Method for producing curdlan through fermentation

Abstract

The invention provides a method for producing curdlan through fermentation, and belongs to the technical field of fermentation. The method comprises the steps of seed culture, fermentation culture and post-treatment. The culture step comprises the following steps: inoculating a seed culture solution into a fermentation culture medium, culturing at 28-32 DEG C, supplementing sucrose with the supplementing amount of 28-33 g / L when the residual sugar is less than 10 g / L, supplementing twice, and adding a multifunctional complexing agent with the adding amount of 0.23-0.28 g / L when the fermentation is carried out for 35th h; according to the fermentation method disclosed by the invention, the sucrose is used as a carbon source, so that the fermentation time is shortened to 62.4-63.3 hours, the yield of the curdlan is improved to 51.1-51.7 g / L, the gel strength of the curdlan is effectively improved, and the phenomena of unstable production, tank inversion and the like are avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation, and in particular relates to a method for producing curdlan by fermentation. Background Art

[0002] Curdlan is a new type of high molecular polymer produced by microbial fermentation of sugar raw materials. It is a neutral, water-insoluble microbial extracellular polysaccharide with thermogelatinization properties, called thermogel, and its chemical name is β-1,3-glucan. Curdlan is an odorless, free-flowing white powder with an unbranched linear glucan structure. It is a water-insoluble dietary fiber that is dispersed in cold water and whipped at high speed to form a relatively viscous hydrated liquid. Curdlan can form a gel in the pH range of 2-11. Compared with gelatin, agar, etc., it has stronger acid and alkali resistance and good freeze-thaw resistance. Freezing and thawing have little effect on the strength of the gel.

[0003] China approved curdlan as a food additive in May 2006. It has been widely used in various fields of the food industry. It can be used in various foods as a stabilizer, coagulant, thickener, water-retaining agent, adhesive, film-forming agent, etc. In soy products, it can enhance heat resistance and freeze resistance, improve the taste and shape; in meat and aquatic cooked meat products, konjac gel products, and surimi products, it can reproduce the taste of various food materials, improve heat resistance and freeze resistance, and can replace protein and soy protein; in artificial fruit particles, it can prevent high-temperature extravasation of pigments.

[0004] The fermentation production of curdlan is a process in which soil bacteria synthesize polysaccharides from small molecule sugars such as glucose and sucrose. The traditional production method usually uses glucose as the carbon source, but glucose usually needs to be decomposed into starch, and its production process is relatively complicated, which increases the production process steps and energy consumption. In addition, the resource supply of starch is affected by various factors such as agricultural production, and there is a lot of instability. In comparison, sucrose is obtained from beets or sugarcane by squeezing, extraction and crystallization, and has a wide source, which is of great significance for large-scale industrial production.

[0005] However, when sucrose is used as a carbon source, during the fermentation process, the OD of the bacteria will be less than 18 and the OD will no longer increase. In addition, the bacteria will continue to produce acid, requiring continuous alkali supplementation, slowing down sugar consumption and failing to produce gum normally, causing metabolic disorders in the bacteria and abnormal fermentation process, ultimately leading to unstable production and even tank tipping, resulting in large economic losses.

[0006] CN114686545A discloses a method for improving the yield and gel strength of curdlan, specifically by adding FeSO4 to the fermentation medium. The components of the medium include: 60 - 120 g / L sucrose, 1 - 3 g / L KH2PO4, 1 - 3 g / L (NH4)2HPO4, 0.5 - 2 g / L MgSO4·7H2O, 2 - 6 g / L yeast powder, 1 - 3 g / L calcium carbonate, 0.01% - 0.04% (w / v) FeSO4; by adding different concentrations of FeSO4 to the medium in this patent, when the content of FeSO4 is 0.02% (w / v), the yield of curdlan is increased to 58.37 g / L and the gel strength is increased to 691.48 g / cm 2 ; The above patent uses sucrose as a carbon source in the fermentation medium and improves the yield and strength of curdlan by adding the inorganic salt FeSO4. However, its gum production is still low, the fermentation rate is slow, and the gel strength of curdlan is low, thus affecting its application. Summary of the Invention

[0007] In order to solve the technical problems existing in the prior art, the present invention provides a method for fermentatively producing curdlan, which uses sucrose as a carbon source in the fermentation medium, avoids production instability and phenomena such as fermentation tank collapse, improves the yield and production efficiency of curdlan, enhances the gel strength of curdlan, and improves the quality of curdlan.

[0008] In view of the above technical problems, the present invention adopts the following technical solutions: A method for fermentatively producing curdlan, including seed culture, fermentation culture and post-treatment steps, specifically as follows: 1. Seed culture Place Agrobacterium radiobacter in the seed medium and conduct seed culture at 29 - 32 °C. After 6 h of seed culture, control the dissolved oxygen to be 46 - 48%, and when the OD value of the seed liquid > 10, obtain the seed culture solution; The Agrobacterium radiobacter is obtained by purchase; The seed medium includes 18 - 22 g / L glucose, 9 - 11 g / L yeast extract powder, 1.7 - 2.2 g / L dipotassium hydrogen phosphate, 0.8 - 1.1 g / L magnesium sulfate, 2.7 - 3.2 g / L diammonium hydrogen phosphate, 0.33 - 0.38 g / L α-ketoglutaric acid, with a pH of 6.8 - 7.2, and is sterilized at 120 - 122 °C for 28 - 32 min.

