CN110819543A - Aureobasidium pullulans for producing polymalic acid by using starch and application thereof - Google Patents
Aureobasidium pullulans for producing polymalic acid by using starch and application thereof Download PDFInfo
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Abstract
The invention discloses aureobasidium pullulans for producing polymalic acid by using starch and application thereof, wherein the classification of the aureobasidium pullulans is named as aureobasidium pullulans (A)Aureobasidium pullulans) GXL-1 with a preservation number of CCTCC NO: m2018519, preservation date of 2018, 08 and 01 months, preservation unit: china center for type culture Collection, collection address: wuhan, Wuhan university. Aureobasidium pullulans of the present invention (Aureobasidium pullulans) GXL-1 can directly utilize raw starch or starch liquefied liquid as a carbon source, 57g/L polymalic acid can be obtained by batch fermentation at 30 ℃ under the condition of not adding a fermentation promoter and a growth factor, and melanin is not generated in the fermentation process.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and particularly relates to aureobasidium pullulans for producing polymalic acid by using starch and application thereof.
Background
Poly (β -L-malic acid), PMA]The method for synthesizing the polymalic acid by the biological method has the advantages of complex steps, more byproducts, smaller molecular weight of products and higher cost, and the operation for synthesizing the polymalic acid by the chemical method is simple, the byproducts are less, the molecular weight of the products is larger and the cost is lower, so the method for synthesizing the polymalic acid by the biological method is the research hotspot at presentPhysarum polycephalum) And Aureobasidium pullulans: (Aureobasidium pullulans) Have been proved to be useful for the fermentative production of polymalic acid. Wherein the output of polymalic acid produced by fermentation of the multi-headed fungus is about 3.3g/L, and the molecular weight can reach 50-300 kDa; the aureobasidium pullulans has strong capacity of synthesizing polymalic acid, the single-batch fermentation yield is 12-60 g/L, and the molecular weight is 4-11 kDa. Compared with the polymalic acid with higher molecular weight, the low molecular weight polymalic acid has better water solubility, biodegradability and biocompatibility. Therefore, the synthesis of the polymalic acid by using the aureobasidium pullulans has development and application values in the aspects of polymalic acid yield and molecular weight.
Aureobasidium pullulans, a class of fungi belonging to the Deuteromycetes, is a polymorphic fungus with a yeast type and a hyphal type, and is widely distributed throughout the world. Usually most of the Aureobasidium pullulans produce melanin, commonly known as the black yeast fungus. Aureobasidium pullulans can be metabolized to produce various products such as polymalic acid, pullulan, enzyme preparations, melanin, aureobasidin and the like. The strain is the core of the fermentation industry, so researchers at home and abroad do a lot of work in the breeding aspect of aureobasidium pullulans strains for producing polymalic acid. For example, patent CN 102220248 screens a strain of aureobasidium pullulans ZD-3D from the surface of a plant leaf, uses glucose (12%, w/v) as a carbon source, and performs batch fermentation for 168 hours in a shake flask at 25 ℃ under the condition of adding a fermentation accelerator sodium fumarate, wherein the yield of polymalic acid is 62.27 g/L; the patent CN 102827778 obtains an aureobasidium pullulans strain FMT1801 by natural breeding, molasses (12%, w/v) is used as a carbon source, batch fermentation is carried out in a fermentation tank at 25 ℃ for 96h, and the yield of polymalic acid is 34.4 g/L; the patent CN 101979499 obtains an aureobasidium pullulans TKPM00006 strain through ultraviolet mutagenesis technology, glucose (12%, w/v) is used as a carbon source, batch fermentation is carried out for 7-12 days in a shake flask at 25 ℃, and the yield of polymalic acid is 18.4 +/-1.3 g/L.
