Recombinant pichia pastoris and application thereof in synthesis of stachyose and verbascose

Abstract

The invention provides recombinant pichia pastoris and application thereof in synthesis of stachyose and verbascose, and belongs to the technical field of biology. According to the invention, the recombinant pichia pastoris capable of obtaining the alpha-1, 6-galactoside transferase with high transglycosylation activity through fermentation culture is obtained for the first time; the recombinant pichia pastoris BLCY-014 takes sucrose and raffinose as substrates, alpha-galactoside is transferred to sucrose molecules to synthesize stachyose and verbascose, and fermentation and conversion conditions of alpha-1, 6-galactoside transferase are explored through a single factor experiment, so that stachyose and verbascose have a relatively high conversion rate.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a recombinant Pichia yeast and application thereof in synthesizing stachyose and verbascose. Background Art

[0002] Stachyose is a naturally occurring tetrasaccharide with a molecular formula of C 24 H 42 O 21 It is a non-reducing functional oligosaccharide formed by connecting two galactoses on the glucose side of sucrose with an α-1,6 glycosidic bond. The sweetness is 22% of sucrose, with a refreshing taste, no odor, and good thermal stability. In addition, stachyose is not decomposed by human gastrointestinal digestive juices and is a water-soluble dietary fiber. Stachyose can absorb gastrointestinal toxic substances and pathogens, regulate human intestinal flora, increase the proliferation of beneficial bacteria, inhibit lead absorption, improve the environment in the digestive tract, promote the absorption of trace elements such as calcium and magnesium, and enhance the body's immunity. As a functional oligosaccharide, stachyose is widely used in functional foods.

[0003] Currently, the main method for preparing stachyose is plant extraction. Because the material contains salt, protein, fiber, carbohydrates and other substances that are difficult to remove, the process is more complicated and the yield is low.

[0004] Verbascose, a galacto-oligosaccharide, is a pentasaccharide composed of three galactose molecules bonded to one side of the glucose residue of sucrose by an α-1,6 glycosidic bond. It is a member of the raffinose family with a molecular formula of C 30 H 52 O 26 Verbascum sugar can promote the proliferation of bifidobacteria, improve the intestinal microecological environment to prevent constipation, promote mineral absorption, improve the body's immunity, and also has physiological functions such as preventing tooth decay and lowering cholesterol.

[0005] There are two ways to obtain verbascose: one is enzymatic synthesis, but the enzyme activity and selectivity currently used are poor; the other is natural extraction. Verbascose is mainly distributed in leguminous plants, but leguminous plants contain many types of soluble oligosaccharides and many large molecular impurities, and the refining process is complex and energy-intensive.

[0006] Currently, α-galactosidase in enzymatic synthesis is an exoglycosidase that catalyzes the hydrolysis of α-galactosidic bonds and generally has no or only weak transglycosidation activity.

[0007] Therefore, there is an urgent need for a method for biosynthesizing stachyose and verbascose with high transglycosidic activity. Summary of the invention

[0008] In view of this, the purpose of the present invention is to provide a recombinant Pichia pastoris and its use in the synthesis of stachyose and verbascose. The present invention is the first to obtain a recombinant Pichia pastoris that can obtain an α-1,6-galactosyltransferase with high transglycosidation activity through fermentation culture; the recombinant Pichia pastoris BLCY-014 uses sucrose and raffinose as substrates to transfer α-galactoside to the sucrose molecule to synthesize stachyose and verbascopentaose.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] In a first aspect, the present invention provides a recombinant Pichia pastoris BLCY-014, wherein the Pichia pastoris ( Pichia pastoris ) BLCY-014 was deposited in the China Center for Type Culture Collection on December 4, 2024, with the address at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the strain collection number is CCTCC NO: M 20242723.

[0011] The present invention uses recombinant Pichia pastoris as a fermentation strain, and its gene is derived from Aspergillus niger.

[0012] In a second aspect, the present invention provides the use of the above-mentioned recombinant Pichia pastoris BLCY-014 in the synthesis of stachyose and verbascose.

[0013] The present invention discovers through experiments that the recombinant Pichia pastoris BLCY-014 obtains α-1,6-galactosyltransferase with high transglycosidation activity after fermentation and culture, and uses sucrose and raffinose as substrates to transfer α-galactoside to sucrose molecules to synthesize stachyose and verbascos pentose.