[0009] 2. Fermentation culture (1) Continuous sterilization Add sucrose and the multifunctional compound agent to the first ingredient tank, perform continuous sterilization at 122 - 126 °C for 13 - 17 min, then add magnesium sulfate and inorganic salts, and perform continuous sterilization at 122 - 126 °C for 13 - 17 min; add yeast extract powder, diammonium hydrogen phosphate, potassium dihydrogen phosphate and defoaming agent to the second ingredient tank, perform continuous sterilization at 122 - 126 °C for 13 - 17 min, mix the first ingredient tank and the second ingredient tank and make up the volume to obtain the fermentation medium; The fermentation medium includes 48 - 52 g / L of sucrose, 4.7 - 5.2 g / L of yeast extract powder, 3.8 - 4.3 g / L of diammonium hydrogen phosphate, 1.4 - 1.6 g / L of potassium dihydrogen phosphate, 0.8 - 1.1 g / L of magnesium sulfate, 0.4 - 0.6 g / L of multifunctional compound agent, 0.08 - 0.12 g / L of defoaming agent, 36 - 43 mg / L of inorganic salts, and the pH is 6.5 - 6.8; The inorganic salts include cobalt chloride, zinc chloride, and calcium chloride, and the mass ratio is 1 - 2:1 - 2:1 - 2; The defoaming agent is polyoxypropylene glycerol ether; The preparation method of the multifunctional compound agent includes the steps of preparing amino-functionalized silica, preparing chitosan complex and mixing, specifically as follows: The step of preparing amino-functionalized silica is to put silica into an ethanol solution, stir evenly, add γ-aminopropyltriethoxysilane, raise the temperature to 60 - 65 °C, keep stirring for 3.2 - 3.8 h, after the stirring ends, lower the temperature to 50 - 54 °C, add L-lysine, then add ammonia water to adjust the pH to 8.4 - 8.6, keep stirring at 210 - 230 rpm for 2.4 - 2.6 h, after the stirring ends, centrifuge at 7800 - 8200 rpm for 12 - 18 min to collect the solid matter, and obtain amino-functionalized silica after washing and drying; The particle size of the silica is 70 - 100 nm; The mass ratio of the silica, ethanol solution, γ-aminopropyltriethoxysilane and L-lysine is 8 - 12:100:1.4 - 1.6:1.1 - 1.3; The mass concentration of the ethanol solution is 47 - 52%; The steps for preparing the chitosan complex are as follows: Add acetic acid solution and chitosan into a reaction vessel. After stirring evenly, add betaine and N,N'-dicyclohexylcarbodiimide. Raise the temperature to 47 - 52 °C and stir for 1.8 - 2.3 h. Then add the mixed acid solution, control the pH to 4.8 - 5.2, and raise the temperature to 62 - 66 °C at a rate of 0.3 - 0.5 °C / min. Keep the temperature and stir for 4.3 - 4.8 h. After the stirring ends, add isopropanol and 3-chloro-2-hydroxypropyltrimethylammonium chloride. Add an 8 - 12 wt% sodium hydroxide solution to adjust the pH to 8.5 - 8.8, and raise the temperature to 66 - 70 °C at a rate of 0.1 - 0.3 °C / min. Keep the temperature and stir for 5.8 - 6.2 h under a nitrogen atmosphere. After the reaction ends, filter and wash, then freeze-dry at -28 to -24 °C for 9 - 11 h and at -42 to -38 °C for 10 - 14 h, and naturally return to room temperature to obtain the chitosan complex; The mass-to-volume ratio of the acetic acid solution, chitosan, betaine, N,N'-dicyclohexylcarbodiimide, mixed acid solution, isopropanol, and 3-chloro-2-hydroxypropyltrimethylammonium chloride is 280 - 320 g: 9.5 - 10.4 g: 1.6 - 1.8 g: 0.4 - 0.6 g: 17 - 22 g: 38 - 42 mL: 3.4 - 3.8 g; The mass concentration of the acetic acid solution is 3.0 - 3.5%; The mixed acid solution is a mixture of a 3.0 - 3.5 wt% acetic acid solution, a 78 - 82 wt% phosphoric acid solution, and phytic acid. The mass ratio of the 3.0 - 3.5 wt% acetic acid solution, 78 - 82 wt% phosphoric acid solution, and phytic acid is 100: 1.0 - 1.4: 1.6 - 2.0; The mixing steps are as follows: Add the chitosan complex into the acetic acid solution. After stirring evenly, add amino-functionalized silica and perform ultrasonic treatment. The ultrasonic treatment time is 28 - 34 min, the ultrasonic power is 100 - 120 W, and the ultrasonic frequency is 30 - 34 kHz. After the ultrasonic treatment ends, centrifuge and wash, then freeze-dry at -38 to -34 °C for 23 - 25 h, and naturally return to room temperature to obtain the multifunctional composite agent; The mass ratio of the chitosan complex, acetic acid solution, and amino-functionalized silica is 4.8 - 5.2: 200: 4.7 - 5.1; The mass concentration of the acetic acid solution is 2.8 - 3.2%.

[0010] (2)Cultivation Inoculate the seed culture solution into the fermentation medium and culture it at 28 - 32 °C with an inoculation amount of 8 - 12%. During the culture process, when the residual sugar < 10 g / L, add sucrose with an addition amount of 28 - 33 g / L, and add it twice in total. When the pH is lower than 5.5, supplement 10 wt% sodium hydroxide solution to maintain the pH at 5.5 - 5.8. When fermenting to the 35th hour, add a multifunctional compound agent with an addition amount of 0.23 - 0.28 g / L. Take samples every 1 h to detect the fermentation process. When the fermentation reaches a residual sugar amount < 5.0 g / L and the curdlan content no longer increases, end the fermentation to obtain the curdlan fermentation broth.

