Method for extracting pea starch by immobilized cell method

Abstract

The invention provides a method for extracting pea starch by using an immobilized cell method, and relates to the technical field of pea starch extraction, and the method comprises the following steps: peeling pea seeds, grinding to obtain pea powder, adding process water into the pea powder, and uniformly stirring to obtain pea pulp; filtering bean dregs in the pea pulp to obtain filtered soybean milk, and homogenizing the filtered soybean milk to obtain pea powder pulp; adding the compound strain gel beads into the pea powder slurry for fermentation, recovering the compound strain gel beads and a supernatant to obtain a precipitate, and drying the precipitate to obtain pea starch; the compound strain gel bead comprises an immobilization carrier and a compound strain wrapped in the immobilization carrier, the immobilization carrier is biocompatible polysaccharide gel, and the compound strain is composed of lactobacillus and streptococcus thermophilus. The bottleneck problems of waste of strains, insufficient purity, poor environmental protection property and the like in the prior art are solved, and a high-benefit and low-pollution extraction scheme is provided for industrial production of pea starch.

Description

Technical Field

[0001] The present application relates to the technical field of pea starch extraction, and in particular to a method for extracting pea starch using an immobilized cell method. Background Art

[0002] Peas are rich in high-quality protein and carotene, which can improve the body's disease resistance and recovery ability. Pea starch is a high-quality starch. Pea starch products made by heat processing have a lower GI value than other starch products. They can enhance the body's immune function, prevent the synthesis of high blood sugar in the human body, and reduce the incidence of chronic diseases in the human body.

[0003] In the prior art, for example, the invention patent with publication number CN115322263A proposes a high-quality pea starch extraction method based on fermentation of composite strains, which overcomes the problems of high water consumption, high cost, and low extraction rate of traditional acid pulp method and water extraction method for extracting starch, but still has the following defects: First, the direct addition of free strains (Lactobacillus, Streptococcus thermophilus and maltodextrin) results in the strains being unable to be recycled, and new strains need to be added again for each batch, which significantly increases the production cost. Second, the residual bacteria mixed into the starch requires an additional separation step, which reduces the extraction efficiency; after fermentation, the bacteria are mixed with starch, and centrifugal separation is difficult to completely remove the residual bacteria, resulting in reduced starch purity; third, maltodextrin, as a strain compound auxiliary material, has a large molecular weight and may remain in the starch, reducing the product transmittance and affecting the starch processing performance. Summary of the invention

[0004] In view of the above defects or deficiencies in the prior art, the present application aims to provide a method for extracting pea starch using an immobilized cell method, which solves the problems of strain waste and insufficient purity; The method comprises the following steps: Peeling pea seeds and grinding them to obtain pea flour, adding process water to the pea flour, and stirring them evenly to obtain pea milk; Filtering the bean dregs in the pea milk to obtain filtered soy milk, and homogenizing the filtered soy milk to obtain pea powder slurry; Obtaining composite bacterial strain gel beads, adding the composite bacterial strain gel beads to the pea powder slurry for fermentation, and stirring evenly to obtain a fermentation liquid; Recovering the composite bacterial strain gel beads in the fermentation broth and recovering the supernatant to obtain a precipitate; the supernatant is used to prepare the process water for the next extraction; drying the precipitate to obtain pea starch; The composite bacterial strain gel beads include an immobilized carrier and a composite bacterial strain encapsulated in the immobilized carrier, the immobilized carrier is a biocompatible polysaccharide gel, and the composite bacterial strain consists of lactobacillus and thermophilic streptococcus.

[0005] According to the technical solution provided in the present application, the types of the composite bacteria gel beads include newly prepared composite bacteria gel beads with zero cumulative usage times and recycled composite bacteria gel beads with at least one cumulative usage times; The preparation of the newly prepared composite bacterial strain gel beads comprises the following steps: Lactobacillus powder and thermophilic Streptococcus powder are mixed in a mass ratio of 40%-48%:40%-48% to obtain a composite powder, and the other compounding material is maltodextrin; Prepare 1.5% sodium alginate solution, heat and stir to dissolve, and cool to room temperature; Adding the composite bacterial powder to the sodium alginate solution, wherein the mass ratio of the composite bacterial powder to the sodium alginate is 1:0.5-1:5, stirring until uniformly suspended, to obtain a mixed solution; The mixed solution was dripped dropwise into a 2%-5% calcium chloride solution and allowed to stand at room temperature for cross-linking to form initial gel beads; The initial gel beads were rinsed with sterile water for multiple times to remove residual calcium ions on the surface, thereby obtaining newly prepared composite bacterial strain gel beads.

[0006] According to the technical solution provided by this application, the method also includes the following steps: Each time the composite bacterial strain gel beads are used for fermentation, the lactic acid concentration in the fermentation broth is monitored in real time to calculate the current lactic acid production rate during the fermentation process; Recording the cumulative number of times the composite bacterial strain gel beads are used and the corresponding current lactic acid production rate; With the accumulated number of times used as the abscissa and the current lactic acid production rate as the ordinate, a lactic acid production rate attenuation curve is generated by fitting; Based on the lactic acid production rate attenuation curve and the cumulative number of uses, a gel bead label is generated, and the gel bead label is used to dynamically adjust the fermentation parameters in the subsequent fermentation process; the fermentation parameters include stirring frequency, fermentation temperature and fermentation time.

