Calcium alginate ball immobilized penicillium chrysogenum and application thereof in production of forsythiaside by converting forsythin

Abstract

The invention discloses calcium alginate ball immobilized penicillium chrysogenum and application thereof in production of forsythiaside by converting forsythin, and belongs to the technical field of enzyme engineering. According to the method, a sodium alginate solution and a penicillium chrysogenum solution are mixed and cross-linked and solidified in a calcium chloride solution to form immobilized microspheres with high enzyme activity and stability, and through pore channels are formed in the immobilized microspheres, so that fungi are effectively intercepted, and a substrate and a product are allowed to freely diffuse. The immobilized bacterium ball can efficiently convert forsythin under optimized conditions to produce forsythiaside with higher medicinal value, the conversion efficiency is high, and immobilized cells can be repeatedly used in multiple batches. The phillygenin, as a key active component in fructus forsythiae, has higher antibacterial, anti-inflammatory and antioxidant activity than forsythin. The method has mild reaction conditions, avoids the use of a large amount of organic solvents in traditional chemical synthesis, and accords with the green chemical principle.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to calcium alginate spheres for immobilizing Penicillium chrysogenum and application thereof in converting forsythiaside to produce forsythiaside. Background Art

[0002] As a traditional Chinese medicine, Forsythia suspensa has a history of clinical application for thousands of years. Its dried fruits are widely used to treat diseases such as wind-heat colds, carbuncles, sores and ulcers, and have core effects such as clearing away heat and detoxifying, reducing swelling and dispersing nodules. Modern pharmacological studies have shown that forsythiaside, the main active ingredient of Forsythia suspensa, is a diepoxylignan glycoside compound, but its bioavailability after oral administration is extremely low due to the presence of glycosidic bonds, which seriously limits its pharmacological activity. It is worth noting that forsythiaside must be hydrolyzed into forsythiaside by intestinal flora or exogenous enzymes in the body before it can be effectively absorbed. The latter, as an aglycone form, has a bioavailability that is more than three times that of forsythiaside and a wider spectrum of pharmacological activity.

[0003] However, traditional methods for preparing forsythiaside have significant drawbacks. For example, chemical hydrolysis can easily cause forsythin to isomerize and produce by-products, and the amount of free enzyme required for conversion is large and difficult to separate from the reactants. In recent years, microbial conversion technology has become a research hotspot due to its high efficiency and specificity. Penicillium chrysogenum, a eukaryotic microorganism with a metabolic system similar to that of humans, can efficiently and specifically catalyze the conversion of forsythiaside to forsythiaside, and is not prone to producing by-products. At the same time, the fungal cells and the conversion products are easy to separate. Preliminary laboratory studies have confirmed that Penicillium chrysogenum has this conversion ability, and immobilization technology can further achieve the reuse of the fungus, reduce production costs, and improve conversion efficiency.

[0004] The natural polymer material sodium alginate exhibits unique advantages in the field of immobilization. Its biocompatibility and non-toxic properties make it an ideal carrier for embedding fungi. The gel network formed by cross-linking sodium alginate and calcium ions has a controllable porous structure, which can effectively embed the fungus of Penicillium chrysogenum without hindering the contact between forsythiaside and glycosidase in the fungus, thereby maintaining the conversion efficiency of the fungus. The material can be formed under mild conditions, and its gel microspheres can optimize mass transfer performance by adjusting the sodium alginate concentration. The low cost of sodium alginate and its stability in repeated use, as well as the fact that it can be separated from the reactants by filtration, eliminating the complicated separation steps, make it an advantageous material for immobilizing fungi.

[0005] In terms of the choice of immobilization method, the encapsulation method has become the preferred technology for immobilizing fungi due to its simple operation, mild reaction conditions and little damage to cells. Compared with the adsorption method or the cross-linking method, the encapsulation method can prevent the loss of bacteria and maintain their metabolic activity by entrapping microbial cells in a polymer network, which is particularly suitable for the immobilization of fungi. The immobilized microspheres prepared by this method have high mechanical strength and can withstand the fluid shear force in the reactor. At the same time, their three-dimensional network structure allows the free diffusion of substrates and oxygen, ensuring the efficient conversion of forsythiaside to forsythiaside. This technology also avoids the common problem of by-product generation in chemical hydrolysis methods, and the separation efficiency of immobilized bacterial balls and products is significantly improved compared to free bacteria. These technological breakthroughs provide new ideas for the industrial transformation of forsythiaside, and also provide a reference paradigm for the biotransformation of other low-potency traditional Chinese medicine ingredients. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for immobilizing Penicillium chrysogenum with calcium alginate balls by an embedding method, so as to obtain highly stable and reusable calcium alginate balls immobilized Penicillium chrysogenum, and to use them for the conversion of forsythin and other β-glucosidic bond hydrolysis reactions.