[0011] 3. Post-treatment Add 70 mL of 2.8 - 3.2 wt% sodium hydroxide solution to 14 - 16 mL of the curdlan fermentation broth, stir for 13 - 18 min and then let it stand for 27 - 35 min. Then centrifuge at 8000 - 9000 rpm for 20 - 30 min, take the supernatant, add 8.0 - 12.0 wt% hydrochloric acid solution to adjust the pH to 6.8 - 7.2. After filtration and washing, add 3 times the mass of absolute ethanol, filter and wash again, and then dry. After pulverizing and sieving, obtain curdlan.

[0012] Compared with the prior art, the present invention has achieved the following beneficial effects: 1. The present invention uses Agrobacterium radiobacter as the fermentation strain. In the seed culture step, different dissolved oxygen levels are controlled at different seed culture stages, and α-ketoglutaric acid is added to the seed medium, which can promote the rapid proliferation of the bacterial cells, provide highly active seeds for the subsequent fermentation process, shorten the fermentation cycle, and improve the fermentation efficiency; in the fermentation step, sucrose is first continuously sterilized, and then inorganic salts and magnesium sulfate are added for continuous sterilization, avoiding the phenomenon that impurity components in sucrose directly contact and react with metal ions during continuous sterilization, thereby effectively ensuring the stability of the fermentation process and avoiding the occurrence of tank collapse; in the fermentation step of the present invention, a multifunctional composite agent is introduced, which is composed of silica and chitosan. Specifically, silica is aminated with an amino-silane coupling agent and an amino acid component. On the one hand, it improves the dispersibility and compatibility of silica, enabling it to stably exist in the fermentation broth. On the other hand, it can better adsorb impurities such as pigments and polyphenols in sucrose, promote the formation of a gel network, and thus improve the gel strength; the chitosan complex first introduces betaine on the surface of chitosan, which can improve the tolerance of the cell membrane to high osmotic pressure, and then a mixed acid solution and 3-chloro-2-hydroxypropyltrimethylammonium chloride are added, which can better chelate metal ions, thereby avoiding the reaction between metal ions and sucrose at high temperatures, and can also better adsorb impurities in sucrose and inhibitors during the fermentation process. In the later stage of fermentation, the quaternary ammonium group can be complexed with curdlan through electrostatic interaction, thereby reducing the viscosity of the fermentation broth, improving the mass transfer efficiency, and better promoting the fermentation effect; the multifunctional composite agent is added to the fermentation medium and at 35 h of fermentation in the present invention, which can stabilize the fermentation process, shorten the fermentation cycle, increase the fermentation yield, improve the quality of curdlan, effectively improve the gel strength, reduce the ash content, and avoid the occurrence of tank collapse during the fermentation process; 2. Using the production method of the present invention to prepare curdlan, the fermentation time is 62.4 - 63.3 h, and the curdlan yield is 51.1 - 51.7 g / L; 3. The curdlan prepared by using the production method of the present invention, the gel strength of 2.0% is 720 - 727 g / cm 2 , and the ash content is 1.12 - 1.16%. Detailed implementation mode

[0013] For a clearer understanding of the technical features, objectives, and effects of the present invention, the specific implementation mode of the present invention is now described.

[0014] Example 1 1. Seed culture Agrobacterium radiobacter is placed in the seed medium and cultured at 30 °C. After 6 h of seed culture, the dissolved oxygen is controlled at 47%. When the OD value of the seed liquid > 10, the seed culture solution is obtained; The Agrobacterium radiobacter was obtained by purchase; The seed culture medium includes 20 g / L of glucose, 10 g / L of yeast extract powder, 2.0 g / L of dipotassium hydrogen phosphate, 1.0 g / L of magnesium sulfate, 3.0 g / L of diammonium hydrogen phosphate, 0.35 g / L of α-ketoglutaric acid, with a pH of 7.0, and is sterilized at 121 °C for 30 min.