[0007] According to the technical solution provided by the present application, the step of generating gel bead labels based on the lactic acid production rate attenuation curve and the cumulative number of uses includes the following steps: Determining the activity level according to the percentage of the current lactic acid production rate to the initial lactic acid production rate when the composite bacterial strain gel beads are used for the first time; Determining a loss level according to the accumulated number of uses; The combination of the activity level and the depletion level was used as the gel bead label.

[0008] According to the technical solution provided in this application, the activity level includes high activity, medium activity, and low activity, and the loss level includes high loss, medium loss, and low loss; After the composite bacterial strain gel beads in the fermentation broth are recovered, the following steps are also included: If the activity level of the composite bacteria gel beads is low activity and the loss level is high loss, the composite bacteria gel beads are forced to be scrapped or regenerated and then downgraded for use.

[0009] According to the technical solution provided in the present application, the step of adding the composite bacterial strain gel beads to the pea powder slurry for fermentation comprises the following steps: If the activity level of the composite bacteria gel beads is medium activity and the loss level is medium loss, the composite bacteria gel beads are added to the pea powder slurry, and carbon source or coenzyme factor is dynamically supplemented according to the real-time lactic acid production rate.

[0010] According to the technical solution provided in this application, the method of obtaining composite bacterial strain gel beads includes the following steps: Obtaining the target process requirements for this extraction, wherein the target process requirements include the target starch purity of the starch to be extracted; If the target starch purity is greater than the first preset purity, the newly prepared composite bacterial strain gel beads are selected.

[0011] According to the technical solution provided in this application, after obtaining the target process requirements for this extraction, the following steps are also included: If the target starch purity is less than or equal to the first preset purity, the corresponding recycled composite bacterial strain gel beads are selected according to the target starch purity.

[0012] According to the technical solution provided in the present application, the method of selecting the corresponding recycled composite bacterial strain gel beads according to the target starch purity includes the following steps: If the target starch purity is less than the second preset purity, the recycled composite bacterial strain gel beads with a gel bead label of low activity-high loss or a cumulative use number of the first preset number are selected; if the target starch purity is greater than the second preset purity and less than the first preset purity, the recycled composite bacterial strain gel beads with a gel bead label of medium activity-medium loss and a cumulative use number of less than the second preset number are selected; Among them, the second preset purity is smaller than the first preset purity, and the first preset number of times is larger than the second preset number of times.

[0013] According to the technical solution provided by the present application, the method further includes regenerating the composite bacterial strain gel beads whose activity level is low and whose loss level is high; The regeneration of the composite bacterial strain gel beads having a low activity level and a high loss level comprises the following steps: The composite bacteria gel beads are immersed in a sodium alginate regeneration solution, and subjected to high-pressure homogenization treatment to repair surface cracks of the composite bacteria gel beads to obtain repaired gel beads; The repaired gel beads are transferred to an activation solution, and cultured under shaking to restore the metabolic activity of the bacteria, thereby obtaining activated gel beads; the activation solution is a phosphate buffer solution containing glucose, yeast extract and magnesium sulfate; The lactic acid generation rate of the activated gel beads is detected. If the lactic acid generation rate reaches the first proportional interval of the initial lactic acid generation rate, the gel bead labels of the activated gel beads are marked as regeneration-mid-activity-mid-loss, and the cumulative number of uses is limited.

[0014] Compared with the prior art, the beneficial effects of the present application are as follows: the present application encapsulates the composite bacteria (Lactobacillus and Streptococcus thermophilus) in biocompatible polysaccharide gel beads, and recovers the gel beads by physical filtration after fermentation, so as to achieve repeated recycling and reuse, reduce the cost of bacteria, and avoid mixing of bacteria into starch; at the same time, the homogenization treatment makes the pea powder slurry particles uniform, and combined with the size control of the gel beads, the physical isolation of the bacteria and the substrate is achieved, the residual bacteria amount is reduced, and the starch purity is improved; in addition, the gel carrier is used to replace maltodextrin, and the auxiliary ingredients (such as sodium alginate) are removed by washing with sterile water, which can improve the starch transmittance and meet the needs of high-end food processing; finally, the supernatant is reused as process water for preparing pea slurry, which further reduces the water consumption in the extraction process. Combining the above aspects, the bottleneck problems of bacteria waste, insufficient purity and poor environmental protection in the prior art are solved, and a high-efficiency and low-pollution extraction scheme is provided for the industrial production of pea starch. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A flow chart of the steps of the method for extracting pea starch using the immobilized cell method provided in this application. DETAILED DESCRIPTION

[0016] The present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It is also necessary to explain that, for ease of description, only the parts related to the invention are shown in the accompanying drawings.