As can be seen, at present, many researchers obtain a plurality of aureobasidium pullulans strains capable of synthesizing polymalic acid through natural breeding or mutation breeding, but through comparison, the strains have some defects in the process of producing the polymalic acid through fermentation: high-concentration glucose is used as a carbon source, so that on one hand, the cost of raw materials is high, and on the other hand, the high-concentration glucose has a substrate inhibition effect on the growth of thalli so as to limit the improvement of the yield of the polymalic acid; when molasses is used as a carbon source, the molasses has a dark color and a high impurity content, so that the cost of raw materials is reduced, but the cost of extraction and purification of polymalic acid and treatment of fermentation waste liquid is increased. And (II) although the yield of the polymalic acid is high and can reach 62.27g/L, sodium fumarate is required to be added into the culture medium as a fermentation promoter, so that the cost of fermentation raw materials is increased. And (III) a certain amount of corn steep liquor or corn leachate is required to be added into the fermentation culture media of the three aureobasidium pullulans, so that the cost of fermentation raw materials is increased, and the influence on the fermentation process is uncertain due to the complex components and difficult quality control of the corn steep liquor or the corn leachate. And (IV) the fermentation temperature of the three aureobasidium pullulans is 25 ℃, and the microbial fermentation is an exothermic process, so that the lower fermentation temperature needs to be maintained by providing a large amount of cooling water, thereby increasing the running cost of fermentation equipment.
In order to solve the problems of the fermentation production of polymalic acid by using Aureobasidium pullulans, the applicant has screened and separated an Aureobasidium pullulans (Aureobasidium pullulans) GXZ-6 which can ferment high-yield polymalic acid by using cassava starch, corn starch, wheat starch, soluble starch, starch saccharification liquid and maltose syrup as a carbon source in earlier researches, and filed patent applications, the application date of which is as follows: 11/20/2017, application No.: 201711159740.X, title of the invention: an Aureobasidium pullulans for high-yield polymalic acid and its application are disclosed. Although Aureobasidium pullulans GXZ-6 can be used for producing polymalic acid by fermenting cassava starch, corn starch, wheat starch, soluble starch, starch saccharification liquid and maltose syrup as carbon sources and can be fermented in batches at 30 ℃ to obtain more than 60g/L of polymalic acid, melanin is produced in the fermentation process, and the melanin produced in the fermentation process usually causes difficulty in downstream separation and extraction of metabolites and influences the yield and the appearance of the polymalic acid product.
Disclosure of Invention
In order to solve the problems of the fermentation production of polymalic acid by using aureobasidium pullulans, the invention aims to provide aureobasidium pullulans (A) (or (B)) for efficiently producing the polymalic acid by using raw starch or starch liquefaction liquidAureobasidium pullulans) GXL-1 and its use in the production of polymalic acid. Aureobasidium pullulans: (Aureobasidium pullulans) The GXL-1 can be fermented in batches to obtain 57g/L polymalic acid at most without adding a fermentation promoter and a growth factor, and melanin is not generated in the fermentation process, so that the method has the advantages of low fermentation cost, easy product purification and simple process, and has great industrial production potential.
Aureobasidium pullulans for producing polymalic acid by using starch is classified and named as Aureobasidium pullulans (A.pullulans)Aureobasidium pullulans) GXL-1 with a preservation number of CCTCC NO: m2018519, preservation date of 2018, 08 and 01 months, preservation unit: china center for type culture Collection, collection address: wuhan, Wuhan university.
Aureobasidium pullulans: (Aureobasidium pullulans) GXL-1 has the following properties:
1. and (3) colony morphology characteristics:
culturing on PDA culture medium plate at 30 deg.C, wherein the colony surface is white viscous, gradually turns into light pink, and hypha extending into the agar appears at the edge, and the whole colony turns into pink. The PDA culture medium is prepared from 200g of potato, 20g of glucose and 20g of agar, and distilled water is added to the mixture to reach a constant volume of 1L.
2. Physiological and biochemical characteristics:
the strain GXL-1 can grow quickly at 25-34 ℃, and the optimal culture temperature is 30 ℃; growth factors do not need to be added into the culture medium; can grow in basic or semi-synthetic culture medium such as Chase, Saccharum sinensis Roxb, yeast sucrose, starch, oil, citrate, casein, etc., but can not grow in gelatin and glucose acetate culture medium.
3. ITS sequence analysis:
the ITS sequences are amplified and sequenced by using fungus ITS universal amplification primers ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCCTCC GCT TAT TGA TAT GC-3'), and the sequence length is 540 bp. Submitting the obtained sequence to GenBank database to obtain sequence number GenBank ID: MK041290, and performing Blast comparison analysis on the sequence and the gene sequence provided by GenBank to obtain the strain GXL-1 and Aureobasidium pullulans (A), (B), (C) and (C)Aureobasidium pullulans) The homology was 100%. Combined with colony morphological characteristics, physiological and biochemical characteristics and ITS sequence analysis, the strain GXL-1 can be classified and identified as aureobasidium pullulans, in particular to aureobasidium pullulans (A)Aureobasidium pullulans）GXL-1。
Aureobasidium pullulans: (Aureobasidium pullulans) The ITS nucleotide sequence of GXL-1 is a nucleotide sequence shown in a DNA sequence table.
