A method for producing curdlan by fermentation

Abstract

The invention provides a method for producing curdlan gum by fermentation, belonging to the field of fermentation technology. The method comprises seed culture, fermentation culture and post-processing steps. The culture step comprises: inoculating seed culture fluid into a fermentation medium and culturing at 28-32°C; adding sucrose in an amount of 28-33 g / L when residual sugar is less than 10 g / L, for a total of two additions; and adding a multifunctional composite agent in an amount of 0.23-0.28 g / L when fermentation reaches 35 hours. By adopting the fermentation method of the invention and using sucrose as a carbon source, the fermentation time is shortened to 62.4-63.3 hours, the curdlan gum yield is increased to 51.1-51.7 g / L, and the gel strength of the curdlan gum is effectively improved, thereby avoiding unstable production and the occurrence of phenomena such as tank tipping.

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 thermo-gelling properties, known as thermogel. Its chemical name is β-1,3-glucan. Curdlan is an odorless, tasteless, freely flowing white powder with an unbranched linear glucan structure. It is a water-insoluble dietary fiber that forms a viscous hydrated liquid when dispersed in cold water and whipped at high speed. 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 gel strength.

[0003] China approved curdlan as a food additive in May 2006, and 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-binding agent, adhesive, film-forming agent, etc.; in soy products, it can enhance heat resistance and freeze resistance, improve the taste, and improve the molding; in meat, aquatic cooked meat products, konjac gel products, and surimi products, it can reproduce the taste of various food ingredients, 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] Curdlan fermentation production is a process in which soil bacteria synthesize small molecular sugars such as glucose and sucrose into polysaccharides; traditional production methods usually use glucose as a carbon source, but glucose usually needs to be obtained through starch decomposition, and its production process is relatively complicated, increasing the production process steps and energy consumption. In addition, the resource supply of starch is affected by various factors such as agricultural production and is more unstable. In comparison, sucrose is obtained by squeezing, extracting and crystallizing from beets or sugarcane, 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, the OD of the bacteria will be less than 18 during the fermentation process, and the OD will no longer increase. In addition, the bacteria will continue to produce acid, requiring continuous alkali supplementation, slowing 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 overturning, resulting in large economic losses.

[0006] CN114686545A discloses a method for increasing the yield and gel strength of curdlan gum. Specifically, FeSO4 is added to a fermentation medium, wherein 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 extract, 1-3 g / L calcium carbonate, and 0.01%-0.04% (w / v) FeSO4. The patent discloses that by adding different concentrations of FeSO4 to the culture medium, when the FeSO4 content is 0.02% (w / v), the yield of curdlan gum is increased to 58.37 g / L and the gel strength is increased to 691.48 g / cm3. 2 ;

[0007] The above patent uses sucrose as a carbon source in the fermentation medium and increases the yield and strength of curdlan by adding the inorganic salt FeSO4. However, the yield is still low, the fermentation rate is slow, and the gel strength of curdlan is low, which affects its application. Summary of the Invention

[0008] In order to solve the technical problems existing in the prior art, the present invention provides a method for producing curdlan by fermentation, which uses sucrose as the carbon source in the fermentation medium, avoids production instability and the occurrence of phenomena such as tank tipping, improves the yield and production efficiency of curdlan, enhances the strength of the curdlan gel, and improves the quality of curdlan.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] A method for producing curdlan by fermentation, comprising seed culture, fermentation culture and post-processing steps, specifically as follows:

[0011] 1. Seed Culture

[0012] Placing Agrobacterium radiobacterium in a seed culture medium, culturing the seeds at 29-32° C., controlling the dissolved oxygen to 46-48% after the 6th hour of seed culture, and culturing the seeds until the OD value of the seed solution is greater than 10, thereby obtaining a seed culture solution;

[0013] The radiobacterium was purchased;

[0014] The seed culture medium comprises 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, and 0.33-0.38 g / L of α-ketoglutaric acid, has a pH of 6.8-7.2, and is sterilized at 120-122° C. for 28-32 minutes.

[0015] 2. Fermentation Culture

[0016] (1) Consecutive elimination

[0017] Add sucrose and a multifunctional compound to a No. 1 batching tank, and incubate at 122-126° C. for 13-17 minutes. Then, add magnesium sulfate and an inorganic salt, and incubate at 122-126° C. for 13-17 minutes. Add yeast extract powder, diammonium hydrogen phosphate, potassium dihydrogen phosphate, and a defoaming agent to a No. 2 batching tank, and incubate at 122-126° C. for 13-17 minutes. Mix the contents of the No. 1 and No. 2 batching tanks and adjust the volume to obtain a fermentation medium.