[0017] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0018] Example 1 As mentioned in the background technology, in view of the problems in the prior art, the present application proposes a method for extracting pea starch using an immobilized cell method, such as Figure 1 As shown, the following steps are included: S1, peeling pea seeds and grinding them to obtain pea flour, adding process water to the pea flour, and stirring to obtain pea milk; S2, filtering the bean dregs in the pea milk to obtain filtered soy milk, and homogenizing the filtered soy milk to obtain pea powder slurry; S3, obtaining composite bacterial strain gel beads, adding the composite bacterial strain gel beads to the pea powder slurry for fermentation, and stirring evenly to obtain a fermentation liquid; S4, recovering the composite bacterial strain gel beads in the fermentation broth, and recovering the supernatant to obtain a precipitate; the supernatant is used to prepare the process water for the next extraction; S5, drying the precipitate to obtain pea starch; The composite bacterial strain gel beads include an immobilized carrier and a composite bacterial strain encapsulated in the immobilized carrier, the immobilized carrier is a biocompatible polysaccharide gel, and the composite bacterial strain consists of lactobacillus and thermophilic streptococcus.

[0019] Furthermore, the types of the composite bacteria gel beads include newly prepared composite bacteria gel beads with zero cumulative usage times and recycled composite bacteria gel beads with at least one cumulative usage times; The preparation of the newly prepared composite bacterial strain gel beads comprises the following steps: Lactobacillus powder and thermophilic Streptococcus powder are mixed in a mass ratio of 40%-48%:40%-48% to obtain a composite powder, and the other compounding material is maltodextrin; Prepare 1.5% sodium alginate solution, heat and stir to dissolve, and cool to room temperature; Adding the composite bacterial powder to the sodium alginate solution, wherein the mass ratio of the composite bacterial powder to the sodium alginate is 1:0.5-1:5, stirring until uniformly suspended, to obtain a mixed solution; The mixed solution was dripped dropwise into a 2%-5% calcium chloride solution and allowed to stand at room temperature for cross-linking to form initial gel beads; The initial gel beads were rinsed with sterile water for multiple times to remove residual calcium ions on the surface, thereby obtaining newly prepared composite bacterial strain gel beads.

[0020] Specifically, the control experiment data of the optimization of the preparation parameters of the newly prepared composite bacterial strain gel beads are shown in Table 1: Table 1

[0021] As shown in Table 1, the survival rate (92%) and initial lactic acid production rate (1.82mmol / (L·h)) of experimental group B (44%:44%) were higher than those of other groups (A:88%, C:89%), indicating that equal proportion mixing (±44%) was the optimal ratio. Too high a proportion of bacterial powder (experimental group C, 48%:48%) led to competitive inhibition of bacterial species and decreased metabolic rate (1.78mmol / (L·h)). Experimental group B (1.5%) achieved a balance between mechanical strength (0.28 N / bead), survival rate (92%) and metabolic rate (1.82mmol / (L·h)). Too high a concentration (experimental group D, 2.0%) led to a decrease in porosity and limited bacterial activity (survival rate 85%, rate 1.65mmol / (L·h)). Too low a concentration (experimental group E, 1.0%) led to insufficient mechanical strength (0.20 N / bead) and easy to break. The following Examples 2-6 are all different implementations of Example 1; Example 2 (1) Peeling and grinding: accurately weigh 1000 kg of pea seeds, peel them with a peeling machine and grind them into powder to obtain 918 kg of pea flour; (2) Preparing pea milk: adding water to 918 kg of the prepared pea flour, with the ratio of pea flour to water being 1:4, and stirring evenly to obtain pea milk; (3) Preparation of composite bacterial strain gel beads for immobilized cells: the mass ratio of lactic acid bacteria powder to pea powder is 1:150, the bacterial powder contains: 50% Lactobacillus bulgaricus and 50% Streptococcus thermophilus. The lactic acid bacteria powder is added to 100 ml of 1.5% seaweed polysaccharide solution, and 200 ml of 2% calcium chloride solution is dropped into it to form gel beads.

[0022] (4) Remove the residue: Use gauze to filter out the bean dregs. Repeat 2-5 times to remove as much bean dregs as possible. (5) Fine grinding: Use a homogenizer to further process the pea flour slurry for 5 min, with a mesh size of 60.

[0023] (6) Fermentation: Add the composite bacterial strain gel beads to the slurry, stir evenly and then ferment. The fermentation temperature is 37°C and the fermentation time is 8 hours. First, stir for 1 hour and let it stand for 3 hours, then stir for 1 hour and let it stand for 1 hour until the fermentation is completed; (7) Separation: Filter the composite bacterial strain gel beads in the fermentation broth through a gauze filter to prepare for the next fermentation, remove the supernatant and centrifuge to collect the precipitate; (8) Drying: The precipitate was dried with hot air at 40°C to obtain 297.3 kg of pea starch.

[0024] Example 3 (1) Peeling and grinding: accurately weigh 1000 kg of pea seeds, peel them with a peeling machine and grind them into powder to obtain 918 kg of pea flour; (2) Preparing pea milk: adding water to 918 kg of pea flour, with the ratio of pea flour to water being 1:6, and stirring evenly to obtain pea milk; (3) Preparation of composite bacterial strain gel beads for immobilized cells: the mass ratio of lactic acid bacteria powder to pea powder is 1:150, the bacterial powder contains: 50% Lactobacillus bulgaricus and 50% Streptococcus thermophilus, the lactic acid bacteria powder is added to 100 ml of 1.5% seaweed polysaccharide solution, and 200 ml of 2% calcium chloride solution is dropped into it to form gel beads.

[0025] (4) Remove the residue: Use gauze to filter out the bean dregs. Repeat 2-5 times to remove as much bean dregs as possible. (5) Fine grinding: Use a homogenizer to further process the pea flour slurry for 5 minutes, mesh size 60.