Aureobasidium pullulans: (Aureobasidium pullulans) GXL-1 utilizes starch to produce polymalic acid, and comprises the following steps:
(1) activating strains: aureobasidium pullulans preserved in glycerol (A)Aureobasidium pullulans) GXL-1 is streaked on a PDA slant culture medium, and is cultured for 2-3 days at a constant temperature of 28-30 ℃.
(2) Seed culture: scraping a ring on the activated strain slant, and inoculating the ring into a 500mL shake flask filled with 80-100 mL liquid seed culture medium. And culturing for 36-48 h at the rotating speed of 160-220 rpm of the shaking table and the temperature of 25-30 ℃ to obtain a seed solution.The liquid seed culture medium is 80-100 g/L glucose, 1-2g/L ammonium chloride, 0.4-0.6 g/L KCl, KH2PO40.05～0.2g/L，MgSO4·7H2O 0.1～0.3g/L，ZnSO4·7H2O 0.05～0.2g/L，CaCO310～30g/L。
(3) Fermentation culture: inoculating the seed solution obtained by culturing into a 250mL triangular flask filled with 30-40 mL liquid fermentation medium according to the inoculation amount of 8-10% (v/v), and culturing for 8-9 days at the conditions of the rotating speed of a shaking table of 160-220 rpm and the temperature of 25-30 ℃ to obtain a fermentation solution. The liquid fermentation medium is a carbon source of 120-160 g/L, ammonium chloride of 1-3 g/L, KCl of 0.4-0.6 g/L and KH2PO40.05～0.2g/L，MgSO4·7H2O 0.2～0.6g/L，ZnSO4·7H2O 0.05～0.2g/L，CaCO330～50g/L。
(4) Extracting polymalic acid: centrifuging the fermentation liquor to remove thalli and residual calcium carbonate, collecting supernatant, adding equal volume of absolute ethyl alcohol, slightly stirring, standing for 1h, centrifuging to remove polysaccharide precipitate, collecting supernatant, continuously adding equal volume of absolute ethyl alcohol, standing overnight at 4 ℃, centrifuging, collecting precipitate, washing the precipitate with 80% ethanol, and freeze-drying to obtain the polymalic acid.
The carbon source of the liquid fermentation medium is any one or the combination of more than two of cassava starch, wheat starch, corn starch and starch liquefied liquid.
The starch liquefaction liquid is prepared by gelatinizing cassava starch, wheat starch or corn starch and then liquefying the gelatinized starch by α -amylase, wherein the specific process conditions are that the starch and water are prepared according to the mass-volume ratio of 1: 5, the pH value is adjusted to 5.5 by hydrochloric acid, the mixture is heated and continuously stirred to be pasty, the temperature is reduced to 60 ℃, the commercially available α -amylase (more than or equal to 3700 activity units/g) is added according to the mass ratio of the enzyme to the starch of 1: 400, and the starch liquefaction liquid is obtained by stirring and enzymolysis for 60min at 60 ℃.
The invention has the beneficial effects that:
aureobasidium pullulans of the present invention (Aureobasidium pullulans) GXL-1 can be used for producing polymalic acid by fermentation of raw starch or starch liquefaction liquid, compared with the fermentation of grapeFor producing polymalic acid by glucose fermentation, the cost of raw materials is lower, and the problem that high-concentration glucose generates inhibition effect on the growth of thalli is solved; compared with the method for producing polymalic acid by utilizing molasses fermentation, the method has the advantages of less raw material impurities and contribution to reducing the cost of extracting and purifying polymalic acid and treating fermentation waste liquid. In addition, Aureobasidium pullulans (A), (B), (C), (Aureobasidium pullulans) In the process of producing polymalic acid by GXL-1 fermentation, a metabolic promoter (such as sodium fumarate) or a growth factor (such as corn steep liquor) is not required to be added, the strain has simple nutritional requirement and low fermentation cost, can meet the requirement of industrial large-scale production, and has remarkable economic benefit.
Aureobasidium pullulans of the present invention (Aureobasidium pullulans) GXL-1 can be used for producing polymalic acid by fermentation at a higher temperature of 30 ℃, and the highest yield can reach 57 g/L. Compared with the production of the polymalic acid by fermenting at 25 ℃ of most aureobasidium pullulans, the yield of the polymalic acid is higher, the consumption of cooling water in the fermentation process can be greatly reduced, and the industrial production value is better.