[0007] To achieve the above object, the present invention adopts the following technical solution: a calcium alginate sphere immobilized Penicillium chrysogenum, which is made by immobilizing Penicillium chrysogenum with the natural polymer material sodium alginate by an embedding method.

[0008] The preparation method of the above-mentioned calcium alginate ball immobilized Penicillium chrysogenum includes the following steps: preparing sodium alginate-bacteria liquid, adding the natural polymer material sodium alginate-bacteria liquid dropwise into a calcium chloride solution for static cross-linking, filtering and separating to obtain calcium alginate balls, and washing to obtain immobilized Penicillium chrysogenum.

[0009] The above-mentioned method for preparing the immobilized Penicillium chrysogenum by calcium alginate beads, in the step of preparing the immobilized Penicillium chrysogenum, the mass of the sodium alginate carrier material is 0.1g-0.4g, the concentration of the Penicillium chrysogenum liquid is 1.5×10 9 ~2×10 9 CFU / mL, calcium chloride concentration was 2% to 4.5%, the optimal temperature for immobilizing fungi was 23°C to 45°C, and the final rotation speed was 150 r / min.

[0010] In the above-mentioned method for preparing calcium alginate spheres immobilized Penicillium chrysogenum, in the step of preparing immobilized Penicillium chrysogenum, the sodium alginate-bacteria solution is placed in a calcium chloride solution for cross-linking for 3 hours, and the final concentration of the calcium chloride solution used for cross-linking is 4%.

[0011] The above-mentioned method for preparing calcium alginate sphere-immobilized Penicillium chrysogenum and the method for preparing sodium alginate-bacteria solution comprise the following steps:

[0012] 1) Preparation of bacterial suspension: After fully diluting several CGMCC 3.521 liquid culture medium of Penicillium chrysogenum, 1 mL of the diluted sample was spread on potato dextrose agar medium. After incubation, the number of colonies was counted. Based on the dilution factor and the sample inoculum volume, the number of viable bacteria in the sample was calculated to be 1.5 × 10 9 ~2×10 9 CFU / mL, prepared before use.

[0013] 2) Preparation of sodium alginate-bacteria solution: Sodium alginate was dissolved in 9 mL of deionized water and mixed by magnetic stirring. Then, 1 mL of bacteria solution was added and mixed by magnetic stirring until no bubbles were present. This gave the sodium alginate-bacteria solution.

[0014] 3) Preparation of calcium chloride solution: Weigh 0.2 g to 0.45 g of calcium chloride and dissolve it in 10 mL of deionized water. Ultrasonicate until the solution is clear and transparent to obtain calcium chloride solutions of different concentrations.

[0015] 4) Preparation of calcium alginate pellets: Prepare the pellets by uniformly adding the sodium alginate-bacteria mixture dropwise to the calcium chloride solution using a 1 mL syringe while gently shaking the beaker. Be careful to avoid inhaling air during injection. After addition, allow to crosslink for 3 hours, then filter and collect the immobilized pellets. Wash the pellets three times with sterile saline and set aside.

[0016] 7. In the above-mentioned method for preparing immobilized Penicillium chrysogenum, the configurations in steps 1), 2), 3), and 4) are carried out at room temperature.

[0017] The aforementioned calcium alginate sphere-immobilized Penicillium chrysogenum converts forsythiaside to forsythiaside. The method is as follows: 5 g of immobilized Penicillium chrysogenum calcium alginate spheres are added to 10 mL of a 1 mg / mL forsythiaside aqueous solution, and the forsythiaside is hydrolyzed and converted to forsythiaside at a constant temperature of 23°C to 45°C and 150 rpm.

[0018] The beneficial effects of the present invention are as follows:

[0019] 1. Sodium alginate and calcium chloride solution form calcium alginate gel spheres with a three-dimensional network structure through ionic crosslinking. This immobilization carrier not only efficiently retains fungi such as Penicillium chrysogenum within the gel network through physical embedding, but also significantly improves the technical bottleneck of traditional free enzyme or microbial recovery. Compared with traditional centrifugation or sedimentation separation methods, the gel spheres formed by immobilized fungi can be directly filtered through a mesh to achieve solid-liquid separation, shortening the operation time and reducing the bacterial cell loss rate. This characteristic not only avoids mechanical damage to the bacterial structure caused by repeated centrifugation, but also effectively maintains the activity of the fungus's endogenous glycosidase, allowing forsythin conversion to maintain a high efficiency even with repeated use. This technology integrates immobilization and separation processes, providing a highly efficient and economical solution for large-scale biotransformation.

[0020] 2. The present invention adopts a one-step ion crosslinking method to prepare immobilized Penicillium chrysogenum. The specific operation is to mix the bacterial suspension containing Penicillium chrysogenum with sodium alginate solution, and then add it dropwise to the calcium chloride solution. 2 + ionic crosslinking reaction with the carboxyl groups on the sodium alginate molecular chain forms calcium alginate gel spheres at room temperature. This technology has low raw material costs, does not require complex equipment, and avoids the destruction of endogenous fungal glycosidase activity by high temperatures or strong acid and alkaline environments, ensuring the efficient conversion of forsythin. Secondly, the three-dimensional network structure formed by calcium alginate provides effective physical protection for the fungus. It also combines excellent mechanical properties with efficient mass transfer characteristics, and can adapt to conversion reaction systems under different pH and temperature conditions, providing a reliable technical solution for industrial continuous production.

