A carrier-free immobilized yeast cell and its application in the synthesis of galactooligosaccharides

Abstract

The present invention relates to a carrier-free immobilized yeast cell and its application in the synthesis of galactooligosaccharides. It includes: (1) culturing yeast producing β-galactosidase; (2) suspending yeast cells with ethanol, stirring, and centrifuging; (3) adding genipin to the yeast cells treated with ethanol to construct a cross-linking system; (4) reacting the cross-linking system for 0.5 - 4 h and centrifuging to obtain carrier-free immobilized yeast cells. The present invention also provides the carrier-free immobilized yeast cells prepared by this method and the application of the carrier-free immobilized yeast cells in the synthesis of galactooligosaccharides. The present invention first directly cross-links genipin with yeast cells without the need to additionally add carrier materials, having the advantages of low cost, easy operation, and high stability. The present invention first uses carrier-free immobilized yeast cells to synthesize galactooligosaccharides with monosaccharides as substrates. In the reaction system, there is only the remaining monosaccharide substrate besides the product, and there is no hydrolysis side reaction in the lactose reaction system.

Description

Technical Field

[0001] The present invention relates to a carrier-free immobilized yeast cell and its application in the synthesis of galactooligosaccharides, belonging to the technical field of sugar engineering. Background Art

[0002] Galactooligosaccharides are human milk oligosaccharide analogs and are widely used in the food, health product, cosmetic, and feed industries. Such oligosaccharides have excellent prebiotic activity, can selectively stimulate the proliferation of probiotics such as Bifidobacterium and Lactobacillus, inhibit the growth and adhesion of pathogenic bacteria, adjust the intestinal microecological balance, relieve gastrointestinal diseases, regulate the absorption and storage of ions, reduce harmful metabolites, enhance immunity, etc. Galactooligosaccharides are widely used as food additives due to their excellent physicochemical properties and biological activities. It is worth noting that infant formula milk powder added with galactooligosaccharides has beneficial effects on infants similar to those of human milk. In addition, galactooligosaccharides also have great potential in medical applications, can regulate the intestinal flora, and improve neurological diseases such as depression, autism, amyotrophic lateral sclerosis, etc., as well as metabolic diseases such as overweight, diabetes and their complications such as kidney disease through the brain-gut axis system. Clinical experiments have also confirmed the efficacy of galactooligosaccharides in the treatment of allergies and HIV infections.

[0003] Galactooligosaccharides generally consist of 2 - 8 sugar units, where the reducing end is glucose or galactose, and the other sugar units are galactose. Currently, the industrial preparation of galactooligosaccharides is through the transglycosylation synthesis of lactose by microbial β-galactosidase. However, this method has a lactose hydrolysis side reaction, resulting in the synthesis product containing, in addition to the oligosaccharide product, galactose and glucose by-products as well as unreacted lactose substrate, which is not conducive to product purification, and the presence of the by-product glucose is not conducive to use by diabetic patients, and to a certain extent limits the application of galactooligosaccharides in the medical field. Therefore, it is very necessary to find a simple method for synthesizing galactooligosaccharides without side reactions.

[0004] On the other hand, for the industrial-scale synthesis of oligosaccharides, it is also necessary to reduce the cost of enzymes. Since immobilized enzymes can be reused, the preparation cost of enzymes can be greatly reduced. Using whole cells as the enzyme source and not requiring steps such as cell disruption for enzyme purification will further reduce costs. Most of the currently reported cell immobilization methods are carrier immobilization, where cells are adsorbed, embedded, or crosslinked to a carrier, which has problems such as low enzyme activity recovery rate, large mass transfer resistance, few recycling times, and toxicity of crosslinking agents. Therefore, there is an urgent need to develop an effective method for immobilizing cells for the cyclic batch synthesis of galactooligosaccharides.

[0005] Genipin is a natural biological crosslinking agent prepared by hydrolyzing geniposide in Gardenia jasminoides Ellis of Rubiaceae. Its toxicity is 5000 - 10000 times lower than that of the commonly used crosslinking agent glutaraldehyde and can be almost ignored. Currently, there is no report on the carrier - free immobilization of yeast cells with genipin in the prior art, nor is there a report on yeast cells synthesizing galactooligosaccharides using monosaccharides as substrates. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides a carrier - free immobilized yeast cell and its application in the synthesis of galactooligosaccharides.