[0015] 2. Fermentation culture (1) Continuous sterilization Sucrose and a multifunctional compound agent are added to the first ingredient tank, and continuous sterilization is carried out at 124 °C for 15 min. Then, magnesium sulfate and inorganic salts are added, and continuous sterilization is carried out at 124 °C for 15 min. Yeast extract powder, diammonium hydrogen phosphate, potassium dihydrogen phosphate, and an antifoaming agent are added to the second ingredient tank, and continuous sterilization is carried out at 124 °C for 15 min. The first ingredient tank and the second ingredient tank are mixed and made up to volume to obtain the fermentation culture medium; The fermentation culture medium includes 50 g / L of sucrose, 5.0 g / L of yeast extract powder, 4.0 g / L of diammonium hydrogen phosphate, 1.5 g / L of potassium dihydrogen phosphate, 1.0 g / L of magnesium sulfate, 0.5 g / L of multifunctional compound agent, 0.1 g / L of antifoaming agent, 40 mg / L of inorganic salts, with a pH of 6.6; The inorganic salts include cobalt chloride, zinc chloride, and calcium chloride, with a mass ratio of 1:1:2; The antifoaming agent is polyoxypropylene glycerol ether; The preparation method of the multifunctional compound agent includes steps of preparing amino-functionalized silica, preparing chitosan complex, and mixing, specifically as follows: The step of preparing amino-functionalized silica is as follows: 10 g of silica is put into 100 g of 50 wt% ethanol solution, and after stirring evenly, 1.5 g of γ-aminopropyltriethoxysilane is added. The temperature is raised to 63 °C, and stirring is carried out for 3.5 h while maintaining the temperature. After the stirring ends, the temperature is lowered to 52 °C, 1.2 g of L-lysine is added, and then ammonia water is added to adjust the pH to 8.5. Stirring is carried out for 2.5 h while maintaining the temperature at 220 rpm. After the stirring while maintaining the temperature ends, centrifugation is carried out at 8000 rpm for 15 min to collect the solid matter, which is washed and dried to obtain amino-functionalized silica; The particle size of the silica is 80 nm; The steps for preparing the chitosan complex are as follows: Add 300 g of 3.0 wt% acetic acid solution and 10.0 g of chitosan into a reaction vessel. After stirring evenly, add 1.7 g of betaine and 0.5 g of N,N'-dicyclohexylcarbodiimide. Raise the temperature to 50 °C and stir for 2.0 h. Then add 20 g of mixed acid solution, control the pH to 5.0, raise the temperature to 65 °C at a rate of 0.4 °C / min, and keep stirring for 4.5 h. After stirring ends, add 40 mL of isopropanol and 3.6 g of 3-chloro-2-hydroxypropyltrimethylammonium chloride. Add 10 wt% sodium hydroxide solution to adjust the pH to 8.7, raise the temperature to 68 °C at a rate of 0.2 °C / min, and keep stirring for 6.0 h under a nitrogen atmosphere. After the reaction ends, filter and wash, freeze-dry at -26 °C for 10 h, freeze-dry at -40 °C for 12 h, and naturally return to room temperature to obtain the chitosan complex; The mixed acid solution is a mixed solution of acetic acid solution, phosphoric acid, and phytic acid, and the mass ratio of the acetic acid solution, phosphoric acid solution, and phytic acid is 100:1.2:1.8; the mass concentration of the acetic acid solution is 3.0%, and the mass concentration of the phosphoric acid solution is 80%; The mixing steps are as follows: Add 5.0 g of chitosan complex into 200 g of 3.0 wt% acetic acid solution. After stirring evenly, add 5.0 g of amino-functionalized silica, and perform ultrasonic treatment. The ultrasonic treatment time is 30 min, the ultrasonic power is 110 W, and the ultrasonic frequency is 32 kHz. After the ultrasonic treatment ends, centrifuge and wash, freeze-dry at -36 °C for 24 h, and naturally return to room temperature to obtain the multifunctional composite agent.

[0016] (2)Cultivation Inoculate the seed culture solution into the fermentation medium and cultivate at 30 °C. The inoculation amount is 10%. During the cultivation process, when the residual sugar < 10 g / L, add sucrose, and the addition amount is 30 g / L, and add it twice in total. When the pH is lower than 5.5, add 10 wt% sodium hydroxide solution to maintain the pH at 5.7. When fermenting to the 35th h, add the multifunctional composite agent, and the addition amount is 0.25 g / L. Take samples every 1 h to detect the fermentation process. When the fermentation reaches the point where the residual sugar amount < 5.0 g / L and the curdlan content no longer increases, end the fermentation to obtain the curdlan fermentation broth.

[0017] 3. Post-treatment Add 70 mL of 3.0 wt% sodium hydroxide solution to 15 mL of curdlan fermentation broth, stir for 15 min, then let it stand for 30 min, and then centrifuge at 8500 rpm for 25 min. Take the supernatant, add 10.0 wt% hydrochloric acid solution to adjust the pH to 7.0, filter and wash, add 3 times the mass of absolute ethanol, filter and wash again and then dry, and pulverize and sieve to obtain curdlan.

[0018] The curdlan can be prepared by using the production method of Example 1. The fermentation time is 62.4 h, the curdlan yield is 51.7 g / L, and for the prepared curdlan, the gel strength at 2.0% is 727 g / cm 2 , and the ash content is 1.12%.

[0019] Example 2 1. Seed culture The Agrobacterium radiobacter is placed in the seed culture medium, and seed culture is carried out at 32 °C. After 6 h of seed culture, the dissolved oxygen is controlled at 48%. When the OD value of the seed liquid > 10, the seed culture solution is obtained; The Agrobacterium radiobacter is obtained by purchase; The seed culture medium includes 22 g / L of glucose, 11 g / L of yeast extract powder, 2.2 g / L of dipotassium hydrogen phosphate, 1.1 g / L of magnesium sulfate, 3.2 g / L of diammonium hydrogen phosphate, 0.38 g / L of α-ketoglutaric acid, with a pH of 7.2, and is sterilized at 122 °C for 28 min.