[0018] The fermentation medium comprises 48-52 g / L sucrose, 4.7-5.2 g / L yeast extract powder, 3.8-4.3 g / L diammonium hydrogen phosphate, 1.4-1.6 g / L potassium dihydrogen phosphate, 0.8-1.1 g / L magnesium sulfate, 0.4-0.6 g / L multifunctional compound, 0.08-0.12 g / L defoaming agent, 36-43 mg / L inorganic salt, and has a pH of 6.5-6.8;

[0019] The inorganic salts include cobalt chloride, zinc chloride, and calcium chloride in a mass ratio of 1-2:1-2:1-2;

[0020] The defoaming agent is polyoxypropylene glycerol ether;

[0021] The preparation method of the multifunctional composite agent includes the steps of preparing amino-silica, preparing chitosan composite and mixing, which are specifically as follows:

[0022] The step of preparing the amino-silica comprises: adding silica to an ethanol solution, stirring evenly, adding γ-aminopropyltriethoxysilane, raising the temperature to 60-65° C., and stirring at this temperature for 3.2-3.8 hours. After the stirring is completed, lowering the temperature to 50-54° C., adding L-lysine, and then adding ammonia water to adjust the pH to 8.4-8.6. The solution is stirred at this temperature at 210-230 rpm for 2.4-2.6 hours. After the stirring is completed, the solid is collected by centrifugation at 7800-8200 rpm for 12-18 minutes, and washed and dried to obtain the amino-silica;

[0023] The particle size of the silicon dioxide is 70-100 nm;

[0024] The mass ratio of the silicon dioxide, ethanol solution, γ-aminopropyltriethoxysilane and L-lysine is 8-12:100:1.4-1.6:1.1-1.3;

[0025] The mass concentration of the ethanol solution is 47-52%;

[0026] The chitosan complex preparation step comprises the following steps: adding acetic acid solution and chitosan to a reaction container, stirring evenly, adding betaine and N,N'-dicyclohexylcarbodiimide, raising the temperature to 47-52°C, stirring and reacting for 1.8-2.3 hours, then adding a mixed acid solution, controlling the pH to 4.8-5.2, raising the temperature to 62-66°C at a rate of 0.3-0.5°C / min, maintaining the temperature and stirring for 4.3-4.8 hours, and after the stirring is completed, adding isopropyl alcohol and 3-chloro- 2-hydroxypropyltrimethylammonium chloride, adding 8-12wt% sodium hydroxide solution to adjust the pH to 8.5-8.8, raising the temperature to 66-70°C at a rate of 0.1-0.3°C / min, and stirring under a nitrogen atmosphere for 5.8-6.2 hours. After the reaction is completed, filtering and washing, freeze-drying at -28 to -24°C for 9-11 hours, freeze-drying at -42 to -38°C for 10-14 hours, and naturally returning to room temperature to obtain a chitosan complex;

[0027] The mass volume ratio of the acetic acid solution, chitosan, betaine, N,N'-dicyclohexylcarbodiimide, mixed acid solution, isopropyl alcohol and 3-chloro-2-hydroxypropyltrimethylammonium chloride is 280-320g:9.5-10.4g:1.6-1.8g:0.4-0.6g:17-22g:38-42mL:3.4-3.8g;

[0028] The mass concentration of the acetic acid solution is 3.0-3.5%;

[0029] The mixed acid solution is a mixture of 3.0-3.5wt% acetic acid solution, 78-82wt% phosphoric acid solution, and phytic acid, wherein the mass ratio of the 3.0-3.5wt% acetic acid solution, 78-82wt% phosphoric acid solution, and phytic acid is 100:1.0-1.4:1.6-2.0;

[0030] The mixing step comprises adding the chitosan complex to the acetic acid solution, stirring evenly, adding amino-silica, and performing ultrasonic treatment, wherein the ultrasonic treatment time is 28-34 minutes, the ultrasonic power is 100-120W, and the ultrasonic frequency is 30-34kHz. After the ultrasonic treatment, the mixture is centrifuged and washed, and then freeze-dried at -38 to -34°C for 23-25 ​​hours and naturally returned to room temperature to obtain a multifunctional composite agent;

[0031] The mass ratio of the chitosan complex, acetic acid solution, and amino-silica is 4.8-5.2:200:4.7-5.1;

[0032] The mass concentration of the acetic acid solution is 2.8-3.2%.

[0033] (2) Cultivation

[0034] The seed culture medium is inoculated into the fermentation medium and cultured at 28-32° C. with an inoculation size of 8-12%. During the culture process, sucrose is added in an amount of 28-33 g / L when the residual sugar is less than 10 g / L, and the addition is made twice. 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. When the fermentation reaches the 35th hour, a multifunctional composite agent is added in an amount of 0.23-0.28 g / L. The fermentation progress is detected by sampling every 1 hour. When the residual sugar content is less than 5.0 g / L and the curdlan content no longer increases, the fermentation is terminated to obtain a curdlan fermentation liquid.

[0035] 3. Post-processing

[0036] To 14-16 mL of curdlan gum fermentation broth, 70 mL of 2.8-3.2 wt% sodium hydroxide solution was added, the mixture was stirred for 13-18 minutes, and then allowed to stand for 27-35 minutes. The mixture was then centrifuged at 8000-9000 rpm for 20-30 minutes. The supernatant was collected, and the pH was adjusted to 6.8-7.2 by adding 8.0-12.0 wt% hydrochloric acid solution. After filtering and washing, 3 times the mass of anhydrous ethanol was added, and the mixture was filtered and washed again, dried, and crushed and sieved to obtain curdlan gum.