[0014] In a third aspect, the present invention provides a method for synthesizing stachyose and verbascose, comprising the following steps:

[0015] S1. The recombinant Pichia pastoris BLCY-014 described above is subjected to primary seed culture and secondary seed culture to obtain a seed solution;

[0016] S2. The seed solution is cultured in the growth stage and the induction stage in sequence and then filtered to obtain an α-1,6-galactosyltransferase solution;

[0017] S3. mixing the α-1,6-galactosyltransferase solution with the complex sugar solution to perform a conversion reaction to obtain a stachyose / verbascoside crude sugar solution;

[0018] S4. Decolorizing, ion exchanging, concentrating and drying the stachyose / verbascose crude sugar solution in sequence to obtain stachyose / verbascose.

[0019] Preferably, the specific method of primary seed culture in S1 is: first inoculate the recombinant Pichia pastoris BLCY-014 on a plate culture medium for culture, then pick a single colony of the recombinant Pichia pastoris into a YPD liquid culture medium, and culture overnight at 30°C and 200-400 rpm to obtain a culture solution.

[0020] Preferably, the specific method of culturing the secondary seeds in S1 is: aspirating the culture solution into BMGY medium, culturing at 30°C and 200-400 rpm for 12-24 h until the OD 600 When it reaches 4~5, you get seed liquid.

[0021] More preferably, the volume ratio of the culture solution to BMGY medium is 1:100.

[0022] Preferably, the specific method of the growth stage culture in S2 is: adding seed solution to the culture medium and adjusting the pH to 5-8, then adding glycerol while culturing until the glycerol is completely consumed to obtain a growth culture solution;

[0023] The dissolved oxygen content during the culture process is 20-40%; the culture temperature is 28-30°C.

[0024] More preferably, the rotation speed of the culture in the growth stage is 300 rpm and the initial ventilation volume is 1.0-1.5 (v / v).

[0025] The flow rate of the glycerol addition is 12-16 mL / h, and the addition time is 24-72 h.

[0026] Preferably, the culture medium consists of: 1% yeast extract powder, 2% peptone, 0.3% K2HPO4, 1.18% KH2PO4, 1.34% YNB, 0.1% 500× biotin;

[0027] The preparation method of 500× biotin is as follows: dissolve 0.02 g of biotin in distilled water to make up to 100 mL, filter through a sterilized 0.22 μm filter membrane, and store at 4°C.

[0028] Preferably, the specific method of the induction stage culture in S2 is: adding methanol to the growth culture solution under the condition of 20-40% dissolved oxygen for induction culture, and filtering to obtain α-1,6-galactosyltransferase solution after the methanol is completely consumed;

[0029] The method of adding methanol is as follows:

[0030] First, add the solution at a flow rate of 10-14 mL / h for 4 h; then add it at a flow rate of 22-26 mL / h for 3 h; and finally add it at a flow rate of 28-32 mL / h for up to 120 h.

[0031] Preferably, the preparation method of the complex sugar solution in S3 is: prepare 500 g / L of sucrose solution and raffinose solution respectively, then mix the sucrose solution and the raffinose solution in a volume ratio of 1:6-1:2 and adjust the pH to 2.0-8.0.

[0032] Preferably, the amount of α-1,6-galactosyltransferase solution added in S3 is 2-6% of the dry weight of the complex sugar solution;

[0033] The conversion temperature is 40-60°C, the conversion speed is 300 rpm, and the conversion time is 10-20 h.

[0034] Preferably, the specific method of decolorization in S4 is: decolorization by activated carbon adsorption; wherein the amount of activated carbon added is 1-3% of the dry basis mass, the pH value during the decolorization process is 5-7, the decolorization temperature is 60-80°C, and the decolorization time is 1-3h;

[0035] The specific method of ion exchange is: using strong acid cation resin D001 and weak base anion resin D301P in series for desalination; the ratio of strong acid cation resin D001 and weak base anion resin D301P is 2:1, the desalination temperature is 30~40 ℃; the desalination pH is 5~7; the desalination flow time is 0.5~1 h.

[0036] The specific method of concentration is: using the four-effect concentration method to concentrate the sugar solution to 60~70%.

[0037] The specific method of drying is: drying by belt drying method.

[0038] Beneficial technical effects:

[0039] The present invention obtains for the first time a recombinant Pichia pastoris capable of obtaining an α-1,6-galactosyltransferase with high transglycosidation activity through fermentation culture; the recombinant Pichia pastoris BLCY-014 uses sucrose and raffinose as substrates to transfer α-galactoside to the sucrose molecule to synthesize stachyose and verbascosose.