[0020] 2. Fermentation culture (1) Continuous sterilization Sucrose and a multifunctional composite agent are added to the first ingredient tank, and continuous sterilization is carried out at 126 °C for 13 min. Then, magnesium sulfate and inorganic salts are added, and continuous sterilization is carried out at 126 °C for 13 min. Yeast extract powder, diammonium hydrogen phosphate, potassium dihydrogen phosphate, and an antifoaming agent are added to the second ingredient tank, and continuous sterilization is carried out at 126 °C for 13 min. The contents of the first ingredient tank and the second ingredient tank are mixed and made up to volume to obtain the fermentation culture medium; The fermentation culture medium includes 52 g / L of sucrose, 5.2 g / L of yeast extract powder, 4.3 g / L of diammonium hydrogen phosphate, 1.6 g / L of potassium dihydrogen phosphate, 1.1 g / L of magnesium sulfate, 0.6 g / L of multifunctional composite agent, 0.12 g / L of antifoaming agent, 43 mg / L of inorganic salts, with a pH of 6.8; The inorganic salts include cobalt chloride, zinc chloride, and calcium chloride, with a mass ratio of 2:1:1; The antifoaming agent is polyoxypropylene glycerol ether; The preparation method of the multifunctional composite agent includes steps of preparing amino-functionalized silica, preparing a chitosan complex, and mixing, specifically as follows: The step of preparing amino-functionalized silica is as follows: 12 g of silica is put into 100 g of 52 wt% ethanol solution. After stirring evenly, 1.6 g of γ-aminopropyltriethoxysilane is added, the temperature is raised to 65 °C, and stirring is carried out for 3.8 h while maintaining the temperature. After the stirring ends, the temperature is lowered to 54 °C, 1.3 g of L-lysine is added, then ammonia water is added to adjust the pH to 8.6, and stirring is carried out at 230 rpm for 2.6 h while maintaining the temperature. After the stirring at a constant temperature ends, the solid is collected by centrifugation at 8200 rpm for 12 min, and after washing and drying, amino-functionalized silica is obtained; The particle size of the silica is 100 nm; The steps for preparing the chitosan complex are as follows: Add 320 g of 3.5 wt% acetic acid solution and 10.4 g of chitosan into a reaction vessel. After stirring evenly, add 1.8 g of betaine and 0.6 g of N,N'-dicyclohexylcarbodiimide. Raise the temperature to 52 °C and stir for 2.3 h. Then add 22 g of the mixed acid solution, control the pH to 5.2, raise the temperature to 66 °C at a rate of 0.5 °C / min, and keep stirring for 4.8 h. After the stirring ends, add 42 mL of isopropanol and 3.8 g of 3-chloro-2-hydroxypropyltrimethylammonium chloride. Add 12 wt% sodium hydroxide solution to adjust the pH to 8.8, raise the temperature to 70 °C at a rate of 0.3 °C / min, and keep stirring for 6.2 h under a nitrogen atmosphere. After the reaction ends, filter and wash, freeze-dry at -24 °C for 11 h, freeze-dry at -38 °C for 14 h, and naturally return to room temperature to obtain the chitosan complex; The mixed acid solution is a mixed solution of acetic acid solution, phosphoric acid, and phytic acid. The mass ratio of the acetic acid solution, phosphoric acid solution, and phytic acid is 100:1.4:2.0; the mass concentration of the acetic acid solution is 3.5%, and the mass concentration of the phosphoric acid solution is 82%; The mixing steps are as follows: Add 5.2 g of the chitosan complex into 200 g of 3.2 wt% acetic acid solution. After stirring evenly, add 5.1 g of aminated silica, and perform ultrasonic treatment. The ultrasonic treatment time is 34 min, the ultrasonic power is 120 W, and the ultrasonic frequency is 34 kHz. After the ultrasonic treatment ends, centrifuge and wash, freeze-dry at -34 °C for 25 h, and naturally return to room temperature to obtain the multifunctional complex agent.

[0021] (2) Cultivation Inoculate the seed culture solution into the fermentation medium and cultivate at 32 °C. The inoculation amount is 12%. During the cultivation process, when the residual sugar < 10 g / L, add sucrose, and the addition amount is 33 g / L. A total of 2 times are added. When the pH is lower than 5.5, add 10 wt% sodium hydroxide solution to maintain the pH at 5.8. When fermenting to the 35th h, add the multifunctional complex agent, and the addition amount is 0.28 g / L. Take samples every 1 h to detect the fermentation process. When the fermentation reaches the point where the residual sugar content < 5.0 g / L and the curdlan content no longer increases, end the fermentation to obtain the curdlan fermentation broth.

[0022] 3. Post-treatment Add 70 mL of 3.2 wt% sodium hydroxide solution to 16 mL of curdlan fermentation broth, stir for 13 min, let stand for 35 min, then centrifuge at 9000 rpm for 20 min. Take the supernatant, add 12.0 wt% hydrochloric acid solution to adjust the pH to 7.2. After filtration and washing, add 3 times the mass of absolute ethanol. After filtration and washing again, dry, pulverize and sieve to obtain curdlan.

[0023] The curdlan was prepared by the production method of Example 2. The fermentation time was 62.8 h, the curdlan yield was 51.3 g / L, and the gel strength of the prepared curdlan at 2.0% was 723 g / cm 2 , and the ash content was 1.15%.

[0024] Example 3 1. Seed culture Place Agrobacterium radiobacter in the seed medium and conduct seed culture at 29 °C. After 6 h of seed culture, control the dissolved oxygen to be 46%. When the OD value of the seed liquid > 10, obtain the seed culture broth; The Agrobacterium radiobacter was purchased; The seed medium includes 18 g / L of glucose, 9 g / L of yeast extract powder, 1.7 g / L of dipotassium hydrogen phosphate, 0.8 g / L of magnesium sulfate, 2.7 g / L of diammonium hydrogen phosphate, 0.33 g / L of α-ketoglutaric acid, with a pH of 6.8, and sterilized at 120 °C for 32 min.