[0021] 3. The calcium alginate gel-immobilized Penicillium chrysogenum system developed in this invention, combined with high-performance liquid chromatography (HPLC), provides a highly efficient platform for the biotransformation of forsythiaside. This technology offers significant advantages over traditional methods: immobilized Penicillium chrysogenum does not introduce additional groups or produce byproducts during the conversion of forsythiaside, significantly improving conversion efficiency. The entire conversion process is time-efficient, and the immobilized pellets are reusable, significantly reducing conversion costs. This immobilized pellet conversion method is simple to operate, efficient, and environmentally friendly. The conversion product, forsythiaside, is easily extracted and separated, showing promising application prospects in areas such as drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The invention discloses an immobilized Penicillium chrysogenum calcium alginate ball prepared by the invention.

[0023] Figure 2 The effect of sodium alginate solution concentration on the enzyme activity of Penicillium chrysogenum immobilized on calcium alginate.

[0024] Figure 3 The effect of calcium chloride solution concentration on the enzyme activity of Penicillium chrysogenum immobilized on calcium alginate.

[0025] Figure 4 The effect of cross-linking time on the enzyme activity of Penicillium chrysogenum immobilized on calcium alginate.

[0026] Figure 5 It is the enzyme activity of the immobilized Penicillium chrysogenum prepared by the present invention after being reused 8 times.

[0027] Figure 6 The invention discloses an effect of the immobilized Penicillium chrysogenum prepared by the present invention on the conversion of forsythiaside at different pH values.

[0028] Figure 7 The invention discloses an effect of the immobilized Penicillium chrysogenum prepared by the present invention on the conversion of forsythiaside at different temperatures. DETAILED DESCRIPTION

[0029] The present invention will be further described in detail below in conjunction with the embodiments and drawings. The specific embodiments described below are not intended to limit the present invention, but are merely intended to explain the present invention.

[0030] In the following examples, Penicillium chrysogenum was purchased from China General Microorganism Culture Collection Center (CGMCC); unless otherwise specified, the various reagents and operations used in the examples are conventional reagents and operations in the art.

[0031] Example 1: Specific method for immobilizing Penicillium chrysogenum with calcium alginate

[0032] The preparation method of calcium alginate ball immobilized Penicillium chrysogenum comprises the following steps:

[0033] 1. Preparation of bacterial solution

[0034] After fully diluting several CGMCC 3.521 liquid PDB culture medium of Penicillium chrysogenum, 1 mL of the diluted sample was spread on potato dextrose agar medium. After constant temperature incubation at 25°C, the number of colonies was counted. According to the dilution multiple and the sampling inoculum volume, the number of viable bacteria in the sample was calculated to be 1.5 × 10 9 ~2×10 9 CFU / mL.

[0035] Potato dextrose agar medium is prepared by adding 200g of chopped potatoes to an appropriate amount of distilled water and boiling for 30 minutes. After cooling, filter through four layers of gauze. Add 20g of glucose and 20g of agar powder to the filtrate, heating and stirring until completely dissolved. The mixture is brought to a volume of 1000mL and autoclaved at 121°C for 15 minutes. Once the medium has cooled to approximately 50°C, aseptically pour it into a Petri dish. After solidification, place it in a 25°C incubator until ready for use.

[0036] 2. Preparation of sodium alginate-bacteria solution

[0037] Accurately weigh 0.10g to 0.40g of sodium alginate sample and dissolve each sample in 9mL of deionized water. Stir thoroughly with a magnetic stirrer until completely dissolved and free of bubbles, creating a sodium alginate solution with a concentration gradient of 1% to 4%. Subsequently, add 1mL of the pre-prepared bacterial solution to each concentration, stir thoroughly, and set aside.

[0038] 3. Preparation of calcium chloride solution

[0039] Accurately weigh 0.2 g to 0.45 g of calcium chloride and dissolve it in 10 mL of deionized water. Ultrasonicate until the solution becomes clear and transparent to prepare a 2% to 4.5% calcium chloride solution as a cross-linking agent.

[0040] 4. Preparation of Calcium Alginate Balls Immobilized Penicillium chrysogenum

[0041] Prepare bacterial pellets using a 1mL sterile syringe. Add the sodium alginate-bacteria mixture dropwise to the calcium chloride solution at room temperature while shaking. Be careful to avoid inhaling air when aspirating the sodium alginate-bacteria mixture. After the addition is complete, allow the mixture to stand for 1–13 hours to crosslink. Collect the immobilized pellets by filtration and wash three times with sterile saline to obtain calcium alginate-immobilized Penicillium chrysogenum.