[0040] Description of biological deposit:

[0041] The recombinant Pichia pastoris BLCY-014 of the present invention, the Pichia pastoris ( Pichia pastoris ) BLCY-014 was deposited in the China Center for Type Culture Collection on December 4, 2024, with the address at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the strain collection number is CCTCC NO: M 20242723. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1This is a diagram showing the effects of dissolved oxygen, induction time, and induction temperature on enzyme activity during fermentation culture; A is the effect of dissolved oxygen on enzyme activity; B is the effect of induction temperature on enzyme activity; and C is the effect of induction time on enzyme activity.

[0043] Figure 2 Figure 3 is the effect of pH and temperature on enzyme activity during the conversion process; A is the effect of conversion temperature on enzyme activity; B is the effect of conversion pH on enzyme activity. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further illustrated by the following examples.

[0045] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0046] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the main idea or essential features of the present invention. Therefore, from all points of view, the embodiments should be regarded as exemplary and non-restrictive, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention, and any reference numerals in the claims should not be regarded as limiting the claims involved.

[0047] In addition, it should be understood that although this specification is described according to the implementation modes, not every implementation mode includes only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation modes that can be understood by those skilled in the art. These other implementation modes are also covered within the protection scope of the present invention.

[0048] It should also be understood that the specific embodiments described above are only used to explain the present invention, and the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention, which should be covered by the protection scope of the present invention / invention.

[0049] Strain source: The present invention uses recombinant Pichia pastoris as the fermentation strain, whose gene is derived from Aspergillus niger ( Aspergillus niger ).

[0050] Unless otherwise specified, the equipment or raw materials used in the following examples can be commercially available.

[0051] Example 1

[0052] Effects of dissolved oxygen, induction time and induction temperature on enzyme activity during fermentation

[0053] S1. Cultivation process:

[0054] (1) Primary seed cultivation:

[0055] First, the recombinant Pichia pastoris BLCY-014 was inoculated on a plate medium for culture, and then a single colony of the recombinant Pichia pastoris was picked up and placed in 20 mL of YPD liquid medium, and cultured overnight at 30° C. and 300 rpm to obtain a culture solution.

[0056] (2) Secondary seed cultivation:

[0057] Pipette 4 mL of culture solution into a 2 L shake flask containing 400 mL of BMGY medium and continue culturing at 30 °C and 300 rpm for 16 h until the OD reaches 0. 600 When the value reaches 4.5, seed solution is obtained.

[0058] S2. Preparation of α-1,6-galactosyltransferase solution

[0059] (1) Growth stage cultivation:

[0060] Prepare 3.5 L of culture medium, place it in a 10 L fermenter, sterilize it at 121 °C for 20 min, reduce the temperature to 28-32 °C (maintain this temperature for cultivation), add seed solution, adjust the pH to 6 with ammonia water, rotate at 300 rpm, set the initial ventilation volume to 1.3 (v / v), maintain the dissolved oxygen at 20-40%, then add glycerol at a flow rate of 14 mL / h for 24-72 h. After the addition is completed, culture until the glycerol is completely consumed and the dissolved oxygen is greater than 80% to obtain growth culture medium.

[0061] The culture medium consists of: 1% yeast extract powder, 2% peptone, 0.3% K2HPO4, 1.18% KH2PO4, 1.34% YNB, 0.1% 500× biotin;

[0062] The preparation method of 500× biotin is as follows: dissolve 0.02 g of biotin in distilled water to make up to 100 mL, filter through a sterilized 0.22 μm filter membrane, and store at 4°C.

[0063] (2) Induction stage culture:

[0064] Methanol is added to the growth culture solution for induction culture, and after the methanol is completely consumed, the α-1,6-galactosyltransferase solution is obtained by filtration; the culture temperature and dissolved oxygen content are kept consistent with those in the growth stage culture;

[0065] Stream acceleration is performed as follows:

[0066] Add at a flow rate of 12 mL / h for 4 h; then at a flow rate of 24 mL / h for 3 h; then at a flow rate of 30 mL / h for 120 h.