[0025] 2. Fermentation culture (1)Continuous sterilization Add sucrose and multifunctional compound agent to the first ingredient tank, conduct continuous sterilization at 122 °C for 17 min, then add magnesium sulfate and inorganic salts, and conduct continuous sterilization at 122 °C for 17 min; add yeast extract powder, diammonium hydrogen phosphate, potassium dihydrogen phosphate and antifoaming agent to the second ingredient tank, conduct continuous sterilization at 122 °C for 17 min, mix the first ingredient tank and the second ingredient tank and make up the volume to obtain the fermentation medium; The fermentation medium includes 48 g / L of sucrose, 4.7 g / L of yeast extract powder, 3.8 g / L of diammonium hydrogen phosphate, 1.4 g / L of potassium dihydrogen phosphate, 0.8 g / L of magnesium sulfate, 0.4 g / L of multifunctional compound agent, 0.08 g / L of antifoaming agent, 36 mg / L of inorganic salts, with a pH of 6.5; The inorganic salts include cobalt chloride, zinc chloride, calcium chloride, and the mass ratio is 1:2:1; The antifoaming agent is polyoxypropylene glycerol ether; The preparation method of the multifunctional compound agent includes the steps of preparing aminated silica, preparing chitosan complex and mixing, specifically as follows: The steps for preparing aminated silica are as follows: 8 g of silica is put into 100 g of a 47 wt% ethanol solution. After stirring evenly, 1.4 g of γ-aminopropyltriethoxysilane is added. The temperature is raised to 60 °C and kept warm with stirring for 3.2 h. After the stirring ends, the temperature is lowered to 50 °C, 1.1 g of L-lysine is added, and then ammonia water is added to adjust the pH to 8.4. It is kept warm with stirring at 210 rpm for 2.4 h. After the warm stirring ends, the solid is collected by centrifugation at 7800 rpm for 18 min, and after washing and drying, aminated silica is obtained; The particle size of the silica is 70 nm; The steps for preparing the chitosan complex are as follows: 280 g of a 3.2 wt% acetic acid solution and 9.5 g of chitosan are added to a reaction vessel. After stirring evenly, 1.6 g of betaine and 0.4 g of N,N'-dicyclohexylcarbodiimide are added. The temperature is raised to 47 °C and the reaction is stirred for 1.8 h. Then 17 g of a mixed acid solution is added, and the pH is controlled to 4.8. The temperature is raised to 62 °C at a rate of 0.3 °C / min and kept warm with stirring for 4.3 h. After the stirring ends, 38 mL of isopropanol and 3.4 g of 3-chloro-2-hydroxypropyltrimethylammonium chloride are added. 8 wt% sodium hydroxide solution is added to adjust the pH to 8.5. The temperature is raised to 66 °C at a rate of 0.1 °C / min and kept warm with stirring for 5.8 h under a nitrogen atmosphere. After the reaction ends, after filtration and washing, it is freeze-dried at -28 °C for 9 h, freeze-dried at -42 °C for 10 h, and naturally restored to room temperature to obtain the chitosan complex; The mixed acid solution is a mixed solution of acetic acid solution, phosphoric acid, and phytic acid. The mass ratio of the acetic acid solution, phosphoric acid solution, and phytic acid is 100:1.0:1.6; the mass concentration of the acetic acid solution is 3.2%, and the mass concentration of the phosphoric acid solution is 78%; The mixing steps are as follows: 4.8 g of the chitosan complex is added to 200 g of a 2.8 wt% acetic acid solution. After stirring evenly, 4.7 g of aminated silica is added, and ultrasonic treatment is carried out. The ultrasonic treatment time is 28 min, the ultrasonic power is 100 W, and the ultrasonic frequency is 30 kHz. After the ultrasonic treatment ends, after centrifugation and washing, it is freeze-dried at -38 °C for 23 h and naturally restored to room temperature to obtain the multifunctional composite agent.

[0026] (2) Cultivation Inoculate the seed culture solution into the fermentation medium and culture it at 28 °C with an inoculation amount of 8%. During the culture process, sucrose is supplemented when the residual sugar < 10 g / L, and the supplementation amount is 28 g / L, with a total of 2 supplements. When the pH is lower than 5.5, 10 wt% sodium hydroxide solution is supplemented to maintain the pH at 5.5. When fermenting to the 35th hour, a multifunctional compound agent is added, and the addition amount is 0.23 g / L. Samples are taken every 1 h to detect the fermentation process. When the fermentation reaches the point where the residual sugar amount < 5.0 g / L and the curdlan content no longer increases, the fermentation is ended to obtain the curdlan fermentation broth.

[0027] 3. Post-treatment Add 70 mL of 2.8 wt% sodium hydroxide solution to 14 mL of the curdlan fermentation broth, stir for 18 min and then let it stand for 27 min, then centrifuge at 8000 rpm for 30 min, take the supernatant, add 8.0 wt% hydrochloric acid solution to adjust the pH to 6.8, after filtration and washing, add 3 times the mass of absolute ethanol, filter and wash again and then dry, and after pulverizing and sieving, obtain curdlan.

[0028] The curdlan is prepared by the production method of Example 3. The fermentation time is 63.3 h, the curdlan yield is 51.1 g / L, and for the prepared curdlan, the gel strength at 2.0% is 720 g / cm 2 , and the ash content is 1.16%.

[0029] The present invention uses Agrobacterium radiobacter as the fermentation strain. In the seed culture step, different dissolved oxygen levels are controlled at different seed culture stages, and α-ketoglutaric acid is added to the seed culture medium, which can promote the rapid proliferation of the bacterial cells, provide highly active seeds for the subsequent fermentation process, shorten the fermentation cycle, and improve the fermentation efficiency. In the fermentation step, sucrose is first continuously sterilized, and then inorganic salts, magnesium sulfate and other components are added for continuous sterilization, avoiding the phenomenon that the impurity components in sucrose directly contact and react with metal ions during continuous sterilization, thus effectively ensuring the stability of the fermentation process and avoiding the occurrence of tank collapse. In the fermentation step of the present invention, a multifunctional composite agent is introduced, which is composed of silica and chitosan. Specifically, silica is first aminated with an amino-silane coupling agent and an amino acid component. On the one hand, the dispersibility and compatibility of silica are improved, enabling it to stably exist in the fermentation broth. On the other hand, it can preferably adsorb impurities such as pigments and polyphenols in sucrose, promote the formation of a gel network, and then improve the gel strength. The chitosan complex first introduces betaine on the surface of chitosan, which can improve the tolerance of the cell membrane to high osmotic pressure, and then a mixed acid solution and 3-chloro-2-hydroxypropyltrimethylammonium chloride are added, which can preferably chelate metal ions, thus avoiding the reaction between metal ions and sucrose at high temperature, and can also preferably adsorb impurities in sucrose and inhibitors during the fermentation process. In the later stage of fermentation, the quaternary ammonium group can be complexed with curdlan through electrostatic interaction, thereby reducing the viscosity of the fermentation broth, improving the mass transfer efficiency, and better promoting the fermentation effect. The multifunctional composite agent is added to the fermentation medium and at 35 h of fermentation in the present invention, which can stabilize the fermentation process, shorten the fermentation cycle, increase the fermentation yield, improve the quality of curdlan, effectively improve the gel strength, reduce the ash content, and avoid the occurrence of tank collapse during the fermentation process.