[0037] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0038] 1. The present invention adopts radiobacterium as the fermentation strain. In the seed culture step, different dissolved oxygen levels are controlled in different seed culture stages. α-ketoglutaric acid is added to the seed culture medium, which can promote rapid proliferation of the bacteria, 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 digested, and then inorganic salts and magnesium sulfate and other components are added for continuous digestion, thereby avoiding the phenomenon that impurities in sucrose directly contact and react with metal ions during the continuous digestion process, thereby effectively ensuring the stability of the fermentation process and avoiding the occurrence of the tank inversion phenomenon. In the fermentation step, the present invention introduces a multifunctional composite agent, which is a composite of silicon dioxide and chitosan. Specifically, an aminosilane coupling agent and an amino acid component are first used to perform an amino treatment on silicon dioxide, which, on the one hand, improves the dispersibility and compatibility of silicon dioxide, so that it can be stably present in the fermentation liquid, and on the other hand, The invention can effectively adsorb impurities such as pigments and polyphenols in sucrose, promote the formation of gel network, and thus improve the gel strength. The chitosan composite first introduces betaine on the surface of chitosan, which can improve the tolerance of cell membrane to high osmotic pressure. Then, a mixed acid solution and 3-chloro-2-hydroxypropyltrimethylammonium chloride are added, which can effectively chelate metal ions, thereby avoiding the reaction between metal ions and sucrose at high temperature, and can also effectively adsorb impurities in sucrose and inhibitors in the fermentation process. In the late fermentation stage, the quaternary ammonium group can be electrostatically compounded with the curdlan, thereby reducing the viscosity of the fermentation liquid, improving the mass transfer efficiency, and better promoting the fermentation effect. The invention adds a multifunctional composite agent to the fermentation medium and at the 35th hour of fermentation, which can stabilize the fermentation process, shorten the fermentation cycle, increase the fermentation yield, improve the quality of the curdlan, effectively improve the gel strength, reduce the ash content, and avoid the phenomenon of tank collapse during the fermentation process.

[0039] 2. The production method of the present invention is used to prepare curdlan gum, with a fermentation time of 62.4-63.3 hours and a curdlan gum yield of 51.1-51.7 g / L;

[0040] 3. The curdlan produced by the production method of the present invention has a 2.0% gel strength of 720-727 g / cm 2 , the ash content is 1.12-1.16%. DETAILED DESCRIPTION

[0041] In order to more clearly understand the technical features, purposes and effects of the present invention, specific embodiments of the present invention are now described.

[0042] Example 1

[0043] 1. Seed Culture

[0044] The radiobacterium was placed in a seed culture medium and cultured at 30°C. After the 6th hour of seed culture, the dissolved oxygen was controlled to 47%. The seed culture solution was obtained when the OD value of the seed solution was greater than 10.

[0045] The radiobacterium was purchased;

[0046] The seed culture medium includes 20 g / L glucose, 10 g / L yeast extract powder, 2.0 g / L dipotassium hydrogen phosphate, 1.0 g / L magnesium sulfate, 3.0 g / L diammonium hydrogen phosphate, and 0.35 g / L α-ketoglutaric acid, has a pH of 7.0, and is sterilized at 121° C. for 30 minutes.

[0047] 2. Fermentation Culture

[0048] (1) Consecutive elimination

[0049] Add sucrose and a multifunctional compound to a No. 1 batching tank, and sterilize at 124° C. for 15 minutes. Then, add magnesium sulfate and an inorganic salt, and sterilize at 124° C. for 15 minutes. Add yeast extract powder, diammonium hydrogen phosphate, potassium dihydrogen phosphate, and a defoaming agent to a No. 2 batching tank, and sterilize at 124° C. for 15 minutes. Mix the No. 1 batching tank and the No. 2 batching tank, and adjust the volume to obtain a fermentation medium.

[0050] The fermentation medium comprises 50 g / L sucrose, 5.0 g / L yeast extract powder, 4.0 g / L diammonium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, 1.0 g / L magnesium sulfate, 0.5 g / L multifunctional compound, 0.1 g / L defoaming agent, 40 mg / L inorganic salt, and has a pH of 6.6;

[0051] The inorganic salts include cobalt chloride, zinc chloride, and calcium chloride in a mass ratio of 1:1:2;

[0052] The defoaming agent is polyoxypropylene glycerol ether;

[0053] The preparation method of the multifunctional composite agent includes the steps of preparing amino-silica, preparing chitosan composite and mixing, which are specifically as follows:

[0054] The step of preparing the amino-silica comprises: adding 10 g of silica to 100 g of a 50 wt% ethanol solution, stirring evenly, adding 1.5 g of γ-aminopropyltriethoxysilane, raising the temperature to 63° C., and stirring at this temperature for 3.5 hours. After the stirring is completed, lowering the temperature to 52° C., adding 1.2 g of L-lysine, and then adding ammonia water to adjust the pH to 8.5. The mixture is stirred at this temperature at 220 rpm for 2.5 hours. After the stirring is completed, the solid is collected by centrifugation at 8000 rpm for 15 minutes, and washed and dried to obtain the amino-silica;

[0055] The particle size of the silicon dioxide is 80 nm;

[0056] The chitosan complex is prepared by adding 300 g of a 3.0 wt% acetic acid solution and 10.0 g of chitosan to a reaction container, stirring evenly, adding 1.7 g of betaine and 0.5 g of N,N'-dicyclohexylcarbodiimide, raising the temperature to 50° C., stirring for 2.0 h, then adding 20 g of a mixed acid solution, controlling the pH to 5.0, raising the temperature to 65° C. at a rate of 0.4° C. / min, and stirring at this temperature for 4.5 h. After the stirring is completed, adding 40 mL of isopropanol and 3.6 g of 3-chloro-2-hydroxypropyltrimethylammonium chloride, adding 10 wt% sodium hydroxide solution to adjust the pH to 8.7, raising the temperature to 68° C. at a rate of 0.2° C. / min, stirring at this temperature for 6.0 h under a nitrogen atmosphere, filtering and washing, and freeze-drying at -26° C. for 10 h and then at -40° C. for 12 h, and naturally returning to room temperature to obtain the chitosan complex.