[0067] (3) Enzyme activity detection and conversion degree detection:

[0068] The specific detection method of enzyme activity is in accordance with the industry standard "NY / T 4361-2023 Spectrophotometric method for the determination of α-galactosidase activity in feed additives". Enzyme activity definition: The amount of enzyme required to release 1 μmol of p-nitrophenol per minute from a 5 mmol / L p-nitrophenyl-α-D-pyranogalactoside solution at 37°C and pH 5.5.

[0069] Based on the above experimental steps, the single factor experiment parameters were set as follows: ① dissolved oxygen: 20%, 25%, 30%, 35%, 40%; ② induction temperature: 28℃, 29℃, 30℃, 31℃, 32℃; ③ culture time in the growth stage: 24 h, 36 h, 48 h, 60 h, 72 h.

[0070] according to Figure 1 As shown, A is the effect of dissolved oxygen on enzyme activity; B is the effect of induction temperature on enzyme activity; C is the effect of induction time on enzyme activity. When the fixed induction temperature is 29°C and the induction starts at 48h, the effect of dissolved oxygen on enzyme activity after tanking during the culture process is explored. The results show that when the dissolved oxygen is maintained at 35%, the enzyme activity can reach a maximum of U / mL. When the dissolved oxygen is maintained at 35% and the induction is started at 48h, the effect of the induction temperature on the enzyme activity after tanking is explored. The results show that the highest enzyme activity is obtained when induced at 29°C. When the dissolved oxygen is maintained at 35% and the induction is carried out at 29°C, the effect of the induction time on the activity is explored. The results show that the highest enzyme activity is obtained when the induction is carried out at 48h.

[0071] Example 2

[0072] The effects of dissolved oxygen, induction time and induction temperature on the hydrolysis characteristics of the enzyme during fermentation culture were investigated by referring to the preparation method of the α-1,6-galactosyltransferase solution and the single-factor control conditions in Example 1.

[0073] The enzymatic hydrolysis test method is:

[0074] 500 g / L raffinose solution was used as substrate, 3% crude enzyme solution was added, and the reaction was carried out at 45 °C and 300 rpm for 8 h. The enzyme was then inactivated, the salt was removed by ionization, and the components were measured by dilution through a membrane.

[0075] The results of the enzymatic hydrolysis experiments are shown in Table 1.

[0076] Table 1 Effects of dissolved oxygen, induction time and induction temperature on enzyme hydrolysis characteristics during fermentation

[0077]

[0078] Example 3

[0079] The effects of dissolved oxygen, induction time, and induction temperature on the enzyme transglycoside characteristics during fermentation culture were investigated by referring to the α-1,6-galactosyltransferase solution preparation method and single factor control conditions of Example 1.

[0080] The experimental method for enzyme transglycosidation characteristics is:

[0081] Prepare 500 g / L raffinose solution and sucrose solution at a ratio of 4:1 (v / v) to obtain a mixed sugar solution. Use the mixed solution as a substrate, add 3% enzyme solution on a dry basis, and react at 45 °C and 300 rpm. Take samples at 8h and 12h, inactivate the enzyme, desalt by ionization, and dilute to pass through a membrane for component determination.

[0082] The results of the enzyme transamination characteristic experiment are shown in Table 2.

[0083] Table 2 Effects of dissolved oxygen, induction time and induction temperature on enzyme transglycoside characteristics during fermentation

[0084]

[0085] Example 4

[0086] Effects of pH and temperature on enzyme activity during transformation

[0087] The conversion method is:

[0088] 500 g / L raffinose solution and sucrose solution were prepared respectively, and the sucrose solution and the raffinose solution were prepared in a volume ratio of 1:4 to obtain a complex sugar solution, and the pH was adjusted to 2.0-8.0, and 3% α-1,6-galactosyltransferase solution was added to the complex sugar solution at a dry basis of 40-60 ° C and a rotation speed of 300 rpm, and the reaction was carried out for 8 h or 12 h to obtain stachyose or verbascose crude sugar solution.

[0089] The stachyose / verbascose crude sugar liquid is sequentially subjected to decolorization, ion exchange, concentration and drying to obtain stachyose / verbascose.

[0090] The specific method of decolorization is: decolorization by activated carbon adsorption method; the amount of activated carbon added is 1-3% of the dry basis weight, the pH value during the decolorization process is 5-7, the decolorization temperature is 60-80°C, and the decolorization time is 1-3 h;

[0091] The specific method of ion exchange is: using strong acid cation resin D001 and weak base anion resin D301P in series for desalination; the ratio of strong acid cation resin D001 and weak base anion resin D301P is 2:1, the desalination temperature is 30~40 ℃; the desalination pH is 5~7; the desalination flow time is 0.5~1 h.