In the fermentation method of patent application 201711159740.X, although sodium nitrate is added to the fermentation medium, a substantial increase in polymalic acid yield can be achieved without a substantial increase in production costs. However, sodium nitrate is irritating to the skin and mucous membranes, toxic, and strongly oxidizing, and can cause combustion and explosion by contact, friction or impact with organic substances or phosphorus and sulfur. According to the invention, ammonium chloride is used as a nitrogen source instead of sodium nitrate in the liquid seed culture medium and the liquid fermentation culture medium, and although the yield is slightly reduced, the ammonium chloride is safer and has lower cost. Aureobasidium pullulans: (Aureobasidium pullulans) GXL-1 can be directly fermented to obtain high-yield polymalic acid by using starch liquefied liquid prepared by α -amylase liquefaction after gelatinization as a carbon source, and the step of saccharifying the starch liquefied liquid by β -saccharifying enzyme or fungal amylase is not needed, so that the production cost is further reduced, melanin is not generated in the fermentation process, the product is easy to purify, and the obtained product has good appearance and high yield.
Detailed Description
In order to describe the present invention in more detail, the present invention will be further described with reference to the following examples.
Example 1
Aureobasidium pullulans: (Aureobasidium pullulans) The method for producing the polymalic acid by using the cassava starch by the GXL-1 comprises the following steps:
(1) activating strains: aureobasidium pullulans preserved in glycerol (A)Aureobasidium pullulans) GXL-1 was streaked on PDA slant medium and cultured at 30 ℃ for 2 days.
(2) Seed culture: scraping a ring on the activated strain inclined plane, inoculating the ring into a 500mL shaking flask filled with 80mL liquid seed culture medium, and culturing for 36h at the rotating speed of 220rpm of a shaking table and the temperature of 25 ℃ to obtain a seed solution. The liquid seed culture medium is 100g/L glucose, 2g/L ammonium chloride, 0.6g/L KCl, KH2PO40.2g/L，MgSO4·7H2O 0.3g/L，ZnSO4·7H2O 0.2g/L，CaCO330g/L。
(3) Fermentation culture: inoculating the seed solution obtained by the culture into a 250mL triangular flask filled with 30mL of liquid fermentation medium according to the inoculation amount of 8% (v/v), and culturing for 9 days at the rotating speed of a shaking table of 220rpm and the temperature of 25 ℃ to obtain a fermentation liquid. The liquid fermentation culture medium is 120g/L of cassava starch, 1g/L of ammonium chloride, 0.6g/L of KCl and KH2PO40.2g/L，MgSO4·7H2O0.6g/L，ZnSO4·7H2O 0.2g/L，CaCO340g/L。
(4) Extracting polymalic acid: centrifuging the fermentation liquor to remove thalli and residual calcium carbonate, collecting supernatant, adding equal volume of absolute ethyl alcohol, slightly stirring, standing for 1h, centrifuging to remove polysaccharide precipitate, collecting supernatant, continuously adding equal volume of absolute ethyl alcohol, standing overnight at 4 ℃, centrifuging, collecting precipitate, washing the precipitate with 80% ethanol, and freeze-drying to obtain the polymalic acid.
Example 2
Aureobasidium pullulans: (Aureobasidium pullulans) GXL-1 utilizes wheat starch to produce polymalic acid, and comprises the following steps:
(1) activating strains: aureobasidium pullulans preserved in glycerol (A)Aureobasidium pullulans) GXL-1 was streaked on PDA slant medium and cultured at 30 ℃ for 2 days.