[0057] The mixed acid solution is a mixture of acetic acid solution, phosphoric acid, and phytic acid, wherein 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%;

[0058] The mixing step comprises adding 5.0 g of the chitosan complex to 200 g of a 3.0 wt% acetic acid solution, stirring evenly, adding 5.0 g of amino-silica, and performing ultrasonic treatment for 30 min, with an ultrasonic power of 110 W and an ultrasonic frequency of 32 kHz. After the ultrasonic treatment, the mixture is centrifuged and washed, freeze-dried at -36°C for 24 h, and naturally returned to room temperature to obtain a multifunctional composite.

[0059] (2) Cultivation

[0060] The seed culture medium was inoculated into the fermentation medium and cultured at 30°C with an inoculation size of 10%. During the culture process, sucrose was added at a rate of 30 g / L when the residual sugar was less than 10 g / L, and the addition was repeated twice. When the pH was lower than 5.5, 10 wt% sodium hydroxide solution was added to maintain the pH at 5.7. When the fermentation reached the 35th hour, a multifunctional composite agent was added at a rate of 0.25 g / L. Samples were taken every hour to detect the fermentation progress. When the residual sugar content was less than 5.0 g / L and the curdlan content no longer increased, the fermentation was terminated to obtain the curdlan fermentation liquid.

[0061] 3. Post-processing

[0062] To 15 mL of curdlan gum fermentation broth, 70 mL of 3.0 wt% sodium hydroxide solution was added, the mixture was stirred for 15 min, allowed to stand for 30 min, and then centrifuged at 8500 rpm for 25 min. The supernatant was taken and 10.0 wt% hydrochloric acid solution was added to adjust the pH to 7.0. After filtering and washing, 3 times the mass of anhydrous ethanol was added, the mixture was filtered and washed again, and then dried. The mixture was crushed and sieved to obtain curdlan gum.

[0063] Curdlan gum was prepared by the production method of Example 1. The fermentation time was 62.4 h, the yield of curdlan gum was 51.7 g / L, and the 2.0% gel strength of the obtained curdlan gum was 727 g / cm 2 , the ash content is 1.12%.

[0064] Example 2

[0065] 1. Seed Culture

[0066] The radiobacterium was placed in a seed culture medium and cultured at 32°C. After the 6th hour of seed culture, the dissolved oxygen was controlled to 48%, and the seed culture solution was obtained when the OD value of the seed solution was greater than 10.

[0067] The radiobacterium was purchased;

[0068] The seed culture medium includes 22 g / L glucose, 11 g / L yeast extract powder, 2.2 g / L dipotassium hydrogen phosphate, 1.1 g / L magnesium sulfate, 3.2 g / L diammonium hydrogen phosphate, and 0.38 g / L α-ketoglutaric acid, has a pH of 7.2, and is sterilized at 122° C. for 28 minutes.

[0069] 2. Fermentation Culture

[0070] (1) Consecutive elimination

[0071] Add sucrose and a multifunctional compound to a No. 1 batching tank, and incubate at 126° C. for 13 minutes. Then, add magnesium sulfate and an inorganic salt, and incubate at 126° C. for 13 minutes. Add yeast extract powder, diammonium hydrogen phosphate, potassium dihydrogen phosphate, and a defoaming agent to a No. 2 batching tank, and incubate at 126° C. for 13 minutes. Mix the contents of the No. 1 and No. 2 batching tanks and adjust the volume to obtain a fermentation medium.

[0072] The fermentation medium comprises 52 g / L sucrose, 5.2 g / L yeast extract powder, 4.3 g / L diammonium hydrogen phosphate, 1.6 g / L potassium dihydrogen phosphate, 1.1 g / L magnesium sulfate, 0.6 g / L multifunctional compound, 0.12 g / L defoaming agent, 43 mg / L inorganic salt, and has a pH of 6.8;

[0073] The inorganic salts include cobalt chloride, zinc chloride, and calcium chloride in a mass ratio of 2:1:1;

[0074] The defoaming agent is polyoxypropylene glycerol ether;

[0075] The preparation method of the multifunctional composite agent includes the steps of preparing amino-silica, preparing chitosan composite and mixing, which are specifically as follows:

[0076] The step of preparing the amino-silica comprises: adding 12 g of silica to 100 g of a 52 wt% ethanol solution, stirring evenly, adding 1.6 g of γ-aminopropyltriethoxysilane, raising the temperature to 65° C., and stirring at this temperature for 3.8 hours. After the stirring is completed, lowering the temperature to 54° C., adding 1.3 g of L-lysine, and then adding ammonia water to adjust the pH to 8.6. The mixture is stirred at this temperature at 230 rpm for 2.6 hours. After the stirring is completed, the solid is collected by centrifugation at 8200 rpm for 12 minutes, and washed and dried to obtain the amino-silica;