[0007] The technical solution of the present invention is as follows:

[0008] A method for preparing a carrier - free immobilized yeast cell, comprising the following steps:

[0009] (1) Cultivate yeast producing β - galactosidase and collect yeast cells;

[0010] (2) Suspend the yeast cells with an ethanol solution, stir at 20 - 30 °C for 1 - 10 min, and centrifuge to obtain yeast cells after ethanol treatment;

[0011] (3) Add genipin to the yeast cells after ethanol treatment to construct a cross - linking system;

[0012] (4) React the cross - linking system at 20 - 30 °C and 100 - 150 rpm for 0.5 - 4 h, and centrifuge to obtain carrier - free immobilized yeast cells.

[0013] Preferably according to the present invention, in step (1), the yeast producing β - galactosidase is Kluyveromyces lactis, and the preservation number is CGMCC 2.1494.

[0014] Preferably according to the present invention, in step (2), the concentration of the ethanol solution is 40 - 50%.

[0015] Preferably according to the present invention, in step (2), the mass - to - volume ratio of the yeast cells to the ethanol solution is (20 - 30):1, with the unit of mg / mL.

[0016] Preferably according to the present invention, in step (3), the concentration of genipin in the cross - linking system is 0.05%, and the concentration of the yeast cells after ethanol treatment is 0.05 - 0.2 mg / μL.

[0017] The present invention also provides the carrier - free immobilized yeast cell prepared by the above method, and the application of the carrier - free immobilized yeast cell in the synthesis of galactooligosaccharides.

[0018] The above application comprises the following steps:

[0019] (a) Prepare a galactose solution with a concentration of 20-75%.

[0020] (b) Add carrier-free immobilized yeast cells to the galactose solution, react at 30-65 °C and pH 4.0-8.0 for 2-8 hours, and after centrifugation and solid-liquid separation, obtain galactooligosaccharides.

[0021] Preferably according to the present invention, in step (b), the addition amount of the carrier-free immobilized yeast cells is 20-85% of the mass of galactose.

[0022] The yeast producing β-galactosidase in the present invention is yeast that performs a synthesis reaction using galactose as a substrate, and others not specified are carried out according to the prior art.

[0023] Beneficial effects

[0024] 1. The present invention first uses genipin to directly crosslink with yeast cells to prepare carrier-free immobilized yeast cells, which do not require additional addition of carrier materials, and have the advantages of low cost, easy operation, and high stability. Moreover, genipin, as a crosslinking agent from natural sources, has almost negligible toxicity compared with existing crosslinking agents. Therefore, the carrier-free immobilization process of the present invention is non-toxic, harmless, and environmentally friendly.

[0025] 2. The carrier-free immobilized yeast cells prepared by the present invention have a high number of repeated uses and can efficiently recycle and synthesize galactooligosaccharides for more than 25 batches, which is much higher than the number of uses of existing immobilized yeast applied to lactose hydrolysis or synthesis of galactooligosaccharides using lactose as a substrate, greatly reducing costs, being suitable for large-scale industrial production, and improving efficiency.

[0026] 3. The present invention first uses carrier-free immobilized yeast cells to synthesize galactooligosaccharides using monosaccharides as substrates, which is different from the traditional synthesis process using lactose as a substrate. The reaction product of the present invention using monosaccharides as substrates only generates galactooligosaccharides, and only the remaining monosaccharide substrates exist in the reaction system except for the product, without the hydrolysis side reaction in the lactose reaction system. Therefore, there are no by-products, especially no glucose, which is beneficial for use by diabetic patients. In addition, since the reaction products are only galactooligosaccharides and monosaccharides, the product purification is easy, which is beneficial for obtaining high-purity oligosaccharides and promoting the application of galactooligosaccharides in the pharmaceutical field. Description of the drawings

[0027] Figure 1 It is a scanning electron micrograph of carrier-free immobilized yeast cells;

[0028] In the figure: A is the scanning electron micrograph of free yeast cells, and B is the scanning electron micrograph of genipin-crosslinked immobilized yeast cells.

[0029] Figure 2Effect of genipin concentration on the enzymatic activity recovery rate of immobilized yeast cells.

[0030] Figure 3 Effect of temperature on the activities of free and immobilized yeast cells.

[0031] Figure 4 Effect of temperature on the stabilities of free and immobilized yeast cells.

[0032] Figure 5 TLC detection results of the synthesis of galactooligosaccharides by immobilized yeast cells using monosaccharides as substrates;

[0033] In the figure, 1 is galactose and 2 is the galactooligosaccharide product.

[0034] Figure 6 Effect of the cyclic reaction of immobilized yeast cells on the synthesis of galactooligosaccharides.