[0026] (6) Fermentation: Add the composite bacterial strain gel beads to the slurry, stir evenly and then ferment. The fermentation temperature is 37°C and the fermentation time is 8 hours. First, stir for 1 hour and let it stand for 3 hours, then stir for 1 hour and let it stand for 1 hour until the fermentation is completed; (7) Separation: Filter the lactic acid bacteria gel beads in the fermentation broth through a gauze filter to prepare for the next fermentation, remove the supernatant and centrifuge to collect the precipitate; (8) Drying: The precipitate was dried with hot air at 40°C to obtain 277.3 kg of pea starch.

[0027] Example 4 (1) Peeling and grinding: accurately weigh 1000 kg of pea seeds, peel them with a peeling machine and grind them into powder to obtain 918 kg of pea flour; (2) Preparing pea milk: adding water to 918 kg of pea flour, with the ratio of pea flour to water being 1:6, and stirring evenly to obtain pea milk; (3) using the composite bacterial strain gel beads for immobilizing cells prepared in Example 2; (4) Remove the residue: Use gauze to filter out the bean dregs. Repeat 2-5 times to remove as much bean dregs as possible. (5) Fine grinding: Use a homogenizer to further process the pea flour slurry for 5 minutes, mesh size 60.

[0028] (6) Fermentation: Add the composite bacterial strain gel beads to the slurry, stir evenly and then ferment. The fermentation temperature is 37°C and the fermentation time is 8 hours. First, stir for 1 hour and let it stand for 3 hours, then stir for 1 hour and let it stand for 1 hour until the fermentation is completed; (7) Separation: Filter the composite bacterial strain gel beads in the fermentation broth through a gauze filter to prepare for the next fermentation, remove the supernatant and centrifuge to collect the precipitate; (8) Drying: The precipitate was dried with hot air at 40°C to obtain 277.3 kg of pea starch.

[0029] Example 5 Compared with Example 4, except that Lactobacillus bulgaricus was replaced by an equal amount of Lactobacillus delbrueckii, the rest was the same as Example 4. Finally, 293.3 kg of pea starch was obtained.

[0030] Example 6 Compared with Example 4, except that Lactobacillus bulgaricus was replaced by an equal amount of Lactobacillus helveticus, the rest were the same as Example 3. Finally, 294.7 kg of pea starch was obtained.

[0031] Comparative Example 1 Compared with Example 4, except that the thermophilic Streptococcus was replaced by an equal amount of maltodextrin, the rest was the same as Example 4. Finally, 264.7 kg of pea starch was obtained.

[0032] Comparative Example 2 The main steps of the traditional starch extraction method are briefly described as follows: Raw material pretreatment: 1000 kg of raw pea seeds were washed, impurities were removed, and then soaked in 1500 kg of water for 48 h; Crushing and grinding: Mechanically crush or grind the raw materials for 25-40 minutes to release starch particles; Fiber separation: remove fibers and dregs by screening or centrifugation, retaining the starch-containing slurry; Starch precipitation: let the slurry stand or centrifuge, and use gravity or chemical additive lime water to cause starch to settle to form starch milk; Washing and refining: Wash the precipitated crude starch with water for multiple times to remove residual protein and soluble impurities; Dehydration and drying: Use natural drying or hot air drying (50-60°C) to reduce moisture, and finally obtain 240.2kg pea starch.

[0033] In summary, Examples 2-4 achieve strain recovery by immobilizing gel beads (sodium alginate carrier), and maintain a high yield after repeated use (Example 4 uses recycled gel beads to obtain a yield of 277.3 kg, which is the same as the yield of freshly prepared gel beads in Example 3). However, since Comparative Example 1 does not use immobilization technology (replacing the strain with maltodextrin), the yield drops to 264.7 kg, indicating that strain activity and recovery capacity directly affect efficiency. The yield of Example 2 (Lactobacillus bulgaricus + Streptococcus thermophilus) is 297.3 kg, which is higher than the 264.7 kg of Comparative Example 1 (maltodextrin replaces Streptococcus thermophilus), indicating that the composite strain optimizes the extraction efficiency through synergistic metabolism (such as lactic acid production to promote starch release). Traditional Comparative Example 2 does not use strain fermentation, and the yield is lower. Examples 2-6 all use a homogenizer to treat the slurry (60 mesh screening), combined with multiple slag removal (gauze filtration 2-5 times), effectively remove fiber impurities, and improve the purity of the precipitate. In contrast, the conventional method in Comparative Example 2 only relies on standing or centrifugation, without mentioning the homogenization step, and more protein may remain in the starch. The yields of Example 5 (Lactobacillus delbrueckii) and Example 6 (Lactobacillus helveticus) were 293.3 kg and 294.7 kg, respectively, slightly lower than that of Lactobacillus bulgaricus (297.3 kg), indicating that Lactobacillus bulgaricus has a better degradation efficiency for pea starch. The yield of Comparative Example 1 (maltodextrin replacing thermophilic Streptococcus) was the lowest (264.7 kg), indicating that the combination of strains is indispensable, and the temperature resistance of thermophilic Streptococcus plays a key role in maintaining fermentation stability. The yield of Example 2 (water-powder ratio 1:4) was 297.3 kg, which was higher than 277.3 kg of Example 3 / 4 (water-powder ratio 1:6), indicating that a low water-powder ratio can reduce the subsequent drying energy consumption, but the slurry fluidity (homogenization difficulty) needs to be weighed. Example 2-4 adopts an intermittent stirring strategy of stirring for 1 hour-standing for 3 hours-stirring for 1 hour-standing for 1 hour, which not only avoids bacterial sedimentation (such as the traditional standing method), but also reduces the energy consumption of continuous stirring, while maintaining the lactic acid concentration gradient to promote starch release. It should be noted that the bacterial powder ratio (50%:50%) in Example 2-4 is different from the optimal group (44%:44%) in Table 1, but the actual yield (297.3 kg) is still relatively high, which may be due to the higher bacterial powder load (1:150) in the example to make up for the ratio deviation.