[0077] The particle size of the silicon dioxide is 100 nm;

[0078] The chitosan complex is prepared by adding 320 g of a 3.5 wt% acetic acid solution and 10.4 g of chitosan to a reaction container, stirring uniformly, adding 1.8 g of betaine and 0.6 g of N,N'-dicyclohexylcarbodiimide, raising the temperature to 52° C., stirring and reacting for 2.3 hours, then adding 22 g of a mixed acid solution, controlling the pH to 5.2, raising the temperature to 66° C. at a rate of 0.5° C. / min, and stirring at this temperature for 4.8 hours. After the stirring is completed, 42 mL of isopropanol and 3.8 g of 3-chloro-2-hydroxypropyltrimethylammonium chloride are added, and a 12 wt% sodium hydroxide solution is added to adjust the pH to 8.8. The temperature is raised to 70° C. at a rate of 0.3° C. / min, and stirring at this temperature for 6.2 hours under a nitrogen atmosphere. After the reaction is completed, filtering and washing, freeze-drying at -24° C. for 11 hours, freeze-drying at -38° C. for 14 hours, and naturally returning to room temperature to obtain the chitosan complex.

[0079] The mixed acid solution is a mixture of acetic acid solution, phosphoric acid, and phytic acid, wherein 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%;

[0080] The mixing step comprises adding 5.2 g of chitosan complex to 200 g of 3.2 wt% acetic acid solution, stirring evenly, adding 5.1 g of amino-silica, and performing ultrasonic treatment for 34 min, with an ultrasonic power of 120 W and an ultrasonic frequency of 34 kHz. After the ultrasonic treatment, the mixture is centrifuged and washed, freeze-dried at -34°C for 25 h, and naturally restored to room temperature to obtain a multifunctional composite agent.

[0081] (2) Cultivation

[0082] Seed culture medium was inoculated into the fermentation medium and cultured at 32°C with an inoculation size of 12%. During the culture process, sucrose was added when the residual sugar was less than 10 g / L, with an addition amount of 33 g / L, for a total of 2 additions. When the pH was lower than 5.5, 10 wt% sodium hydroxide solution was added to maintain the pH at 5.8. When the fermentation reached the 35th hour, a multifunctional composite agent was added in an amount of 0.28 g / L. Samples were taken every 1 hour to detect the fermentation progress. When the residual sugar content was less than 5.0 g / L and the curdlan content no longer increased, the fermentation was terminated to obtain curdlan fermentation liquid.

[0083] 3. Post-processing

[0084] To 16 mL of curdlan gum fermentation broth, 70 mL of 3.2 wt% sodium hydroxide solution was added, the mixture was stirred for 13 min, allowed to stand for 35 min, and then centrifuged at 9000 rpm for 20 min. The supernatant was taken, and 12.0 wt% hydrochloric acid solution was added to adjust the pH to 7.2. After filtering and washing, 3 times the mass of anhydrous ethanol was added, the mixture was filtered and washed again, and then dried. The mixture was crushed and sieved to obtain curdlan gum.

[0085] Curdlan gum was prepared by the production method of Example 2. The fermentation time was 62.8 h, the yield of curdlan gum was 51.3 g / L, and the 2.0% gel strength of the obtained curdlan gum was 723 g / cm 2 , the ash content is 1.15%.

[0086] Example 3

[0087] 1. Seed Culture

[0088] The radiobacterium was placed in a seed culture medium and cultured at 29°C. After the 6th hour of seed culture, the dissolved oxygen was controlled to 46%. The seed culture solution was obtained when the OD value of the seed solution was greater than 10.

[0089] The radiobacterium was purchased;

[0090] The seed culture medium includes 18 g / L glucose, 9 g / L yeast extract powder, 1.7 g / L dipotassium hydrogen phosphate, 0.8 g / L magnesium sulfate, 2.7 g / L diammonium hydrogen phosphate, and 0.33 g / L α-ketoglutaric acid, has a pH of 6.8, and is sterilized at 120° C. for 32 minutes.

[0091] 2. Fermentation Culture

[0092] (1) Consecutive elimination

[0093] Add sucrose and a multifunctional compound to a No. 1 batching tank, and sterilize at 122° C. for 17 minutes. Then add magnesium sulfate and an inorganic salt, and sterilize at 122° C. for 17 minutes. Add yeast extract powder, diammonium hydrogen phosphate, potassium dihydrogen phosphate, and a defoaming agent to a No. 2 batching tank, and sterilize at 122° C. for 17 minutes. Mix the No. 1 batching tank and the No. 2 batching tank, and adjust the volume to obtain a fermentation medium.