[0035] Figure 7 TLC detection results of the synthesis of galactooligosaccharides by immobilized yeast cells using lactose as a substrate;

[0036] In the figure, 1 is lactose, 2 is galactose, 3 is glucose, and 4 is the galactooligosaccharide product. Detailed implementation mode

[0037] The present invention will be further described below in conjunction with the accompanying drawings of the specification and embodiments, but the scope of protection of the present invention is not limited thereto. Those not clearly defined in the embodiments can be according to the existing technologies in the art. "%" not specially stated in the embodiments all refers to mass percentage. The Kluyveromyces lactis with the preservation number of CGMCC 2.1494 described in the present invention is available from the China General Microbiological Culture Collection Center.

[0038] Example 1: Preparation and ethanol treatment of free yeast cells

[0039] 1. Inoculate Kluyveromyces lactis CGMCC 2.1494 onto a slant medium containing 1% glucose, 1% peptone, 0.5% yeast extract, and 1.5% agar. After growing at 30°C for 24 h, transfer it to a liquid seed medium and culture it at 200 rpm / min and 30°C for 24 h. Then transfer it to a fermentation medium containing 1% glucose, 1% peptone, 0.5% yeast extract, and 1% lactose at a volume ratio of 3% and culture it at 30°C for 32 h. After centrifugation, collect the Kluyveromyces lactis cells.

[0040] 2. Prepare an ethanol solution with a concentration of 50% using a 50 mM potassium phosphate buffer at pH 7.5. Suspend Kluyveromyces lactis cells with the ethanol solution to make the concentration of Kluyveromyces lactis cells 25 mg / mL. Then stir for 1 min at 25 °C and centrifuge to obtain ethanol-treated Kluyveromyces lactis cells. Meanwhile, measure the activity of β-galactosidase in the ethanol-treated Kluyveromyces lactis cells.

[0041] The method for measuring the activity of the β-galactosidase: Take yeast cells, add 50 μL of a 50 mM potassium phosphate buffer at pH 7.5 and mix well. Then add 450 μL of 2 mM o-nitrophenyl-β-galactoside, vortex to mix evenly, react in a water bath at 37 °C for 10 min, add 1 mL of 0.5 M Na2CO3 to terminate the reaction, centrifuge, take 1 mL of the supernatant in a cuvette, and detect the absorbance at 420 nm with an ultraviolet spectrophotometer.

[0042] The definition of the enzyme activity unit of β-galactosidase: The amount of enzyme that hydrolyzes 1 μmol of o-nitrophenol released from o-nitrophenyl-β-galactoside in 1 minute is defined as one enzyme activity unit (U).

[0043] Example 2: Preparation of carrier-free immobilized yeast cells

[0044] 1. Prepare a genipin solution using a 50 mM potassium phosphate buffer at pH 7.5. Then add the genipin solution to the ethanol-treated Kluyveromyces lactis cells obtained in Example 2 to construct a cross-linking system, such that the concentration of genipin in the cross-linking system is 0.05% (2.2 mM), and the concentration of ethanol-treated Kluyveromyces lactis cells is 0.1 mg / μL. Then react at 25 °C and 125 rpm for 4 h, centrifuge at 11,000 revolutions per minute for 2 min to obtain carrier-free immobilized yeast cells.

[0045] 2. Measure the activity of β-galactosidase in free cells and carrier-free immobilized yeast cells. The enzyme activity of free cells is 0.66 U / mg, and the enzyme activity of carrier-free immobilized yeast cells is 0.63 U / mg. Calculate that the enzyme activity recovery rate of carrier-free immobilized cells is 95.5%. Conduct a scanning electron microscopy detection and analysis on the carrier-free immobilized cells, and the results are as Figure 1 shown.

[0046] From Figure 1 it can be seen that compared with free cells, the carrier-free immobilized yeast cells have significantly aggregated to form aggregates, indicating that the carrier-free immobilization is successful.

[0047] The method for measuring the activity of the β-galactosidase is the same as that in Example 1.

[0048] The calculation method of the enzyme activity recovery rate: the percentage of the enzyme activity of the immobilized cells to that of the free cells.

[0049] The analysis method of the scanning electron microscope is as follows: resuspend the yeast cells in a potassium phosphate buffer solution with a concentration of 50 mM and a pH of 7.5 to make the final concentration 0.5 mg / mL. Paste the glass slide on the aluminum sample stage with carbon conductive glue, then drop the cell suspension on the glass slide, dry it under an infrared lamp, spray platinum on the surface, and detect and analyze it with a Sirion field emission scanning electron microscope (Netherlands).