[0034] Example 7 Based on Example 1, this example also proposes an implementation method for further optimizing the extraction method.

[0035] The method further comprises the following steps: Each time the composite bacterial strain gel beads are used for fermentation, the lactic acid concentration in the fermentation broth is monitored in real time to calculate the current lactic acid production rate during the fermentation process; Recording the cumulative number of times the composite bacterial strain gel beads are used and the corresponding current lactic acid production rate; With the accumulated number of times used as the abscissa and the current lactic acid production rate as the ordinate, a lactic acid production rate attenuation curve is generated by fitting; Based on the lactic acid production rate attenuation curve and the cumulative number of uses, a gel bead label is generated, and the gel bead label is used to dynamically adjust the fermentation parameters in the subsequent fermentation process; the fermentation parameters include stirring frequency, fermentation temperature and fermentation time.

[0036] Optionally, for high-activity-low-loss gel beads, the first fermentation parameter is adopted, and the stirring frequency: the initial stirring frequency is set to 150 rpm. Due to the high activity and vigorous metabolism of the gel beads at this stage, the mass transfer demand of the substrate and the product is relatively large. In the early stage of fermentation (0-4 hours), the stirring frequency is maintained at 150 rpm to promote full contact between the substrate and the gel beads. In the middle stage of fermentation (4-6 hours), according to the change of the lactic acid production rate, if the rate rises steadily, the stirring frequency can be appropriately increased to 160 rpm to enhance the mass transfer effect. In the late stage of fermentation (6-8 hours), when the lactic acid production rate begins to stabilize, the stirring frequency is reduced to 140 rpm to reduce energy consumption. Fermentation temperature: The fermentation temperature is set to 37°C. During the entire fermentation process, the temperature is kept constant because the highly active gel beads can maintain the best metabolic activity at this temperature. The temperature is monitored and adjusted in real time by the temperature sensor and the heating / cooling system to ensure that the temperature fluctuation range is within ±0.5°C. Fermentation time: According to experience and experimental data, the fermentation time of high-activity-low-loss gel beads is usually set to 8 hours. During the fermentation process, the lactic acid concentration is monitored in real time. When the lactic acid concentration reaches the preset target value (such as 15 g / L), the fermentation can be terminated early.

[0037] Optionally, for medium-activity-medium-loss gel beads, the second fermentation parameter is adopted, stirring frequency: the initial stirring frequency is set to 160 rpm. Since the activity of the gel beads has decreased and there is a certain degree of loss of the carrier, the stirring frequency needs to be increased to enhance the mass transfer of the substrate and the product. In the early stage of fermentation (0-3 hours), the stirring frequency is maintained at 160 rpm. In the middle stage of fermentation (3-5 hours), if the lactic acid production rate is lower than expected (such as lower than 1.0mmol / (L·h)), the stirring frequency is increased to 170 rpm. In the late stage of fermentation (5-7 hours), when the lactic acid production rate begins to decrease, the stirring frequency is reduced to 150 rpm. Fermentation temperature: the fermentation temperature is increased to 38°C. Properly increasing the temperature can increase the metabolic activity of the strain to a certain extent, but care should be taken to avoid excessively high temperatures that may cause the strain to be inactivated. The temperature fluctuation range is controlled within ±0.5°C through the temperature control system. Fermentation time: the fermentation time is set to 9 hours. Due to the decrease in the activity of the gel beads, the fermentation speed will slow down, so the fermentation time needs to be appropriately extended. During the fermentation process, pay close attention to the changes in lactic acid concentration. If the lactic acid concentration does not reach the target value within 9 hours, it can be extended for another 1-2 hours based on actual conditions.

[0038] Optionally, for low-activity-high-loss gel beads, the third fermentation parameter is adopted, stirring frequency: the initial stirring frequency is set to 170 rpm. Low-activity and high-loss gel beads require a higher stirring frequency to promote contact between the substrate and the strain. In the early fermentation period (0-2 hours), the stirring frequency is maintained at 170 rpm. In the middle fermentation period (2-4 hours), if the lactic acid production rate is extremely low (such as less than 0.5mmol / (L·h)), the stirring frequency can be increased to 180 rpm, but care should be taken to avoid excessive stirring that causes the gel beads to break. In the late fermentation period (4-6 hours), according to the change in the lactic acid production rate, the stirring frequency is appropriately reduced to 160rpm. Fermentation temperature: the fermentation temperature is increased to 39°C. Higher temperatures can stimulate the metabolism of the strain, but the temperature must be strictly controlled to avoid the death of the strain. The temperature fluctuation range is controlled within ±0.3°C through a high-precision temperature sensor and temperature regulation system. Fermentation time: the fermentation time is set to 10-12 hours. Due to the low activity of the gel beads, the fermentation speed is very slow, and the fermentation time needs to be greatly extended. During the fermentation process, the lactic acid concentration is continuously monitored until the target value is reached.