[0042] Example 2: Effect of Sodium Alginate Solution Concentration on the Immobilization of Penicillium chrysogenum with Calcium Alginate Spheres

[0043] 1. Preparation of bacterial solution

[0044] After fully diluting several CGMCC 3.521 liquid cultures of Penicillium chrysogenum, 1 mL of the diluted sample was spread on potato dextrose agar. After incubation, the number of colonies was counted. The number of viable bacteria in the sample was calculated to be 1.5 × 10 9 ~2×10 9 CFU / mL.

[0045] Potato dextrose agar medium is prepared by adding 200g of chopped potatoes to an appropriate amount of distilled water and boiling for 30 minutes. After cooling, filter through four layers of gauze. Add 20g of glucose and 20g of agar powder to the filtrate, heating and stirring until completely dissolved. The mixture is brought to a volume of 1000mL and autoclaved at 121°C for 15 minutes. Once the medium has cooled to approximately 50°C, aseptically pour it into a Petri dish. After solidification, place it in a 25°C incubator until ready for use.

[0046] 2. Preparation of sodium alginate-bacteria solution

[0047] Accurately weigh 0.10g to 0.40g of sodium alginate sample in 0.05g increments and dissolve in 9mL of deionized water. Stir thoroughly with a magnetic stirrer until completely dissolved and free of bubbles, to prepare sodium alginate solutions with concentration gradients ranging from 1% to 4%. Subsequently, add 1mL of the pre-prepared bacterial solution to each concentration solution, stir thoroughly, and set aside. Prepare three replicates for each concentration.

[0048] 3. Preparation of calcium chloride solution

[0049] Accurately weigh 0.3 g of calcium chloride, dissolve it in 10 mL of deionized water, and sonicate until the solution is clear and transparent to ensure that the calcium chloride is completely dissolved to obtain a 3% concentration calcium chloride solution.

[0050] 4. Prepare bacterial pellets using a 1mL sterile syringe. Add the sodium alginate-bacteria mixture dropwise to the calcium chloride solution at a constant rate, taking care to avoid inhaling air. Gently shake the beaker while adding the pellets. After the addition is complete, allow the pellets to stand for 3 hours for crosslinking. Filter and collect the immobilized pellets. Rinse the calcium chloride solution with sterile saline to obtain immobilized Penicillium chrysogenum. Weigh 5g of pellets per group and set aside.

[0051] 5. Determination of enzyme activity of immobilized Penicillium chrysogenum

[0052] Immobilized Penicillium chrysogenum possesses β-glucosidase, which can hydrolyze forsythin to forsythiaside. p-Nitrophenyl-β-D-glucopyranoside, under the action of β-glucosidase, generates the yellow product p-nitrophenol. Under alkaline conditions, p-nitrophenol has a maximum absorbance at 405 nm. Enzyme activity can be quantitatively determined by measuring the absorbance at 405 nm. To 5 g of immobilized Penicillium chrysogenum prepared in step 4 above, add 10 mL of a 5 mmol / L p-nitrophenyl-β-D-glucopyranoside solution as the substrate. Incubate at 25°C with constant shaking at 150 rpm for 1 hour, and filter to separate the calcium alginate spheres. Using a 96-well plate, sample 100 μL of the reaction solution 5 minutes after the addition of the spheres and add 100 μL of a 1 mol / L sodium carbonate solution. This serves as the background value. After the reaction, 100 μL of the reaction solution was taken and added with 100 μL of 1 mol / L sodium carbonate solution. The absorbance value was measured at a wavelength of 405 nm using a microplate reader. After deducting the blank background value, 1% to 4% of the enzyme activity of Penicillium chrysogenum immobilized in the sodium alginate solution was obtained.

[0053] Depend on Figure 2 It can be seen that the enzyme activity of immobilized Penicillium chrysogenum reaches the highest when the sodium alginate concentration is 2.5%. Too high a dense structure is not conducive to the contact between immobilized Penicillium chrysogenum and the substrate, while too low a alginate concentration makes the bacteria easy to leak out. Therefore, the optimal sodium alginate concentration for immobilizing Penicillium chrysogenum is 2.5%.

[0054] Example 3: Effect of Calcium Chloride Solution Concentration on the Immobilization of Penicillium Chrysogenum with Calcium Alginate Spheres

[0055] 1. Preparation of bacterial solution

[0056] After fully diluting several CGMCC 3.521 liquid cultures of Penicillium chrysogenum, 1 mL of the diluted sample was spread on potato dextrose agar. After incubation, the number of colonies was counted. The number of viable bacteria in the sample was calculated to be 1.5 × 10 9 ~2×10 9 CFU / mL.