[0094] The fermentation medium comprises 48 g / L sucrose, 4.7 g / L yeast extract powder, 3.8 g / L diammonium hydrogen phosphate, 1.4 g / L potassium dihydrogen phosphate, 0.8 g / L magnesium sulfate, 0.4 g / L multifunctional compound, 0.08 g / L defoaming agent, 36 mg / L inorganic salt, and has a pH of 6.5;

[0095] The inorganic salts include cobalt chloride, zinc chloride, and calcium chloride in a mass ratio of 1:2:1;

[0096] The defoaming agent is polyoxypropylene glycerol ether;

[0097] The preparation method of the multifunctional composite agent includes the steps of preparing amino-silica, preparing chitosan composite and mixing, which are specifically as follows:

[0098] The step of preparing the amino-silica comprises: adding 8 g of silica to 100 g of a 47 wt% ethanol solution, stirring evenly, adding 1.4 g of γ-aminopropyltriethoxysilane, raising the temperature to 60° C., and stirring at this temperature for 3.2 hours. After the stirring is completed, lowering the temperature to 50° C., adding 1.1 g of L-lysine, and then adding ammonia water to adjust the pH to 8.4. The mixture is stirred at this temperature at 210 rpm for 2.4 hours. After the stirring is completed, the solid is collected by centrifugation at 7800 rpm for 18 minutes, and washed and dried to obtain the amino-silica;

[0099] The particle size of the silicon dioxide is 70 nm;

[0100] The chitosan complex is prepared by adding 280 g of a 3.2 wt% acetic acid solution and 9.5 g of chitosan to a reaction container, stirring evenly, adding 1.6 g of betaine and 0.4 g of N,N'-dicyclohexylcarbodiimide, raising the temperature to 47°C, stirring and reacting for 1.8 hours, then adding 17 g of a mixed acid solution, controlling the pH to 4.8, raising the temperature to 62°C at a rate of 0.3°C / min, and stirring at this temperature for 4.3 hours. After the stirring is completed, 38 mL of isopropanol and 3.4 g of 3-chloro-2-hydroxypropyltrimethylammonium chloride are added, and an 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 stirring at this temperature for 5.8 hours under a nitrogen atmosphere. After the reaction is completed, filtering and washing, freeze-drying at -28°C for 9 hours, freeze-drying at -42°C for 10 hours, and naturally returning to room temperature to obtain the chitosan complex.

[0101] The mixed acid solution is a mixture of acetic acid solution, phosphoric acid, and phytic acid, wherein 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%;

[0102] The mixing step comprises adding 4.8 g of chitosan complex to 200 g of 2.8 wt% acetic acid solution, stirring evenly, adding 4.7 g of amino-silica, and performing ultrasonic treatment for 28 min, an ultrasonic power of 100 W, and an ultrasonic frequency of 30 kHz. After the ultrasonic treatment, the mixture is centrifuged and washed, freeze-dried at -38°C for 23 h, and naturally restored to room temperature to obtain a multifunctional composite agent.

[0103] (2) Cultivation

[0104] Seed culture medium was inoculated into the fermentation medium and cultured at 28°C with an inoculation size of 8%. During the culture process, sucrose was added when the residual sugar was less than 10 g / L, with an addition amount of 28 g / L, for a total of 2 additions. When the pH was lower than 5.5, 10 wt% sodium hydroxide solution was added to maintain the pH at 5.5. When the fermentation reached 35 hours, a multifunctional composite agent was added in an amount of 0.23 g / L. Samples were taken every hour to detect the fermentation progress. When the residual sugar content was less than 5.0 g / L and the curdlan content no longer increased, the fermentation was terminated to obtain curdlan fermentation liquid.

[0105] 3. Post-processing

[0106] To 14 mL of curdlan gum fermentation broth, 70 mL of 2.8 wt% sodium hydroxide solution was added, the mixture was stirred for 18 min, allowed to stand for 27 min, and then centrifuged at 8000 rpm for 30 min. The supernatant was taken, and 8.0 wt% hydrochloric acid solution was added to adjust the pH to 6.8. After filtering and washing, 3 times the mass of anhydrous ethanol was added, the mixture was filtered and washed again, and then dried. The mixture was crushed and sieved to obtain curdlan gum.

[0107] Curdlan gum was prepared by the production method of Example 3. The fermentation time was 63.3 h, the yield of curdlan gum was 51.1 g / L, and the 2.0% gel strength of the obtained curdlan gum was 720 g / cm 2 , the ash content is 1.16%.

[0108] The present invention adopts radiobacterium as the fermentation strain. In the seed culture step, different dissolved oxygen is controlled in different seed culture stages, and an α-ketoglutaric acid component is added to the seed culture medium, which can promote the rapid proliferation of the bacteria, provide high-activity seeds for the subsequent fermentation process, shorten the fermentation cycle, and improve the fermentation efficiency. In the fermentation step, sucrose is first continuously digested, and then inorganic salts and magnesium sulfate and other components are added for continuous digestion, thereby avoiding the phenomenon that impurity components in sucrose directly contact and react with metal ions during the continuous digestion process, thereby effectively ensuring the stability of the fermentation process and avoiding the occurrence of the tank inversion phenomenon. The present invention introduces a multifunctional composite agent in the fermentation step, which is composited by silicon dioxide and chitosan, specifically, firstly amino-treated silicon dioxide with an aminosilane coupling agent and an amino acid component, on the one hand, improving the dispersibility and compatibility of silicon dioxide so that it can be stably present in the fermentation liquid, and on the other hand, The invention can effectively adsorb impurities such as pigments and polyphenols in sucrose, promote the formation of a gel network, and thus improve the gel strength. The chitosan composite first introduces betaine on the surface of chitosan, which can improve the tolerance of the cell membrane to high osmotic pressure, and then adds a mixed acid solution and 3-chloro-2-hydroxypropyltrimethylammonium chloride, which can effectively chelate metal ions, thereby avoiding the reaction of metal ions with sucrose at high temperature, and can also effectively adsorb impurities in sucrose and inhibitors in the fermentation process. In the later stage of fermentation, the quaternary ammonium group can be electrostatically compounded with the curdlan, thereby reducing the viscosity of the fermentation liquid, improving the mass transfer efficiency, and better promoting the fermentation effect. The invention adds a multifunctional composite agent to the fermentation medium and at the 35th hour of fermentation, which can stabilize the fermentation process, shorten the fermentation cycle, increase the fermentation yield, improve the quality of the curdlan, effectively improve the gel strength, reduce the ash content, and avoid the phenomenon of tank collapse during the fermentation process.