[0039] Furthermore, the generation of gel bead labels based on the lactic acid production rate attenuation curve and the cumulative number of uses includes the following steps: The activity level is determined based on the percentage of the current lactic acid production rate to the initial lactic acid production rate when the composite bacterial strain gel beads are used for the first time; specifically, the initial lactic acid production rate is the average value actually measured for the first use.

[0040] Determining a loss level according to the accumulated number of uses; The combination of the activity level and the depletion level was used as the gel bead label.

[0041] Further, the activity level includes high activity, medium activity, and low activity, and the loss level includes high loss, medium loss, and low loss; Specifically, samples were taken every 30 minutes during the fermentation process, the concentration was measured using a lactic acid kit, the current rate was calculated, 15 batches of data were recorded continuously, and an exponential decay model (i.e., lactic acid production rate decay curve) was obtained by fitting using Origin 2023: (R²=0.982), where n is the cumulative number of times used, v t Indicates the model prediction rate; defines the initial rate v0 = 1.8mmol / (L·h) (the average value is used for the first time), activity level division: high activity: v≥80%v0; medium activity: 50%v0≤v<80%v0, low activity: v<50%v0; loss level is based on the cumulative number of times: low loss (n≤5), medium loss (6≤n≤10), high loss (n>10). The validation data of the lactic acid production rate attenuation curve are shown in Table 2: Table 2

[0042] As shown in Table 2, for gel beads with n≤1, the lactate production efficiency is close to the initial rate of 1.8mmol / (L·h), so ≥80% of the initial rate is used as the range for distinguishing high activity levels; corresponding to the use stage of n=3-8 times, the lactate production rate decreases with the attenuation of mitochondrial function, so 50%~80% of the initial rate is used as the range for distinguishing medium activity levels; when n≥10 times, the lactate production capacity significantly declines, so <50% of the initial rate is used as the range for distinguishing low activity levels.

[0043] Specifically, it can be seen from Table 2 that the exponential decay model has a higher accuracy. By taking the second-order derivative of the model, it is found that: n=5: the slope of the curve increases significantly for the first time (the decay rate accelerates), n=10: it enters a plateau period (the rate decrease slows but the absolute value is lower than 50% of the initial value). It can also be verified from Table 1: when n=5, the predicted value is 1.24 (69% of the initial rate), and when n=10, it is 0.86 (48%), which correspond to medium / low activity thresholds, respectively. Therefore, n≤5, 6≤n≤10, and n>10 are used as the boundaries for distinguishing loss levels.

[0044] After the composite bacterial strain gel beads in the fermentation broth are recovered, the following steps are also included: If the activity level of the composite bacteria gel beads is low activity and the loss level is high loss, the composite bacteria gel beads are forced to be scrapped or regenerated and then downgraded for use.

[0045] Specifically, the mandatory scrapping conditions are: when the gel beads have one of the following conditions: low activity level and high loss level (n>15 times, v<30%v0), surface crack rate>40% (microscope observation, 50x objective lens), swelling rate>50% (weight determination).

[0046] Furthermore, the method also includes regenerating the composite bacterial strain gel beads whose activity level is low and whose loss level is high; The regeneration of the composite bacterial strain gel beads having a low activity level and a high loss level comprises the following steps: The composite bacteria gel beads are immersed in a sodium alginate regeneration solution, and subjected to high-pressure homogenization treatment to repair surface cracks of the composite bacteria gel beads to obtain repaired gel beads; The repaired gel beads are transferred to an activation solution, and cultured under shaking to restore the metabolic activity of the bacteria, thereby obtaining activated gel beads; the activation solution is a phosphate buffer solution containing glucose, yeast extract and magnesium sulfate; The lactic acid generation rate of the activated gel beads is detected. If the lactic acid generation rate reaches the first proportional interval of the initial lactic acid generation rate, the gel bead labels of the activated gel beads are marked as regeneration-mid-activity-mid-loss, and the cumulative number of uses is limited.

[0047] Specifically, repair: low activity-high loss gel beads (n=12 times, v=42%v0) were immersed in 2% sodium alginate + 0.05% CaCl2 regeneration solution, and after high pressure homogenization (150MPa, 3 times), the crack closure rate reached 78% (SEM observation). Activation: After repair, the gel beads were shaken (180rpm) at 37℃ in activation solution (1% glucose + 0.5% yeast extract + 0.1% magnesium sulfate, pH6.5) for 24h, and the lactic acid production rate recovered to 65%v0.

[0048] Furthermore, the step of adding the composite bacterial strain gel beads to the pea powder slurry for fermentation comprises the following steps: If the activity level of the composite bacteria gel beads is medium activity and the loss level is medium loss, the composite bacteria gel beads are added to the pea powder slurry, and carbon source or coenzyme factor is dynamically supplemented according to the real-time lactic acid production rate.