[0057] 2. Preparation of sodium alginate-bacteria solution

[0058] Accurately weigh six 0.25g sodium alginate samples and dissolve them in 9mL of deionized water. Stir thoroughly with a magnetic stirrer until completely dissolved and free of bubbles, to prepare a 2.5% sodium alginate solution. Add 1mL of the pre-prepared bacterial solution to each solution, stir thoroughly, and set aside. Prepare three replicates for each group.

[0059] 3. Preparation of calcium chloride solution

[0060] Accurately weigh 0.2 g to 0.45 g of calcium chloride, dissolve it in 10 mL of deionized water, and sonicate until the solution is clear and transparent to ensure that the calcium chloride is completely dissolved to obtain a calcium chloride solution with a concentration of 2% to 4.5%.

[0061] 4. Prepare bacterial pellets using a 1mL sterile syringe. Add the sodium alginate-bacteria mixture dropwise to the calcium chloride solution at a constant rate, taking care to avoid inhaling air. Gently shake the beaker while adding the pellets. After the addition is complete, allow the pellets to stand for 3 hours for crosslinking. Filter and collect the immobilized pellets. Rinse the calcium chloride solution with sterile saline to obtain immobilized Penicillium chrysogenum. Weigh 5g of pellets per group and set aside.

[0062] 5. Determination of enzyme activity of immobilized Penicillium chrysogenum

[0063] Immobilized Penicillium chrysogenum possesses β-glucosidase, which can hydrolyze forsythin to forsythiaside. p-Nitrophenyl-β-D-glucopyranoside, under the action of β-glucosidase, generates the yellow product p-nitrophenol. Under alkaline conditions, p-nitrophenol has a maximum absorbance at 405 nm. Enzyme activity can be quantitatively determined by measuring the absorbance at 405 nm. To 5 g of immobilized Penicillium chrysogenum prepared in step 4 above, add 10 mL of a 5 mmol / L p-nitrophenyl-β-D-glucopyranoside solution as the substrate. Incubate at 25°C with constant shaking at 150 rpm for 1 hour, and filter to separate the calcium alginate spheres. Using a 96-well plate, sample 100 μL of the reaction solution 5 minutes after the addition of the spheres and add 100 μL of a 1 mol / L sodium carbonate solution. This serves as the background value. After the reaction, 100 μL of the reaction solution was taken and added with 100 μL of 1 mol / L sodium carbonate solution. The absorbance value was measured at a wavelength of 405 nm using a microplate reader. After deducting the blank background value, 2% to 4.5% of the enzyme activity of Penicillium chrysogenum immobilized in the sodium alginate solution was obtained.

[0064] Figure 3 Figure 3 is the effect of calcium chloride solution concentration on the enzyme activity of immobilized Penicillium chrysogenum. It can be seen from the figure that the enzyme activity of immobilized Penicillium chrysogenum reaches the maximum when the calcium chloride concentration is 4%. Therefore, the optimal calcium chloride concentration for immobilized Penicillium chrysogenum is 4%.

[0065] Example 4: Effect of cross-linking time on the activity of Penicillium chrysogenum immobilized on calcium alginate

[0066] 1. Preparation of bacterial solution

[0067] After fully diluting several CGMCC 3.521 liquid cultures of Penicillium chrysogenum, 1 mL of the diluted sample was spread on potato dextrose agar. After incubation, the number of colonies was counted. The number of viable bacteria in the sample was calculated to be 1.5 × 10 9 ~2×10 9 CFU / mL.

[0068] 2. Preparation of sodium alginate-bacteria solution

[0069] Accurately weigh seven 0.25g sodium alginate samples and dissolve them in 9mL of deionized water. Stir thoroughly with a magnetic stirrer until completely dissolved and free of bubbles, to prepare a 2.5% sodium alginate solution. Add 1mL of the pre-prepared bacterial solution to each solution, stir thoroughly, and set aside. Prepare three replicates for each group.

[0070] 3. Preparation of calcium chloride solution

[0071] Accurately weigh 0.4 g of calcium chloride, dissolve it in 10 mL of deionized water, and sonicate until the solution is clear and transparent to ensure that the calcium chloride is completely dissolved to obtain a 4% concentration of calcium chloride solution.

[0072] 4. Prepare bacterial pellets using a 1mL sterile syringe. Add the sodium alginate-bacteria mixture dropwise to the calcium chloride solution at a uniform rate, taking care to avoid inhaling air while adding. Gently shake the beaker while adding. After the addition is complete, allow the mixture to stand for crosslinking for 1 hour, 3 hours, 5 hours, 7 hours, 9 hours, 11 hours, and 13 hours. Collect the immobilized bacterial pellets by filtration and rinse the calcium chloride solution with sterile saline to obtain immobilized Penicillium chrysogenum. Weigh 5g of pellets per group for later use.