[0050] Example 3: Effect of genipin concentration on the enzyme activity recovery rate of immobilized cells

[0051] Prepare a genipin solution with a 50 mM, pH 7.5 potassium phosphate buffer solution, and then add the genipin solution to the ethanol-treated Kluyveromyces lactis cells obtained in Example 2 to construct a cross-linking system, so that the genipin concentrations in the cross-linking system are 0.005, 0.01, 0.05, 0.1, and 0.5% respectively, and the concentration of the ethanol-treated Kluyveromyces lactis cells is 0.1 mg / μL. Then react at 25 °C and 125 rpm for 4 h, centrifuge at 11,000 revolutions per minute for 2 min to obtain carrier-free immobilized yeast cells. Measure the activities of β-galactosidase of free cells and carrier-free immobilized yeast cells, and calculate the enzyme activity recovery rate of carrier-free immobilized cells. The results are as Figure 2 shown.

[0052] It can be seen from Figure 2 that when the genipin concentration is 0.05%, the enzyme activity recovery rate of the obtained immobilized yeast cells is the highest, which is 95.5%. When the genipin concentration is lower or higher than 0.05%, the enzyme activity recovery rate decreases significantly.

[0053] The method for measuring the activity of the β-galactosidase is the same as that in Example 1.

[0054] The calculation method of the enzyme activity recovery rate is the same as that in Example 2.

[0055] Example 4: Effect of temperature on the activity and stability of carrier-free immobilized yeast cells

[0056] 1. Selection of the optimal temperature: Take free and carrier-free immobilized yeast cells, suspend them in 50 μL of a potassium phosphate buffer solution with a pH of 7.5 and a concentration of 50 mM, add 450 μL of 2 mM o-nitrophenyl-β-galactoside, vortex and mix evenly, and react at 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C for 10 min respectively. Add 1 mL of a 0.5 M Na2CO3 solution to terminate the reaction, centrifuge, and take the supernatant to detect OD420 with a spectrophotometer. The results are as Figure 3 shown.

[0057] It can be seen fromFigure 3 It can be seen that the optimal temperatures of free and immobilized yeast cells are 35 °C and 40 °C respectively. And in the high-temperature range, the relative enzyme activity of immobilized cells is higher than that of free cells, indicating that immobilized cells are more heat-resistant than free cells.

[0058] 2. Determination of temperature stability: Take free and carrier-free immobilized yeast cells, suspend them in 50 μL of potassium phosphate buffer at pH 7.5 and 50 mM, and place them at 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C for 2 hours respectively. Then add 450 μL of 2 mM o-nitrophenyl-β-galactoside, vortex and mix evenly, react in a water bath at 37 °C for 10 min, add 1 mL of 0.5 M Na2CO3 to terminate the reaction, centrifuge, and take the supernatant for OD420 detection with a spectrophotometer. The results are as Figure 4 shown.

[0059] From Figure 4 it can be seen that the stability of free cells and immobilized cells is better at 35 °C. When the temperature continues to rise above 45 °C, the stability of immobilized cells is slightly higher than that of free cells.

[0060] Example 5: Synthesis of galactooligosaccharides from monosaccharide substrates by carrier-free immobilized yeast cells

[0061] Prepare a galactose solution with a concentration of 54% using a potassium phosphate buffer at 50 mM and pH 7.5. Add the carrier-free immobilized yeast cells prepared in Example 2, with the addition amount being 74% of the mass of galactose. Then react at 37 °C and pH 7.5 for 6 hours, centrifuge at 11,000 rpm for 2 minutes, and the supernatant is galactooligosaccharides. Perform thin-layer chromatography detection on the obtained galactooligosaccharides. The results are as Figure 5 shown.

[0062] From Figure 5 it can be seen that oligosaccharide product spots are generated below the galactose substrate spots, indicating that the immobilized enzyme can efficiently synthesize galactooligosaccharides using galactose as a substrate, and the yield of galactooligosaccharides can reach 40%.

[0063] The method for the thin-layer chromatography detection is as follows: After spotting on the thin-layer chromatography plate, develop it in the developing agent, spray the color-developing agent, and bake it at 120 °C for 5 minutes to make the sugar spots visible; after the sugar spots are visible, scan the thin plate and perform quantitative analysis through the software ImageJ v1.51k (http: / / rsb.info.nih.gov / ij / ).

[0064] The developing agent is prepared by mixing n-butanol, absolute ethanol, and water in a volume ratio of 5:3:2; the color-developing agent is a 0.5% solution of 3,5-dihydroxytoluene prepared with a 20% sulfuric acid solution by volume percentage.

[0065] Example 6: Synthesis of galactooligosaccharides by carrier - free immobilized yeast cells in a cyclic batch process

[0066] According to the reaction system and conditions of Example 5, the carrier - free immobilized yeast cells were mixed with the galactose substrate, and the first - batch synthesis reaction of galactooligosaccharides was carried out. After the reaction, centrifugation was performed. The obtained immobilized enzyme was resuspended in fresh galactose solution, and the second - batch reaction was continued according to Example 5, and so on for the cyclic batch synthesis reaction.