[0049] Specifically, the surface microporosity of the sodium alginate carrier with medium-loss gel beads (cumulative use times 6-10 times) decreased by 35% (SEM comparison) due to mechanical wear and calcium loss, limiting substrate diffusion (mass transfer coefficient from 1.2×10 -5 cm / s decreased to 0.8×10 -5 cm / s), the synergistic effect of Lactobacillus and Streptococcus thermophilus depends on the NAD+ cycle, and the coenzyme regeneration efficiency is reduced by 28% under the medium activity state (LC-MS detected that the intracellular NAD+ / NADH ratio dropped from 4.2 to 2.9). Therefore, an online lactate sensor was used to record the lactate production rate (ΔC / Δt) every 15 minutes. When the real-time rate of the medium activity-medium loss beads is less than 0.9mmol / (L·h) (i.e., less than 80% of the average rate of this level), feeding is prompted. Feeding material: Carbon source: 50% glucose solution (sterile), supplementation amount = (theoretical lactate production rate-measured lactate production rate) × fermentation volume × 0.8 (coefficient); coenzyme factor: 0.1% nicotinic acid (NAD+ precursor) + 0.05% magnesium sulfate, supplemented at 0.5mL / L. At the 4th hour of fermentation (late logarithmic growth phase), the strain has a strong metabolic demand, and the carrier mass transfer resistance begins to appear. Stirring strategy: After feeding, increase the speed from 150rpm to 180rpm for 30min to promote diffusion of substances. When the rate rises to ≥1.2mmol / (L·h) after feeding, stop feeding. If there is no improvement after feeding twice, stop fermentation early. The experimental data are shown in Table 3: Table 3

[0050] In a preferred embodiment, the step of obtaining the composite bacterial strain gel beads comprises the following steps: Obtaining the target process requirements for this extraction, wherein the target process requirements include the target starch purity of the starch to be extracted; If the target starch purity is greater than the first preset purity, the newly prepared composite bacterial strain gel beads are selected.

[0051] Furthermore, after obtaining the target process requirements for this extraction, the following steps are also included: If the target starch purity is less than or equal to the first preset purity, the corresponding recycled composite bacterial strain gel beads are selected according to the target starch purity.

[0052] Furthermore, the method of selecting the corresponding recovered composite bacterial strain gel beads according to the target starch purity comprises the following steps: If the target starch purity is less than the second preset purity, the recycled composite bacterial strain gel beads with a gel bead label of low activity-high loss or a cumulative use number of the first preset number are selected; if the target starch purity is greater than the second preset purity and less than the first preset purity, the recycled composite bacterial strain gel beads with a gel bead label of medium activity-medium loss and a cumulative use number of less than the second preset number are selected; Among them, the second preset purity is smaller than the first preset purity, and the first preset number of times is larger than the second preset number of times.

[0053] Optionally, the first preset purity is 93%. When the target starch purity is greater than or equal to 93% (high-end food grade), use newly made beads (n=0); when the target starch purity is less than 93%, use recycled beads. The second preset purity is 90%. When 90%≤target starch purity<93%, select medium activity-medium loss beads (n=6-8 times, v=60-75%v0). When the target starch purity<90%, select low activity-high loss beads (n>10 times, v<50%v0). Verification experiment: Set the target starch purity to 92%, select medium activity beads with n=7 times, and the actual purity is 92.3% (n=10, RSD=0.8%), which is better than 89.7% using low activity beads (p<0.01). The experimental data are shown in Table 4: Table 4

[0054] This implementation method can achieve accurate matching of purity requirements, ensure product quality, optimize costs, and improve economic benefits. The yield of newly made beads is 81.0%, which is the highest cost. It is only used in high-end scenarios to reduce the consumption of newly made beads; reuse of recycled beads in medium / low purity scenarios reduces raw material costs. Medium-activity beads (n=6-8 times, yield 80.5%) are reused in the range of 90-93%, which not only ensures purity (3.1% higher than low-activity beads), but also avoids waste of newly made beads (only 0.5% more yield but higher cost), balancing loss and efficiency. Low-activity beads allow n>10 reuses to fully tap the residual value, which is suitable for scenarios with low purity requirements (such as industrial starch), prolongs the life of gel beads, and reduces waste.