[0092] The specific method of concentration is: using the four-effect concentration method to concentrate the sugar solution to 60~70%.

[0093] The specific method of drying is: drying by belt drying method.

[0094] Single-factor experiment construction: ① When the conversion temperature was 45℃, different pH conditions were set to 2, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, and 8 to explore the optimal pH of the enzyme; ② When the conversion pH was 4.5, different temperature conditions were set to 30, 35, 40, 45, 50, 55, 60, 65, and 70℃ to explore the optimal temperature of the enzyme.

[0095] The effects of pH and temperature on enzyme activity during the conversion process are shown in Figure 2 .

[0096] according to Figure 2 As shown, A is the effect of conversion temperature on enzyme activity; B is the effect of conversion pH on enzyme activity; pH ​​and temperature have a great influence on enzyme activity. When the temperature is 45°C and the pH is 4.5, the enzyme activity reaches the maximum.

[0097] Example 5

[0098] Effects of enzyme dosage and reaction time on enzymatic hydrolysis characteristics during conversion

[0099] The conversion method is:

[0100] 500 g / L raffinose solution and sucrose solution were prepared respectively, and the sucrose solution and the raffinose solution were prepared in a volume ratio of 1:4 to obtain a complex sugar solution, and the pH was adjusted to 4.5. 1-5% α-1,6-galactosyltransferase solution was added to the complex sugar solution at a dry basis of 45 ° C and a rotation speed of 300 rpm, and the reaction was carried out for 6-14 h to obtain a stachyose / verbascoside crude sugar solution.

[0101] The stachyose / verbascose crude sugar liquid is sequentially subjected to decolorization, ion exchange, concentration and drying to obtain stachyose / verbascose.

[0102] The specific method of decolorization is: decolorization by activated carbon adsorption method; the amount of activated carbon added is 2% of the dry basis weight, the pH value during the decolorization process is 6, the decolorization temperature is 70 °C, and the decolorization time is 2 h;

[0103] The specific method of ion exchange is: using strong acid cation resin D001 and weak base anion resin D301P in series for desalination; the ratio of strong acid cation resin D001 and weak base anion resin D301P is 2:1, the desalination temperature is 35°C; the desalination pH is 6; the desalination flow time is 0.7 h.

[0104] The specific method of concentration is: using the four-effect concentration method to concentrate the sugar solution to 65%.

[0105] The specific method of drying is: drying by belt drying method.

[0106] The single-factor experiment was constructed as follows: ① First, the effect of the amount of enzyme added on the enzymatic hydrolysis characteristics was investigated. Different enzyme concentrations were set: 1, 2, 3, 4, and 5%. Samples were taken after 8 hours of reaction. ② Secondly, the effect of reaction time on the hydrolysis characteristics was investigated. The amount of enzyme added was 2%.

[0107] The experimental results show that when the enzyme dosage is 3%, the raffinose has been completely hydrolyzed into galactose and sucrose after 8 hours of hydrolysis, and no glucose is produced. When the enzyme dosage is 2%, the raffinose has been completely hydrolyzed after 10 hours of hydrolysis. Based on the above results, it can be seen that both the enzyme dosage and the reaction time affect the degree of hydrolysis. Therefore, the optimal group is selected, with 3% enzyme dosage and 8 hours of reaction.

[0108] The effects of enzyme addition amount and reaction time on the enzymatic hydrolysis characteristics during the conversion process were tested. The test results are shown in Table 3

[0109] Table 3 Effects of enzyme dosage and reaction time on enzymatic hydrolysis characteristics during conversion

[0110]

[0111] Example 6

[0112] The effect of enzyme addition amount and reaction time on the enzyme transglycoside characteristics during the conversion process was explored by referring to the conversion method and single factor control conditions of Example 4.

[0113] Single-factor experiment construction: ① First, explore the effect of enzyme addition on the transglycosidation characteristics, set different enzyme addition concentrations: 1, 2, 3, 4, 5%, and take samples after 8h and 12h of reaction; ② Secondly, explore the effect of reaction time on transglycosidation characteristics, take samples after 4, 6, 8, 10, 12, 14, 14h after reaction, and the enzyme addition amount is 3%. ③ Finally, explore the effect of substrate ratio on transglycosidation characteristics, and set the parameters to 1:2, 1:3, 1:4, 1:5, 1:6. The results are shown in Table 4.