(2) Seed culture: scraping a ring on the activated strain inclined plane, inoculating the ring into a 500mL shaking flask filled with 80mL liquid seed culture medium, and culturing for 36h at the rotating speed of 220rpm of a shaking table and the temperature of 25 ℃ to obtain a seed solution. The liquid seed culture medium is 100g/L glucose, 2g/L ammonium chloride, 0.6g/L KCl, KH2PO40.2g/L，MgSO4·7H2O 0.3g/L，ZnSO4·7H2O 0.2g/L，CaCO330g/L。
(3) Fermentation culture: inoculating the seed solution obtained by the culture into a 250mL triangular flask filled with 30mL of liquid fermentation medium according to the inoculation amount of 8% (v/v), and culturing for 9 days at the rotating speed of a shaking table of 220rpm and the temperature of 25 ℃ to obtain a fermentation liquid. The liquid fermentation medium is wheat starch 120g/L, ammonium chloride 1g/L, KCl 0.6g/L, KH2PO40.2g/L，MgSO4·7H2O0.6g/L，ZnSO4·7H2O 0.2g/L，CaCO340g/L。
(4) Extracting polymalic acid: centrifuging the fermentation liquor to remove thalli and residual calcium carbonate, collecting supernatant, adding equal volume of absolute ethyl alcohol, slightly stirring, standing for 1h, centrifuging to remove polysaccharide precipitate, collecting supernatant, continuously adding equal volume of absolute ethyl alcohol, standing overnight at 4 ℃, centrifuging, collecting precipitate, washing the precipitate with 80% ethanol, and freeze-drying to obtain the polymalic acid.
Example 3
Aureobasidium pullulans: (Aureobasidium pullulans) GXL-1 utilizes corn starch to produce polymalic acid, and comprises the following steps:
(1) activating strains: aureobasidium pullulans preserved in glycerol (A)Aureobasidium pullulans) GXL-1 was streaked on PDA slant medium and cultured at 30 ℃ for 2 days.
(2) Seed culture: scraping a ring on the activated strain inclined plane, inoculating the ring into a 500mL shaking flask filled with 80mL liquid seed culture medium, and culturing for 36h at the rotating speed of 220rpm of a shaking table and the temperature of 25 ℃ to obtain a seed solution. The liquid seed culture medium is 100 g/ml glucoseL, ammonium chloride 2g/L, KCl 0.6g/L, KH2PO40.2g/L，MgSO4·7H2O 0.3g/L，ZnSO4·7H2O 0.2g/L，CaCO330g/L。
(3) Fermentation culture: inoculating the seed solution obtained by the culture into a 250mL triangular flask filled with 30mL of liquid fermentation medium according to the inoculation amount of 8% (v/v), and culturing for 9 days at the rotating speed of a shaking table of 220rpm and the temperature of 25 ℃ to obtain a fermentation liquid. The liquid fermentation medium is corn starch 120g/L, ammonium chloride 1g/L, KCl 0.6g/L, KH2PO40.2g/L，MgSO4·7H2O0.6g/L，ZnSO4·7H2O 0.2g/L，CaCO340g/L。
(4) Extracting polymalic acid: centrifuging the fermentation liquor to remove thalli and residual calcium carbonate, collecting supernatant, adding equal volume of absolute ethyl alcohol, slightly stirring, standing for 1h, centrifuging to remove polysaccharide precipitate, collecting supernatant, continuously adding equal volume of absolute ethyl alcohol, standing overnight at 4 ℃, centrifuging, collecting precipitate, washing the precipitate with 80% ethanol, and freeze-drying to obtain the polymalic acid.
Example 4
Aureobasidium pullulans: (Aureobasidium pullulans) The method for producing the polymalic acid by using the cassava starch liquefied liquid by GXL-1 comprises the following steps:
(1) activating strains: aureobasidium pullulans preserved in glycerol (A)Aureobasidium pullulans) GXL-1 was streaked on PDA slant medium and incubated at 28 ℃ for 2 days.
(2) Seed culture: scraping a ring on the activated strain inclined plane, inoculating the ring into a 500mL shaking flask filled with 100mL liquid seed culture medium, and culturing for 36h under the conditions that the rotation speed of a shaking table is 180rpm and the temperature is 30 ℃ to obtain seed liquid. The liquid seed culture medium is 80g/L glucose, 1g/L ammonium chloride, 0.5g/L KCl, KH2PO40.1g/L，MgSO4·7H2O 0.1g/L，ZnSO4·7H2O 0.05g/L，CaCO320g/L。
(3) Inoculating the cultured seed solution into a 250mL triangular flask containing 40mL liquid fermentation medium according to the inoculation amount of 10% (v/v), and rotating on a shaking tableCulturing at 180rpm and 30 deg.C for 8 days to obtain fermentation broth. The liquid fermentation medium is starch liquefied liquid 160g/L, ammonium chloride 3g/L, KCl 0.5g/L, KH2PO40.1g/L，MgSO4·7H2O 0.1g/L，ZnSO4·7H2O 0.2g/L，CaCO330g/L。
(4) Extracting polymalic acid: centrifuging the fermentation liquor to remove thalli and residual calcium carbonate, collecting supernatant, adding equal volume of absolute ethyl alcohol, slightly stirring, standing for 1h, centrifuging to remove polysaccharide precipitate, collecting supernatant, continuously adding equal volume of absolute ethyl alcohol, standing overnight at 4 ℃, centrifuging, collecting precipitate, washing the precipitate with 80% ethanol, and freeze-drying to obtain the polymalic acid.
