquid is centrifuged for 10 min (8000 r / min, 20°C), and the resulting solid is dried at 70°C to obtain calcium gluconate.

[0025] (6) At the same time, the centrifuged liquid obtained in step (4) is subjected to ion exchange treatment using D001 (H type) resin and D354 (OH type) resin (the filling ratio of cation and anion resin is 2:1). The ion exchange liquid is collected at room temperature at 2.5 BV / h. The ion exchange liquid is concentrated at 70℃, 45r / min, and -0.09MPa until the soluble solids content is 75%, thus obtaining fructooligosaccharide syrup.

[0026] Example 3 A method for increasing the content of fructooligosaccharides and co-producing calcium gluconate specifically includes the following steps: (1) Add deionized water to sucrose to prepare 3 kg of sucrose solution with a concentration of 35% (w / v).

[0027] (2) Adjust the pH of the sucrose solution obtained in step (1) to 5.0~6.0 using 0.1M hydrochloric acid solution. Transfer the solution to the reactor and add fructosyltransferase at a ratio of 60U per 100g of sucrose. React for 6 hours at a temperature of 35.5℃, a rotation speed of 250r / min, and an aeration rate of 2.0L / min to obtain the oligofructose solution.

[0028] (3) Add glucose oxidase and catalase to the oligofructose solution described in step (2). The amount of glucose oxidase added is 55U of glucose oxidase per 100g of sucrose, and the amount of catalase added is 85U of catalase per 100g of sucrose. Continue the reaction for 6 hours at a temperature of 40℃, a rotation speed of 250r / min, and an aeration rate of 2.0L / min. During the reaction, use 10% calcium carbonate to adjust the pH value to the range of 5.0~6.0.

[0029] (4) The reaction solution obtained in step (3) is filtered and impurities are removed by using perlite as a filter aid. The filter aid is made into a paste with the reaction solution and evenly coated on the surface of the filter medium. The filter is then filtered to form a sediment layer of 1-5 mm thickness. The reaction solution is then filtered to obtain a filtrate containing fructooligosaccharides and calcium gluconate.

[0030] (5) The filtrate obtained in step (4) is cooled to 20°C at a gradient of 1.5°C / h and crystallized for 3 hours. The crystallized liquid is centrifuged for 10 min (8000 r / min, 20°C), and the resulting solid is dried at 75°C to obtain calcium gluconate.

[0031] (6) At the same time, the centrifuged liquid obtained in step (4) is subjected to ion exchange treatment using D001 (H type) resin and D354 (OH type) resin (the filling ratio of cation and anion resin is 2:1). The ion exchange liquid is collected at room temperature at 3.0 BV / h. The ion exchange liquid is concentrated at 75℃, 50r / min, and -0.08MPa until the soluble solids content is 75%, thus obtaining fructooligosaccharide syrup.

[0032] Comparative Example A method for increasing the content of fructooligosaccharides and co-producing calcium gluconate specifically includes the following steps: (1) Add deionized water to sucrose to prepare 3 kg of sucrose solution with a concentration of 30% (w / v).

[0033] (2) Adjust the pH of the sucrose solution obtained in step (1) to 5.0~6.0 using 0.1M hydrochloric acid solution. Transfer the solution to the reactor and add fructosyltransferase, glucose oxidase and catalase. The amount of fructosyltransferase added is 60U per 100g of sucrose, the amount of glucose oxidase added is 55U per 100g of sucrose, and the amount of catalase added is 85U per 100g of sucrose. React for 48 hours at a temperature of 35.5℃, a rotation speed of 250r / min and an aeration rate of 2.0L / min. During the reaction, use 10% calcium carbonate to adjust the pH value to the range of 5.0~6.0.

[0034] (3) The reaction solution obtained in step (2) is filtered and impurities are removed by using perlite as a filter aid. The filter aid is made into a paste with the reaction solution and evenly coated on the surface of the filter medium. The filter is then filtered to form a sediment layer of 1-5 mm thickness. The reaction solution is then filtered to obtain a filtrate containing fructooligosaccharides and calcium gluconate.

[0035] (4) The filtrate obtained in step (3) is cooled to 20°C at a gradient of 1.5°C / h and crystallized for 3 hours. The crystallized liquid is centrifuged for 10 min (8000 r / min, 20°C), and the resulting solid is dried at 75°C to obtain calcium gluconate.

[0036] (5) At the same time, the centrifuged liquid obtained in step (4) is subjected to ion exchange treatment using D001 (H type) resin and D354 (OH type) resin (the filling ratio of cation and anion resin is 2:1). The ion exchange liquid is collected at room temperature at 3.0 BV / h. The ion exchange liquid is concentrated at 75℃, 50r / min and -0.08MPa until the soluble solids content is 75%, thus obtaining fructooligosaccharide syrup.

[0037] Performance testing: The yield (%) and content (%) of fructooligosaccharides, as well as the conversion rate (%) and content (%) of calcium gluconate, were calculated for Examples 1-3 and the comparative examples. The results are shown in Tables 1-3.

[0038] The content of fructooligosaccharides was determined by high performance liquid chromatography (HPLC) according to GB / T23528.2-2021. The specific detection method is as follows: accurately weigh an appropriate amount of the sample to be tested, dissolve it in water and make up to 100 mL, mix well, filter it using a 0.45 μm microporous membrane, collect the filtrate, dilute the filtrate according to the range of the standard curve of each sugar of fructooligosaccharides, and calculate the percentage content of each sugar using the area normalization method.