[0114] The experimental results show that verbascose begins to be generated at 8h and the stachyose content begins to decrease. Therefore, the stachyose content reaches the maximum at 8h of reaction. As the transglycosidation reaction proceeds, the verbascose content is the maximum at 12h and then begins to decrease. Samples were taken for testing at 8h. When the substrate ratio was 1:4 and the enzyme addition amount was 3%, the stachyose content was the highest. Similarly, samples were taken for testing at 12h. When the substrate ratio was 1:4 and the enzyme addition amount was 3%, the verbascose content was the highest.

[0115] Table 4 Effects of substrate ratio, enzyme dosage and reaction time on enzyme transglycoside characteristics during transformation

[0116]

[0117] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant Pichia pastoris BLCY-014, characterized in that: The Pichia pastoris ( Pichia pastoris ) BLCY-014 was deposited in the China Center for Type Culture Collection on December 4, 2024, with the address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the strain collection number is CCTCC NO: M 20242723.

2. Use of the recombinant Pichia pastoris BLCY-014 according to claim 1 in the synthesis of stachyose and verbascose.

3. The use according to claim 2, characterized in that: The method for synthesizing stachyose and verbascose comprises the following steps: S1. The recombinant Pichia pastoris BLCY-014 according to claim 1 is subjected to primary seed culture and secondary seed culture to obtain a seed solution; S2. The seed solution is cultured in the growth stage and the induction stage in sequence and then filtered to obtain an α-1,6-galactosyltransferase solution; S3. mixing the α-1,6-galactosyltransferase solution with the complex sugar solution to perform a conversion reaction to obtain a stachyose / verbascoside crude sugar solution; S4. Decolorizing, ion exchanging, concentrating and drying the stachyose / verbascose crude sugar solution in sequence to obtain stachyose / verbascose.

4. The method according to claim 3, characterized in that The specific method of the primary seed culture in S1 is: first inoculate the recombinant Pichia pastoris BLCY-014 on a plate culture medium for culture, then pick a single colony of the recombinant Pichia pastoris into a YPD liquid culture medium, and culture overnight at 30° C. and 200-400 rpm to obtain a culture solution.

5. The method according to claim 4, characterized in that The specific method of culturing the secondary seeds in S1 is as follows: aspirate the culture solution into BMGY medium, and culture at 30°C and 200-400 rpm for 12-24 h until the OD 600 When it reaches 4~5, you get seed liquid.

6. The method according to claim 5, characterized in that The specific method of the growth stage culture in S2 is: adding seed solution to the culture medium and adjusting the pH to 5-8, then adding glycerol while culturing until the glycerol is completely consumed to obtain a growth culture solution; The dissolved oxygen content during the culture process is 20-40%; the culture temperature is 28-30°C; The flow rate of the glycerol addition is 12-16 mL / h, and the addition time is 24-72 h.

7. The method according to claim 6, characterized in that The culture medium consists of: 1% yeast extract powder, 2% peptone, 0.3% K2HPO4, 1.18% KH2PO4, 1.34% YNB, 0.1% 500× biotin; The preparation method of 500× biotin is as follows: dissolve 0.02 g of biotin in distilled water to make up to 100 mL, filter through a sterilized 0.22 μm filter membrane, and store at 4°C.

8. The method according to claim 6, characterized in that The specific method of the induction stage culture in S2 is: adding methanol to the growth culture solution for induction culture, and filtering to obtain α-1,6-galactosyltransferase solution after the methanol is completely consumed; The culture temperature and dissolved oxygen content are consistent with those in the growth stage; The method of adding methanol is as follows: First, add the solution at a flow rate of 10-14 mL / h for 4 h; then add it at a flow rate of 22-26 mL / h for 3 h; and finally add it at a flow rate of 28-32 mL / h for up to 120 h.

9. The method according to claim 3, characterized in that: The preparation method of the composite sugar solution in S3 is: prepare 500 g / L of sucrose solution and raffinose solution respectively, then mix the sucrose solution and the raffinose solution in a volume ratio of 1:6-1:2 and adjust the pH to 2.0-8.

0.

10. The method according to claim 3, characterized in that: The amount of α-1,6-galactosyltransferase solution added in S3 is 2-6% of the dry weight of the complex sugar solution; The conversion temperature is 40-60°C, the conversion speed is 300 rpm, and the conversion time is 10-20 h.

Images