[0067] The supernatants of all the above batches were detected by thin - layer chromatography, and the yield of galactooligosaccharides was quantitatively analyzed. The reusability of the immobilized cells was evaluated based on the yield of galactooligosaccharides after batch reactions and the enzyme activity of the immobilized yeast cells. The synthesis results of the immobilized enzyme batch reactions are as Figure 6 shown.

[0068] As Figure 6 can be seen, calculated by relative yield, taking the yield of galactooligosaccharides in the first reaction batch as 100%, the relative yield of galactooligosaccharides can reach more than 80% after the carrier - free immobilized yeast cells are recycled 25 times. Taking the initial enzyme activity as 100%, the relative enzyme activity of the immobilized cells still remains above 60% after 25 batch reactions. The above results confirm that the immobilized yeast cells have good repeated reaction activity.

[0069] The above thin - layer chromatography detection and quantitative analysis are the same as in Example 5.

[0070] Comparative Example 1: Preparation of carrier - free immobilized yeast cells using glutaraldehyde as a cross - linker

[0071] 1. A 25% glutaraldehyde solution was added to the ethanol - treated Kluyveromyces lactis cells obtained in Example 2 to construct a cross - linking system, such that the concentration of glutaraldehyde in the cross - linking system was 0.11% (2.2 mM), and the concentration of ethanol - treated Kluyveromyces lactis cells was 0.1 mg / μL. Then the reaction was carried out at 25 °C and 125 rpm for 4 h, and centrifuged at 11000 revolutions per minute for 2 min to obtain carrier - free immobilized yeast cells.

[0072] 2. The activities of β - galactosidase in free cells and carrier - free immobilized yeast cells were measured. The enzyme activity of free cells was 0.66 U / mg, and the enzyme activity of carrier - free immobilized yeast cells was 0.6 U / mg. The calculated enzyme activity recovery rate of carrier - free immobilized cells was 90.9%, which was lower than that of genipin cross - linking.

[0073] Comparative Example 2: Synthesis of galactooligosaccharides using lactose as a substrate by carrier - free immobilized yeast cells

[0074] Prepare a lactose solution with a concentration of 54% using a potassium phosphate buffer at 50 mM and pH 7.5. Then add the carrier-free immobilized yeast cells prepared in Example 2, with the addition amount being 74% of the lactose mass. React at 37 °C and pH 7.5 for 6 h, and then centrifuge at 11,000 revolutions per minute for 2 min. The supernatant is galactooligosaccharide. The obtained galactooligosaccharide is detected by thin-layer chromatography, and the results are as Figure 7 shown.

[0075] It can be Figure 7 seen that oligosaccharide product spots are generated below the lactose substrate spot, and at the same time, spots of glucose and galactose hydrolysis by-products are generated above the lactose spot. However, when using galactose as the substrate in the present invention, no other sugar by-products are generated.

Claims

1. A method for synthesizing galactooligosaccharides by using carrier-free immobilized yeast cells, characterized in that, It includes the following steps: (1) Cultivate yeast producing β-galactosidase and collect yeast cells; (2) Suspend the yeast cells with an ethanol solution, stir at 20 - 30 °C for 1 - 10 min, and centrifuge to obtain yeast cells after ethanol treatment; Among them, the concentration of the ethanol solution is 40 - 50%, and the mass-volume ratio of yeast cells to the ethanol solution is (20 - 30):1, with the unit of mg / mL; (3) Add genipin to the yeast cells after ethanol treatment to construct a cross-linking system; Among them, the concentration of genipin in the cross-linking system is 0.05%, and the concentration of yeast cells after ethanol treatment is 0.05 - 0.2 mg / μL; (4) React the cross-linking system at 20 - 30 °C and 100 - 150 rpm for 0.5 - 4 h, and centrifuge to obtain carrier-free immobilized yeast cells; (5) Prepare a galactose solution with a concentration of 20 - 75%; (6) Add the carrier-free immobilized yeast cells to the galactose solution, react at 30 - 65 °C and pH 4.0 - 8.0 for 2 - 8 h, and after centrifugation and solid-liquid separation, obtain galactooligosaccharides.

2. The method according to claim 1, wherein In step (1), the yeast producing β-galactosidase is Kluyveromyces lactis, with the preservation number of CGMCC 2.1494.

3. The method according to claim 1, wherein In step (6), the addition amount of the carrier-free immobilized yeast cells is 20 - 85% of the mass of galactose.

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