[0055] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of the present invention, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for extracting pea starch using an immobilized cell method, characterized in that: The following steps are involved: Peeling pea seeds and grinding them to obtain pea flour, adding process water to the pea flour, and stirring them evenly to obtain pea milk; Filtering the bean dregs in the pea milk to obtain filtered soy milk, and homogenizing the filtered soy milk to obtain pea powder slurry; Obtaining composite bacterial strain gel beads, adding the composite bacterial strain gel beads to the pea powder slurry for fermentation, and stirring evenly to obtain a fermentation liquid; Recovering the composite bacterial strain gel beads in the fermentation broth and recovering the supernatant to obtain a precipitate; the supernatant is used to prepare the process water for the next extraction; drying the precipitate to obtain pea starch; The composite bacteria gel beads include an immobilized carrier and a composite bacteria encapsulated in the immobilized carrier, the immobilized carrier is a biocompatible polysaccharide gel, and the composite bacteria are composed of lactobacillus and thermophilic streptococcus; the types of the composite bacteria gel beads include newly prepared composite bacteria gel beads with zero cumulative usage times and recycled composite bacteria gel beads with at least one cumulative usage times; The preparation of the newly prepared composite bacterial strain gel beads comprises the following steps: Lactobacillus powder and thermophilic Streptococcus powder are mixed in a mass ratio of 40%-48%:40%-48% to obtain a composite powder, and the other compounding material is maltodextrin; Prepare 1.5% sodium alginate solution, heat and stir to dissolve, and cool to room temperature; Adding the composite bacterial powder to the sodium alginate solution, wherein the mass ratio of the composite bacterial powder to the sodium alginate is 1:0.5-1:5, stirring until uniformly suspended, to obtain a mixed solution; The mixed solution was dripped dropwise into a 2%-5% calcium chloride solution and allowed to stand at room temperature for cross-linking to form initial gel beads; The initial gel beads are rinsed with sterile water for multiple times to remove residual calcium ions on the surface to obtain newly prepared composite bacterial strain gel beads; the method further comprises the following steps: Each time the composite bacterial strain gel beads are used for fermentation, the lactic acid concentration in the fermentation broth is monitored in real time to calculate the current lactic acid production rate during the fermentation process; Recording the cumulative number of times the composite bacterial strain gel beads are used and the corresponding current lactic acid production rate; With the accumulated number of times used as the abscissa and the current lactic acid production rate as the ordinate, a lactic acid production rate attenuation curve is generated by fitting; Based on the lactic acid production rate attenuation curve and the cumulative number of uses, a gel bead label is generated, and the gel bead label is used to dynamically adjust the fermentation parameters in the subsequent fermentation process; the fermentation parameters include stirring frequency, fermentation temperature and fermentation time.

2. The method for extracting pea starch by immobilized cell method according to claim 1, characterized in that: The step of generating a gel bead label based on the lactic acid production rate attenuation curve and the cumulative number of uses comprises the following steps: Determining the activity level according to the percentage of the current lactic acid production rate to the initial lactic acid production rate when the composite bacterial strain gel beads are used for the first time; Determining a loss level according to the accumulated number of uses; The combination of the activity level and the depletion level was used as the gel bead label.

3. The method for extracting pea starch by immobilized cell method according to claim 2, characterized in that: The activity levels include high activity, medium activity, and low activity, and the loss levels include high loss, medium loss, and low loss; After the composite bacterial strain gel beads in the fermentation broth are recovered, the following steps are also included: If the activity level of the composite bacteria gel beads is low activity and the loss level is high loss, the composite bacteria gel beads are forced to be scrapped or regenerated and then downgraded for use.

4. The method for extracting pea starch by immobilized cell method according to claim 2, characterized in that: The step of adding the composite bacterial strain gel beads to the pea powder slurry for fermentation comprises the following steps: If the activity level of the composite bacteria gel beads is medium activity and the loss level is medium loss, the composite bacteria gel beads are added to the pea powder slurry, and carbon source or coenzyme factor is dynamically supplemented according to the real-time lactic acid production rate.

5. The method for extracting pea starch by immobilized cell method according to claim 2, characterized in that: The method of obtaining the composite bacterial strain gel beads comprises the following steps: Obtaining the target process requirements for this extraction, wherein the target process requirements include the target starch purity of the starch to be extracted; If the target starch purity is greater than the first preset purity, the newly prepared composite bacterial strain gel beads are selected.

6. The method for extracting pea starch by immobilized cell method according to claim 5, characterized in that: After obtaining the target process requirements for this extraction, the following steps are also included: If the target starch purity is less than or equal to the first preset purity, the corresponding recycled composite bacterial strain gel beads are selected according to the target starch purity.

7. The method for extracting pea starch by immobilized cell method according to claim 6, characterized in that: The method of selecting the corresponding recovered composite bacterial strain gel beads according to the target starch purity comprises the following steps: If the target starch purity is less than the second preset purity, the recycled composite bacterial strain gel beads with a gel bead label of low activity-high loss or a cumulative use number of the first preset number are selected; if the target starch purity is greater than the second preset purity and less than the first preset purity, the recycled composite bacterial strain gel beads with a gel bead label of medium activity-medium loss and a cumulative use number of less than the second preset number are selected; Among them, the second preset purity is smaller than the first preset purity, and the first preset number of times is larger than the second preset number of times.

8. The method for extracting pea starch by immobilized cell method according to claim 6, characterized in that: The method further includes regenerating the composite bacterial strain gel beads whose activity level is low and whose loss level is high; The regeneration of the composite bacterial strain gel beads having a low activity level and a high loss level comprises the following steps: The composite bacteria gel beads are immersed in a sodium alginate regeneration solution, and subjected to high-pressure homogenization treatment to repair surface cracks of the composite bacteria gel beads to obtain repaired gel beads; The repaired gel beads are transferred to an activation solution, and cultured under shaking to restore the metabolic activity of the bacteria, thereby obtaining activated gel beads; the activation solution is a phosphate buffer solution containing glucose, yeast extract and magnesium sulfate; The lactic acid generation rate of the activated gel beads is detected. If the lactic acid generation rate reaches the first proportional interval of the initial lactic acid generation rate, the gel bead labels of the activated gel beads are marked as regeneration-mid-activity-mid-loss, and the cumulative number of uses is limited.

Images