[0109] Comparative Example 1-1

[0110] Based on Example 1, the following changes were made:

[0111] 1. During the seed culture step, replace an equal amount of α-ketoglutarate in the seed culture medium with glucose;

[0112] 2. In the fermentation culture step, (1) the inorganic salt components in the fermentation medium are replaced with calcium chloride in equal amounts;

[0113] (2) Omitting the chitosan complex preparation and mixing steps in the preparation method of the multifunctional composite agent and replacing the multifunctional composite agent with an equal amount of amino-silica;

[0114] The rest of the operations are the same.

[0115] Comparative Example 1-1 omits the α-ketoglutaric acid component in the seed culture step, which will lead to slower bacterial proliferation and affect the metabolic activity of the fermentation process. It also only uses calcium chloride as an inorganic salt, which will lead to slower bacterial growth, reduced enzyme activity, and inhibition of the synthesis rate of curdlan. Comparative Example 1-1 also only uses amino-silica as a multifunctional composite agent, which lacks the chelating and adsorption properties of the chitosan complex and cannot effectively regulate the ion concentration and metabolic waste in the culture medium. The accumulation efficiency of curdlan is reduced in the later stage of fermentation, ultimately extending the fermentation time, reducing production efficiency, and affecting product quality.

[0116] Curdlan was prepared by the fermentation production method of Comparative Example 1-1. The fermentation time was 70.3 h, the yield of curdlan was 43.2 g / L, and the curdlan had a 2.0% gel strength of 664 g / cm 2 , the ash content is 1.34%.

[0117] Comparative Example 1-2

[0118] On the basis of Example 1, the changes are as follows:

[0119] In the fermentation culture step, the steps of preparing amino-treated silicon dioxide and preparing chitosan complex are omitted, and silicon dioxide and chitosan without any treatment are directly mixed;

[0120] The multifunctional composite agent is prepared by adding 5.0 g of chitosan to 200 g of a 3.0 wt% acetic acid solution, stirring evenly, adding 5.0 g of silicon dioxide, and performing ultrasonic treatment for 30 minutes, an ultrasonic power of 110 W, and an ultrasonic frequency of 32 kHz. After the ultrasonic treatment, the mixture is centrifuged and washed, freeze-dried at -36°C for 24 hours, and naturally returned to room temperature to obtain the multifunctional composite agent.

[0121] The particle size of the silicon dioxide is 80 nm;

[0122] (2) Omit the operation step of "adding the multifunctional compound at 0.25 g / L at the 35th hour of fermentation";

[0123] The rest of the operations are the same.

[0124] Comparative Example 1-2 directly uses untreated silica and chitosan as functional composite agents. The interaction between the two is weak, and the agglomeration of silica affects the growth of the bacteria. Chitosan cannot be evenly dispersed in the fermentation liquid and cannot effectively exert its adsorption properties. The multifunctional composite agent of Comparative Example 1-2 has a weak promoting effect on bacterial metabolism and curdlan synthesis, which greatly affects the fermentation efficiency and product quality.

[0125] Curdlan was prepared by the fermentation production method of Comparative Example 1-2. The fermentation time was 73.5 h, the yield of curdlan was 41.9 g / L, and the curdlan had a 2.0% gel strength of 641 g / cm 2 , the ash content is 1.48%.

[0126] Unless otherwise specified, all ratios and percentages described in the present invention are by mass.

[0127] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for producing curdlan by fermentation, characterized in that: Including seed culture, fermentation culture and post-processing steps; The seed culture step comprises culturing Agrobacterium radiobacterium in a seed culture medium; The seed culture medium comprises 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, and 0.33-0.38 g / L α-ketoglutaric acid, has a pH of 6.8-7.2, and is sterilized at 120-122° C. for 28-32 minutes. The fermentation and culturing step includes continuous digestion and culturing steps; The culturing step comprises: inoculating the seed culture solution into the fermentation medium and culturing at 28-32° C., adding sucrose when the residual sugar is less than 10 g / L, the added amount being 28-33 g / L, and adding sucrose twice in total; and adding the multifunctional compound at 0.23-0.28 g / L when the fermentation reaches the 35th hour; The fermentation medium comprises 48-52 g / L sucrose, 4.7-5.2 g / L yeast extract powder, 3.8-4.3 g / L diammonium hydrogen phosphate, 1.4-1.6 g / L potassium dihydrogen phosphate, 0.8-1.1 g / L magnesium sulfate, 0.4-0.6 g / L multifunctional compound, 0.08-0.12 g / L defoaming agent, 36-43 mg / L inorganic salt, and has a pH of 6.5-6.8; The preparation method of the multifunctional composite agent includes the steps of preparing amino-silica, preparing chitosan composite and mixing; The step of preparing the amino-silica comprises: adding silica to an ethanol solution, stirring evenly, adding γ-aminopropyltriethoxysilane, raising the temperature to 60-65° C., and stirring at this temperature for 3.2-3.8 hours. After the stirring is completed, lowering the temperature to 50-54° C., adding L-lysine, and then adding ammonia water to adjust the pH to 8.4-8.