[0073] 5. Determination of enzyme activity of immobilized Penicillium chrysogenum

[0074] Immobilized Penicillium chrysogenum possesses β-glucosidase, which can hydrolyze forsythin to forsythiaside. p-Nitrophenyl-β-D-glucopyranoside, under the action of β-glucosidase, generates the yellow product p-nitrophenol. Under alkaline conditions, p-nitrophenol has a maximum absorbance at 405 nm. Enzyme activity can be quantitatively determined by measuring the absorbance at 405 nm. To 5 g of immobilized Penicillium chrysogenum prepared in step 4 above, add 10 mL of a 5 mmol / L p-nitrophenyl-β-D-glucopyranoside solution as the substrate. Incubate at 25°C with constant shaking at 150 rpm for 1 hour, and filter to separate the calcium alginate spheres. Using a 96-well plate, sample 100 μL of the reaction solution 5 minutes after the addition of the spheres and add 100 μL of a 1 mol / L sodium carbonate solution. This serves as the background value. After the reaction, 100 μL of the reaction solution was taken and added with 100 μL of 1 mol / L sodium carbonate solution. The absorbance value was measured at a wavelength of 405 nm using a microplate reader. After deducting the blank background value, the effect of different cross-linking times on the activity of immobilized Penicillium chrysogenum enzyme was obtained.

[0075] Figure 4 This figure shows the effect of cross-linking time on the enzyme activity of immobilized Penicillium chrysogenum. It can be seen from the figure that the enzyme activity of immobilized Penicillium chrysogenum is the highest when the cross-linking time is 3 hours. The cross-linking time is a key influencing parameter affecting the immobilization of fungi by calcium alginate. An appropriate time is required to form a fungal ball that can not only firmly embed the fungus body but also does not hinder the reaction between the fungus and the substrate. Therefore, the optimal cross-linking time for immobilized Penicillium chrysogenum is 3 hours.

[0076] Example 5: Enzyme activity of immobilized Penicillium chrysogenum after 8 repeated uses

[0077] Methods: Two groups of 1 mL bacterial suspension were prepared: one group was immobilized with 2.5% sodium alginate and 4% calcium chloride, and crosslinked for 3 hours. The other group served as a free bacterial control, with triplicate replicates per group. 5 g of immobilized bacterial pellets and 5 g of free bacterial pellets were added to 10 mL of a 5 mmol / mL solution of p-nitrophenyl-β-D-pyranoglucopyranoside. The reaction was carried out at 25°C and 150 rpm for 6 hours. The free bacterial pellets were collected by centrifugation at 10,000 rpm for 10 minutes. Repeated reactions were performed using the pellets after centrifugation. Immobilized bacterial pellets were stored at 4°C in 20% glycerol-water and washed three times with sterile saline before each experiment. After the reaction, enzyme activity was measured. For free bacterial enzyme activity, the following steps were performed: centrifuge the reaction solution, take 100 μL of the supernatant, and add 100 μL of 1 mol / L sodium carbonate solution. For immobilized bacterial pellet activity, take 100 μL of the reaction solution and add 100 μL of 1 mol / L sodium carbonate solution. For background value, 5 minutes after the addition of bacterial pellets, take 100 μL of the reaction solution and add 100 μL of 1 mol / L sodium carbonate solution. The enzyme activity was measured in a 96-well plate reader for 8 consecutive days, with the free bacterial enzyme activity on the first day as 100%. Results were expressed as OD 405.

[0078] The results are as follows Figure 5 As shown in the results, after eight reuses, the relative activity of immobilized Penicillium chrysogenum remained above 53%, while the free fungus required centrifugation each time, resulting in severe cell loss and reduced enzyme activity. Calcium alginate spheres can effectively immobilize Penicillium chrysogenum.

[0079] Example 6: Effect of immobilized Penicillium chrysogenum on the conversion of forsythiaside at different pH values

[0080] 1. Preparation of bacterial solution

[0081] After fully diluting several CGMCC 3.521 liquid cultures of Penicillium chrysogenum, 1 mL of the diluted sample was spread on potato dextrose agar. After incubation, the number of colonies was counted. The number of viable bacteria in the sample was calculated to be 1.5 × 10 9 ~2×10 9 CFU / mL.

[0082] 2. Preparation of sodium alginate-bacteria solution

[0083] Accurately weigh seven 0.25g sodium alginate samples and dissolve them in 9mL of deionized water. Stir thoroughly with a magnetic stirrer until completely dissolved and free of bubbles, to prepare a 2.5% sodium alginate solution. Add 1mL of the pre-prepared bacterial solution to each solution, stir thoroughly, and set aside. Prepare three replicates for each group.

[0084] 3. Preparation of calcium chloride solution

[0085] Accurately weigh 0.4 g of calcium chloride, dissolve it in 10 mL of deionized water, and sonicate until the solution is clear and transparent to ensure that the calcium chloride is completely dissolved to obtain a 4% concentration of calcium chloride solution.