[0030] Comparative Example 1-1 Based on Example 1, the following changes are made: 1. In the seed culture step, α-ketoglutaric acid in the seed culture medium is replaced with glucose in equal amounts; 2. In the fermentation culture step, (1) the inorganic salt components in the fermentation medium are all replaced with calcium chloride in equal amounts; (2) The preparation method of the multifunctional composite agent omits the preparation and mixing steps of the chitosan complex, and the multifunctional composite agent is replaced with aminated silica in equal amounts; The remaining operations are the same.

[0031] In Comparative Example 1-1, the α-ketoglutaric acid component was omitted in the seed culture step, which led to slower proliferation of the bacterial cells, affecting the metabolic activity during the fermentation process. Moreover, only calcium chloride was used as the inorganic salt, which caused the growth of the bacterial cells to slow down, the enzyme activity to decrease, and the synthesis rate of curdlan to be inhibited. In Comparative Example 1-1, only aminated silica was used as the multifunctional composite agent, lacking the chelating and adsorption properties of the chitosan complex, unable to effectively regulate the ion concentration and metabolic waste in the culture medium, reducing the accumulation efficiency of curdlan in the later stage of fermentation, ultimately prolonging the fermentation time, reducing the production efficiency, and affecting the product quality.

[0032] Using the fermentation production method of Comparative Example 1-1 to prepare curdlan, the fermentation time was 70.3 h, the curdlan yield was 43.2 g / L, and for the prepared curdlan, the 2.0% gel strength was 664 g / cm 2 , and the ash content was 1.34%.

[0033] Comparative Example 1-2 Based on Example 1, the changes were as follows: In the fermentation culture step, the steps of preparing aminated silica and preparing the chitosan complex were omitted, and the untreated silica and the untreated chitosan were directly mixed; The preparation method of the multifunctional composite agent was as follows: 5.0 g of chitosan was added to 200 g of 3.0 wt% acetic acid solution, and after stirring evenly, 5.0 g of silica was added, followed by ultrasonic treatment. The ultrasonic treatment time was 30 min, the ultrasonic power was 110 W, the ultrasonic frequency was 32 kHz. After the ultrasonic treatment was completed, it was centrifuged and washed, and then freeze-dried at -36 °C for 24 h and naturally restored to room temperature to obtain the multifunctional composite agent; The particle size of the silica was 80 nm; (2) The operation step of "when fermenting to the 35th h, adding the multifunctional composite agent with an addition amount of 0.25 g / L" was omitted; The remaining operations were the same.

[0034] In Comparative Example 1-2, directly using the untreated silica and chitosan as the functional composite agent, the interaction force between them was weak, and the aggregation of silica would affect the growth of the bacterial cells. Chitosan could not be evenly dispersed in the fermentation broth and could not effectively exert its adsorption properties. The multifunctional composite agent in Comparative Example 1-2 had a weak promoting effect on the bacterial cell metabolism and curdlan synthesis, greatly affecting the fermentation efficiency and product quality.

[0035] Using the fermentation production method of Comparative Example 1-2 to prepare curdlan, the fermentation time was 73.5 h, the curdlan yield was 41.9 g / L, and for the prepared curdlan, the 2.0% gel strength was 641 g / cm 2 , and the ash content was 1.48%.

[0036] Unless otherwise specified, the ratios described in the present invention are all mass ratios, and the percentages are all mass percentages.

[0037] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for fermentatively producing curdlan, characterized in that, It includes a seed culture step, a fermentation culture step, and a post-treatment step; In the seed culture step, Agrobacterium radiobacter is cultured in a seed medium; The seed medium includes 18 - 22 g / L of glucose, 9 - 11 g / L of yeast extract powder, 1.7 - 2.2 g / L of dipotassium hydrogen phosphate, 0.8 - 1.1 g / L of magnesium sulfate, 2.7 - 3.2 g / L of diammonium hydrogen phosphate, 0.33 - 0.38 g / L of α-ketoglutaric acid, with a pH of 6.8 - 7.2, and is sterilized at 120 - 122 °C for 28 - 32 min; The fermentation culture step includes a continuous sterilization step and a culture step; In the culture step, the seed culture solution is inoculated into the fermentation medium and cultured at 28 - 32 °C. When the residual sugar < 10 g / L, sucrose is supplemented, and the supplementation amount is 28 - 33 g / L, and it is supplemented 2 times in total. When fermenting to the 35th h, a multifunctional composite agent is added, and the addition amount is 0.23 - 0.28 g / L; The fermentation medium includes 48 - 52 g / L of sucrose, 4.7 - 5.2 g / L of yeast extract powder, 3.8 - 4.3 g / L of diammonium hydrogen phosphate, 1.4 - 1.6 g / L of potassium dihydrogen phosphate, 0.8 - 1.1 g / L of magnesium sulfate, 0.4 - 0.6 g / L of multifunctional composite agent, 0.08 - 0.12 g / L of antifoaming agent, 36 - 43 mg / L of inorganic salts, with a pH of 6.5 - 6.8; The preparation method of the multifunctional composite agent includes a step of preparing amino-functionalized silica, a step of preparing a chitosan complex, and a mixing step; In the step of preparing the chitosan complex, an acetic acid solution and chitosan are added to a reaction vessel, stirred evenly, betaine and N,N'-dicyclohexylcarbodiimide are added, reacted at 47 - 52 °C for 1.8 - 2.3 h, a mixed acid solution is added, stirred at 62 - 66 °C for 4.3 - 4.8 h, isopropanol and 3-chloro-2-hydroxypropyltrimethylammonium chloride are added, and stirred at 66 - 70 °C under a nitrogen atmosphere for 5.8 - 6.2 h to obtain the chitosan complex.