6. The solution is stirred at this temperature at 210-230 rpm for 2.4-2.6 hours. After the stirring is completed, the solid is collected by centrifugation at 7800-8200 rpm for 12-18 minutes, and washed and dried to obtain the amino-silica; The chitosan complex preparation step comprises the following steps: adding acetic acid solution and chitosan to a reaction container, stirring evenly, adding betaine and N,N'-dicyclohexylcarbodiimide, raising the temperature to 47-52°C, stirring and reacting for 1.8-2.3 hours, then adding a mixed acid solution, controlling the pH to 4.8-5.2, raising the temperature to 62-66°C at a rate of 0.3-0.5°C / min, maintaining the temperature and stirring for 4.3-4.8 hours, and after the stirring is completed, adding isopropyl alcohol and 3-chloro- 2-hydroxypropyltrimethylammonium chloride, adding 8-12wt% sodium hydroxide solution to adjust the pH to 8.5-8.8, raising the temperature to 66-70°C at a rate of 0.1-0.3°C / min, and stirring under a nitrogen atmosphere for 5.8-6.2 hours. After the reaction is completed, filtering and washing, freeze-drying at -28 to -24°C for 9-11 hours, freeze-drying at -42 to -38°C for 10-14 hours, and naturally returning to room temperature to obtain a chitosan complex; The mixing step comprises adding the chitosan complex to the acetic acid solution, stirring evenly, adding amino-silica, and performing ultrasonic treatment, wherein the ultrasonic treatment time is 28-34 minutes, the ultrasonic power is 100-120W, and the ultrasonic frequency is 30-34kHz. After the ultrasonic treatment, the mixture is centrifuged and washed, and then freeze-dried at -38 to -34°C for 23-25 ​​hours and naturally restored to room temperature to obtain the multifunctional complex.

2. The method for producing curdlan by fermentation according to claim 1, characterized in that: The seed culture step comprises culturing Agrobacterium radiobacterium in a seed culture medium at 29-32° C., controlling the dissolved oxygen to 46-48% after the 6th hour of seed culture, and culturing until the OD value of the seed liquid is greater than 10 to obtain a seed culture liquid.

3. The method for producing curdlan by fermentation according to claim 1, characterized in that: In the fermentation and culturing step, the continuous digestion step comprises the following steps: adding sucrose and a multifunctional compound to a No. 1 batching tank, continuously digesting at 122-126° C. for 13-17 minutes, then adding magnesium sulfate and an inorganic salt, and continuously digesting at 122-126° C. for 13-17 minutes; adding yeast extract powder, diammonium hydrogen phosphate, potassium dihydrogen phosphate, and a defoaming agent to a No. 2 batching tank, continuously digesting at 122-126° C. for 13-17 minutes, mixing the No. 1 batching tank and the No. 2 batching tank, and fixing the volume to obtain a fermentation medium.

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

5. The method for producing curdlan by fermentation according to claim 1, wherein: In the step of preparing the amino-silica, the particle size of the silica is 70-100 nm; The mass ratio of the silicon dioxide, 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. The method for producing curdlan by fermentation according to claim 1, characterized in that: In the step of preparing the chitosan complex, the mass volume ratio of the acetic acid solution, chitosan, betaine, N,N'-dicyclohexylcarbodiimide, mixed acid solution, isopropyl alcohol 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 3.0-3.5wt% acetic acid solution, 78-82wt% phosphoric acid solution and phytic acid, and the mass ratio of the 3.0-3.5wt% acetic acid solution, 78-82wt% phosphoric acid solution and phytic acid is 100:1.0-1.4:1.6-2.

0.

7. The method for producing curdlan by fermentation according to claim 1, characterized in that: In the mixing step, the mass ratio of the chitosan complex, the acetic acid solution, and the amino-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. The method for producing curdlan by fermentation according to claim 1, characterized in that: The fermentation and culturing step comprises the following steps: in the culturing step, when the pH is lower than 5.5, adding 10 wt % sodium hydroxide solution to maintain the pH at 5.5-5.8, sampling every hour to detect the fermentation progress, and ending the fermentation when the residual sugar content is less than 5.0 g / L and the curdlan content no longer increases, thereby obtaining a curdlan fermentation liquid.

9. The method for producing curdlan by fermentation according to claim 8, characterized in that: The post-treatment step comprises adding 70 mL of 2.8-3.2 wt% sodium hydroxide solution to 14-16 mL of curdlan gum fermentation broth, stirring for 13-18 minutes, standing for 27-35 minutes, and then centrifuging at 8000-9000 rpm for 20-30 minutes. The supernatant is collected, and an 8.0-12.0 wt% hydrochloric acid solution is added to adjust the pH to 6.8-7.

2. After filtering and washing, three times the mass of anhydrous ethanol is added, filtering and washing again, drying, and crushing and sieving to obtain curdlan gum.