[0086] 4. Prepare bacterial pellets using a 1mL sterile syringe. Add the sodium alginate-bacteria mixture dropwise to the calcium chloride solution at a constant rate, taking care to avoid inhaling air. Gently shake the beaker while adding the pellets. After the addition is complete, allow the pellets to stand for 3 hours for crosslinking. Filter and collect the immobilized pellets. Rinse the calcium chloride solution with sterile saline to obtain immobilized Penicillium chrysogenum. Weigh 5g of pellets per group and set aside.

[0087] 5. Immobilized Penicillium chrysogenum to convert forsythiaside to forsythiaside

[0088] Immobilized Penicillium chrysogenum hydrolyzes forsythin by removing the β-glucosidic bond to produce forsythiaside. High-performance liquid chromatography (HPLC) can quantitatively detect the reduction of forsythin and the production of forsythiaside. Three parallel solutions of forsythiaside (1.0 mg / mL) in saline were prepared at pH values ​​of 5, 6, 7, 8, 9, and 10. The pH was adjusted with 1 mol / L HCl and NaOH. After complete sonication for 10 minutes, the solution was heated in a 60°C water bath for 5 minutes to promote dissolution. Immobilized bacterial pellets were prepared according to the above-described ratios. 5 g of each solution was added to the forsythiaside solutions at different pH values ​​and the reaction was incubated at 25°C and 150 rpm for 48 hours. After completion of the reaction, 30 mL of methanol was added to terminate the reaction and sonication was performed for 10 minutes. The pellets were removed by filtration, concentrated under reduced pressure, and extracted three times with ethyl acetate at a ratio of 1:3 (v / v). The organic phases were combined and concentrated under reduced pressure. The residue was dissolved in 10 mL of chromatographic methanol, filtered through a 0.22 μm filter, and analyzed using a Shimadzu 2030plus. Liquid phase conditions: Diamonsil-C 18 Column (4.6 mm × 250 mm, 5 μm); injection volume: 10 μL; mobile phase: acetonitrile-0.2% formic acid aqueous solution, gradient elution; flow rate: 1 mL / min; column temperature: 30°C; detection wavelength: 277 nm; HPLC gradient elution conditions: 0 min: 10% acetonitrile, 90% 0.2% formic acid aqueous solution; 30 min: 100% acetonitrile, 0% 0.2% formic acid aqueous solution; 40 min: 10% acetonitrile, 90% 0.2% formic acid aqueous solution. The experimental results were calculated using the peak area according to the following formula:

[0089]

[0090] Figure 6 The effect of immobilized Penicillium chrysogenum on the conversion of forsythin under different pH conditions was observed. The yield of Penicillium chrysogenum was the highest at pH = 7. The results showed that the optimal conversion pH for Penicillium chrysogenum was 7.

[0091] Example 7: Effect of immobilized Penicillium chrysogenum on the conversion of forsythiaside at different temperatures

[0092] 1. Preparation of bacterial solution

[0093] After fully diluting several CGMCC 3.521 liquid cultures of Penicillium chrysogenum, 1 mL of the diluted sample was spread on potato dextrose agar. After incubation, the number of colonies was counted. The number of viable bacteria in the sample was calculated to be 1.5 × 10 9 ~2×10 9 CFU / mL.

[0094] 2. Preparation of sodium alginate-bacteria solution

[0095] Accurately weigh seven 0.25g sodium alginate samples and dissolve them in 9mL of deionized water. Stir thoroughly with a magnetic stirrer until completely dissolved and free of bubbles, to prepare a 2.5% sodium alginate solution. Add 1mL of the pre-prepared bacterial solution to each solution, stir thoroughly, and set aside. Prepare three replicates for each group.

[0096] 3. Preparation of calcium chloride solution

[0097] Accurately weigh 0.4 g of calcium chloride, dissolve it in 10 mL of deionized water, and sonicate until the solution is clear and transparent to ensure that the calcium chloride is completely dissolved to obtain a 4% concentration of calcium chloride solution.

[0098] 4. Prepare bacterial pellets using a 1mL sterile syringe. Add the sodium alginate-bacteria mixture dropwise to the calcium chloride solution at a constant rate, taking care to avoid inhaling air. Gently shake the beaker while adding the pellets. After the addition is complete, allow the pellets to stand for 3 hours for crosslinking. Filter and collect the immobilized pellets. Rinse the calcium chloride solution with sterile saline to obtain immobilized Penicillium chrysogenum. Weigh 5g of pellets per group and set aside.