[0039] The calcium gluconate content was calculated using the determination method specified in GB15571-2012. The specific detection procedure was as follows: Take approximately 0.5g of the sample to be tested, accurate to 0.0001g, add 100mL of water to dissolve it, let it stand at room temperature, add 15mL of sodium hydroxide solution and 0.1g of calcium violet indicator, and titrate with disodium ethylenediaminetetraacetate standard solution until the solution changes from purple to pure blue. The titration result was corrected with a blank test. The mass fraction of calcium gluconate, w1, expressed as a percentage, was calculated using the following formula:

[0040] In the formula: V – The volume of disodium ethylenediaminetetraacetate standard titrant consumed by the laboratory sample, in milliliters (mL); V0 — The volume of disodium ethylenediaminetetraacetate standard titrant consumed in the blank test, in milliliters (mL); c — The accurate value of the concentration of the disodium ethylenediaminetetraacetate standard titration solution, in moles per liter (mol / L); m1 — The numerical value of the laboratory sample mass, in grams (g). w3—Mass fraction of sample loss on drying, expressed as a percentage; M – The numerical value of the molar mass of calcium gluconate, expressed in grams per mole (g / mol).

[0041] Table 1. Fructooligosaccharide Index

[0042] Note: Fructooligosaccharide yield = Actual amount of fructooligosaccharides produced / Theoretical amount of fructooligosaccharides produced.

[0043] Taking Example 1 as an example: the actual amount of fructooligosaccharide produced was 1495.8g × 75% = 1121.8g; the theoretical amount produced was the amount of sucrose substrate, i.e., 3000g × 45% = 1350g; the yield of fructooligosaccharide was 1121.8g / 1350g = 83.1%.

[0044] Table 2 Calcium gluconate index

[0045] Calcium gluconate conversion rate = Amount of glucose in calcium gluconate / Total amount of glucose.

[0046] Taking Example 1 as an example: the amount of glucose in calcium gluconate is 142g × 90.7% = 128.79g (90.7% is the percentage of glucose in calcium gluconate), the total amount of glucose is the amount of sucrose substrate after removing fructooligosaccharides, that is, 3000g × 45% - 1495.8 × 75% = 228.15g, and the conversion rate of calcium gluconate is 128.79g / 228.15g = 56.4%.

[0047] Table 3 Total Product Yield

[0048] Total product yield = (Amount of fructooligosaccharides + Amount of glucose in calcium gluconate) / Amount of sucrose substrate.

[0049] Taking Example 1 as an example: Total yield = (1495.8g × 75% + 142g × 90.7%) / 3000g × 45% = 92.6%.

[0050] As shown in Table 1, the fructooligosaccharide content obtained in Examples 1-3 was all above 90%, with a yield of over 83%. In Example 3, the fructooligosaccharide content was increased to 90.8% by controlling the reaction temperature, calcium carbonate concentration, and pH value. Furthermore, as shown in Table 2, the calcium gluconate content obtained through simultaneous co-production was all above 99%, meeting national standards, and the total yield of the two products was over 92% (Table 3).

[0051] In the comparative example, the content of fructooligosaccharides was only 88.1%. Although the finished syrup was prepared and the relevant indicators were tested, the content was less than 90%, which could not meet the content requirements of fructooligosaccharides in type 90 fructooligosaccharide syrup. In other words, the fructooligosaccharide syrup product obtained by this process could not meet the standards.

[0052] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for increasing the content of fructooligosaccharides and co-producing calcium gluconate, characterized in that, Includes the following steps: (1) Dissolve sucrose in deionized water to prepare a sucrose solution with a concentration of 30-45% (w / v); (2) Adjust the pH of the sucrose solution obtained in step (1) to 5.0~7.0, add fructosyltransferase, and react for 4~6 hours at a temperature of 34.5~35.5℃, a rotation speed of 150~250r / min, and an aeration rate of 1.0~2.0L / min to obtain an oligofructose solution; The amount of fructosyltransferase added is 40-60 U of fructosyltransferase per 100g of sucrose; (3) Add glucose oxidase and catalase to the oligofructose solution obtained in step (2), and continue the reaction for 6 to 8 hours at a temperature of 30 to 40°C, a rotation speed of 150 to 250 r / min and an aeration rate of 1.0 to 2.0 L / min. During the reaction, add calcium carbonate to adjust the pH value to the range of 5.0 to 7.

0. The amount of glucose oxidase added is 35-55 U of glucose oxidase per 100g of sucrose; the amount of catalase added is 65-85 U of catalase per 100g of sucrose. (4) Filter the reaction solution obtained in step (3) to remove impurities and obtain a filtrate containing fructooligosaccharides and calcium gluconate; (5) Crystallize, centrifuge and dry the filtrate obtained in step (4) to obtain calcium gluconate; at the same time, concentrate the centrifuged liquid to obtain fructooligosaccharide syrup.

2. The method for increasing the content of fructooligosaccharides and co-producing calcium gluconate according to claim 1, characterized in that, Step (3) Add a calcium carbonate solution with a concentration of 5-10% to adjust the pH value to the range of 5.0-7.

0.

3. A method for increasing the content of fructooligosaccharides and co-producing calcium gluconate according to claim 1 or 2, characterized in that, The specific steps of crystallization, centrifugation, and drying described in step (5) are as follows: The filtrate obtained in step (4) is placed in a reaction vessel and cooled to 15-25℃ at a gradient of 1.0-1.5℃ / h for 2-4 hours to crystallize. The crystallized liquid is centrifuged to obtain the centrifuged liquid and solid. The obtained solid is dried at 65-75℃ to obtain calcium gluconate. The obtained centrifuged liquid was subjected to ion exchange treatment with D001 resin and D354 resin. The ion exchange solution was collected at room temperature at 2.0~3.0 BV / h. The ion exchange solution was concentrated at 65~75℃, 40~50 r / min, and -0.08~-0.1 MPa until the soluble solids content was 75% to obtain fructooligosaccharide syrup.

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