2. A method for fermentatively producing curdlan according to claim 1, characterized in that In the seed culture step, Agrobacterium radiobacter is cultured in a seed medium, and the seed culture is carried out at 29 - 32 °C. After the 6th h of the seed culture, the dissolved oxygen is controlled at 46 - 48%. When the OD value of the seed liquid > 10, the seed culture solution is obtained.

3. A method for fermentatively producing curdlan according to claim 1, characterized in that In the fermentation culture step, in the continuous sterilization step, sucrose and the multifunctional composite agent are added to a first ingredient tank, continuously sterilized at 122 - 126 °C for 13 - 17 min, then magnesium sulfate and inorganic salts are added, and continuously sterilized at 122 - 126 °C for 13 - 17 min; yeast extract powder, diammonium hydrogen phosphate, potassium dihydrogen phosphate, and antifoaming agent are added to a second ingredient tank, continuously sterilized at 122 - 126 °C for 13 - 17 min, and the first ingredient tank and the second ingredient tank are mixed and made up to volume to obtain the fermentation medium.

4. A method for fermentatively producing curdlan according to claim 3, characterized in that In the fermentation medium, the inorganic salts include cobalt chloride, zinc chloride, and calcium chloride, and the mass ratio is 1-2:1-2:1-2; The defoaming agent is polyoxypropylene glycerol ether.

5. A method for fermentatively producing curdlan according to claim 1, characterized in that The steps for preparing amino-functionalized silica are as follows: Put silica into an ethanol solution, stir evenly, add γ-aminopropyltriethoxysilane, raise the temperature to 60-65 °C, keep stirring for 3.2-3.8 h. After stirring, lower the temperature to 50-54 °C, add L-lysine, then add ammonia water to adjust the pH to 8.4-8.6, keep stirring at 210-230 rpm for 2.4-2.6 h. After the end of the holding and stirring, centrifuge at 7800-8200 rpm for 12-18 min to collect the solid matter, wash and dry to obtain amino-functionalized silica; The particle size of the silica is 70-100 nm; The mass ratio of the silica, ethanol solution, γ-aminopropyltriethoxysilane, and L-lysine is 8-12:100:1.4-1.6:1.1-1.3; The mass concentration of the ethanol solution is 47-52%.

6. A method for fermentatively producing curdlan according to claim 1, characterized in that In the steps for preparing the chitosan complex, the mass-to-volume ratio of the acetic acid solution, chitosan, betaine, N,N'-dicyclohexylcarbodiimide, mixed acid solution, isopropanol, and 3-chloro-2-hydroxypropyltrimethylammonium chloride is 280-320 g:9.5-10.4 g:1.6-1.8 g:0.4-0.6 g:17-22 g:38-42 mL:3.4-3.8 g; The mass concentration of the acetic acid solution is 3.0-3.5%; The mixed acid solution is a mixture of a 3.0-3.5 wt% acetic acid solution, a 78-82 wt% phosphoric acid solution, and phytic acid, and the mass ratio of the 3.0-3.5 wt% acetic acid solution, 78-82 wt% phosphoric acid solution, and phytic acid is 100:1.0-1.4:1.6-2.

0.

7. A method for fermentatively producing curdlan according to claim 1, characterized in that The mixing step is as follows: Add the chitosan complex to the acetic acid solution, stir evenly, add amino-functionalized silica, and perform ultrasonic treatment. The ultrasonic treatment time is 28-34 min, the ultrasonic power is 100-120 W, and the ultrasonic frequency is 30-34 kHz. After the ultrasonic treatment, after centrifugation and washing, freeze-dry at -38 to -34 °C for 23-25 h, and naturally return to room temperature to obtain a multifunctional composite agent; The mass ratio of the chitosan complex, acetic acid solution, and amino-functionalized silica is 4.8-5.2:200:4.7-5.1; The mass concentration of the acetic acid solution is 2.8-3.2%.

8. A method for fermentatively producing curdlan according to claim 1, characterized in that In the fermentation and culture step, during the culture step, when the pH is lower than 5.5, a 10 wt% sodium hydroxide solution is added to maintain the pH at 5.5 - 5.

8. Samples are taken every 1 h to detect the fermentation progress. Fermentation ends when the residual sugar content < 5.0 g / L and the curdlan content no longer increases, obtaining a curdlan fermentation broth.

9. A method for fermentatively producing curdlan according to claim 8, characterized in that in the post-treatment step, 70 mL of a 2.8 - 3.2 wt% sodium hydroxide solution is added to 14 - 16 mL of the curdlan fermentation broth, stirred for 13 - 18 min and then left to stand for 27 - 35 min, then centrifuged at 8000 - 9000 rpm for 20 - 30 min. The supernatant is taken, and the pH is adjusted to 6.8 - 7.2 with an 8.0 - 12.0 wt% hydrochloric acid solution. After filtration and washing, 3 times the mass of absolute ethanol is added, and after filtration and washing again and drying, it is pulverized and sieved to obtain curdlan.