[0099] 5. Immobilized Penicillium chrysogenum converts forsythiaside into forsythiaside

[0100] Under the action of the fungal β-glucosidase, immobilized Penicillium chrysogenum hydrolyzes forsythin to produce forsythiaside, and the production of forsythiaside can be quantitatively detected by high-performance liquid chromatography. Prepare 6 portions of 1.0 mg / mL forsythiaside physiological saline solution (10 mL each) and adjust the pH to 7.0. Ultrasonicate for 10 minutes and heat in a water bath for 5 minutes. 5 g of the fungus pellets prepared according to the above ratio method were placed at 23°C, 27°C, 31°C, 37°C, 41°C, and 45°C and converted at 150 r / min for 48 hours. After the reaction, 30 mL of methanol was added to terminate the reaction and ultrasonicate for 10 minutes. The fungus pellets were removed by filtration, concentrated under reduced pressure, and extracted three times with ethyl acetate in a ratio of 1:3 (v / v). The organic phases were combined and concentrated. The residue was dissolved in 10 mL of methanol, filtered through a 0.22 μm filter membrane, and subjected to high-performance liquid chromatography. The liquid phase conditions were: Diamonsil-C 18Column (4.6 mm × 250 mm, 5 μm); injection volume: 10 μL; mobile phase: acetonitrile-0.2% formic acid aqueous solution, gradient elution; flow rate: 1 mL / min; column temperature: 30°C; detection wavelength: 277 nm; HPLC gradient elution conditions: 0 min: 10% acetonitrile, 90% 0.2% formic acid aqueous solution; 30 min: 100% acetonitrile, 0% 0.2% formic acid aqueous solution; 40 min: 10% acetonitrile, 90% 0.2% formic acid aqueous solution. The experimental results were calculated using the peak area according to the following formula:

[0101]

[0102] Figure 7 The study examined the effects of immobilized Penicillium chrysogenum on the conversion of forsythiaside at different temperatures. P. chrysogenum exhibited optimal conversion efficiency at 41°C. Temperatures that were too high or too low affected enzyme activity, thereby affecting forsythiaside yield. The results confirmed that the optimal conversion temperature for P. chrysogenum was 41°C.

Claims

1. A calcium alginate ball immobilized Penicillium chrysogenum, characterized in that: The method is to mix Penicillium chrysogenum CGMCC 3.521 fungal liquid with sodium alginate solution, add the mixture dropwise into calcium chloride solution, and cross-link and solidify the mixture to form calcium alginate balls to immobilize Penicillium chrysogenum.

2. The method for preparing calcium alginate sphere-immobilized Penicillium chrysogenum according to claim 1, characterized in that: The steps include: mixing 1% to 4% sodium alginate solution with 1.5×10 9 ~2×10 9 The method comprises mixing a 1% to 2% CFU / mL Penicillium chrysogenum liquid, performing cross-linking in a 2% to 4.5% calcium chloride solution for 1 hour to 13 hours, filtering and separating, and washing the calcium chloride solution with deionized water to obtain calcium alginate ball-immobilized Penicillium chrysogenum.

3. The method for preparing calcium alginate sphere-immobilized Penicillium chrysogenum according to claim 2, wherein: By volume ratio, sodium alginate solution: Penicillium chrysogenum fungus solution: calcium chloride solution = 9:1:

10.

4. The method for preparing calcium alginate sphere-immobilized Penicillium chrysogenum according to claim 2, wherein: The preparation method of the calcium alginate ball-immobilized Penicillium chrysogenum comprises the following steps: 1) Preparation of bacterial suspension: After fully diluting several samples of Penicillium chrysogenum CGMCC 3.521 liquid culture medium, 1 mL of the diluted sample was spread on potato dextrose agar medium. After incubation, the number of colonies was counted. Based on the dilution factor and the sample inoculum volume, the number of viable bacteria in the sample was calculated to be 1.5 × 10 9 ~2×10 9 CFU / mL, prepared before use; 2) Preparation of Sodium Alginate-Bacteria Solution: Sodium alginate was dissolved in deionized water and mixed by magnetic stirring. 1 mL of the bacteria solution was then added and mixed by magnetic stirring until no bubbles were present. This resulted in the sodium alginate-bacteria solution. 3) Preparation of calcium chloride solution: Weigh 0.2 g to 0.45 g of calcium chloride and dissolve it in 10 mL of deionized water. Ultrasonicate until the solution is clear and transparent to obtain calcium chloride solutions of different concentrations. 4) Preparation of calcium alginate spheres: Prepare the spheres using a 1 mL syringe. Add the sodium alginate-bacteria mixture dropwise into the calcium chloride solution at a constant rate while gently shaking the beaker. Be careful not to inhale air during the addition. After the addition is complete, allow to crosslink for 3 h. Filter and collect the immobilized spheres, wash, and set aside.

5. The method for preparing calcium alginate sphere-immobilized Penicillium chrysogenum according to claim 4, characterized in that: The configurations described in step 1), step 2), step 3), and step 4) are performed at room temperature.

6. Use of the calcium alginate sphere-immobilized Penicillium chrysogenum according to claim 1 in converting forsythiaside to produce forsythiaside.

7. The use according to claim 6, characterized in that The method is as follows: 5 g of calcium alginate balls immobilized with Penicillium chrysogenum are added to 10 mL of a 1 mg / mL forsythin aqueous solution, and the forsythin is hydrolyzed and converted to produce forsythiaside at a constant temperature of 23° C. to 45° C. and 150 r / min.

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