The cassava starch liquefied liquid is prepared by gelatinizing cassava starch and then liquefying the cassava starch by α -amylase, wherein the specific process conditions are that after the cassava starch and water are prepared according to the mass-volume ratio of 1: 5, hydrochloric acid is used for adjusting the pH value to 5.5, the mixture is heated and continuously stirred to be pasty, when the temperature is reduced to 60 ℃, the commercially available α -amylase (more than or equal to 3700 activity units/g) is added according to the mass ratio of the enzyme to the cassava starch of 1: 400, and the mixture is stirred and enzymolyzed for 60min at 60 ℃ to obtain the cassava starch liquefied liquid.
Example 5
Aureobasidium pullulans: (Aureobasidium pullulans) The method for producing the polymalic acid by using the wheat starch liquefied liquid by GXL-1 comprises the following steps:
(1) activating strains: aureobasidium pullulans preserved in glycerol (A)Aureobasidium pullulans) GXL-1 was streaked on PDA slant medium and incubated at 28 ℃ for 2 days.
(2) Seed culture: scraping a ring on the activated strain inclined plane, inoculating the ring into a 500mL shaking flask filled with 100mL liquid seed culture medium, and culturing for 36h under the conditions that the rotation speed of a shaking table is 180rpm and the temperature is 30 ℃ to obtain seed liquid. The liquid seed culture medium is 80g/L glucose, 1g/L ammonium chloride, 0.5g/L KCl, KH2PO40.1g/L，MgSO4·7H2O 0.1g/L，ZnSO4·7H2O 0.05g/L，CaCO320g/L。
(3) The seed liquid obtained by the culture was mixed in an amount of 10% (v/v)Inoculating the inoculum size into a 250mL triangular flask filled with 40mL liquid fermentation medium, and culturing for 8 days at the conditions of the rotating speed of a shaking table of 180rpm and the temperature of 30 ℃ to obtain fermentation liquor. The liquid fermentation medium is starch liquefied liquid 160g/L, ammonium chloride 3g/L, KCl 0.5g/L, KH2PO40.1g/L，MgSO4·7H2O 0.1g/L，ZnSO4·7H2O 0.2g/L，CaCO330g/L。
(4) Extracting polymalic acid: centrifuging the fermentation liquor to remove thalli and residual calcium carbonate, collecting supernatant, adding equal volume of absolute ethyl alcohol, slightly stirring, standing for 1h, centrifuging to remove polysaccharide precipitate, collecting supernatant, continuously adding equal volume of absolute ethyl alcohol, standing overnight at 4 ℃, centrifuging, collecting precipitate, washing the precipitate with 80% ethanol, and freeze-drying to obtain the polymalic acid.
The wheat starch liquefied liquid is prepared by gelatinizing wheat starch and then liquefying with α -amylase, wherein the specific process conditions comprise that after the wheat starch and water are prepared according to the mass-volume ratio of 1: 5, the pH value is adjusted to 5.5 by hydrochloric acid, the mixture is heated and continuously stirred to be pasty, when the temperature is reduced to 60 ℃, the commercially available α -amylase (more than or equal to 3700 activity units/g) is added according to the mass ratio of 1: 400 of the enzyme and the wheat starch, and the mixture is stirred and enzymolyzed for 60min at 60 ℃ to obtain the wheat starch liquefied liquid.
Example 6
Aureobasidium pullulans: (Aureobasidium pullulans) The method for producing the polymalic acid by using the liquefied corn starch by GXL-1 comprises the following steps:
(1) activating strains: aureobasidium pullulans preserved in glycerol (A)Aureobasidium pullulans) GXL-1 was streaked on PDA slant medium and incubated at 28 ℃ for 2 days.
(2) Seed culture: scraping a ring on the activated strain inclined plane, inoculating the ring into a 500mL shaking flask filled with 100mL liquid seed culture medium, and culturing for 36h under the conditions that the rotation speed of a shaking table is 180rpm and the temperature is 30 ℃ to obtain seed liquid. The liquid seed culture medium is 80g/L glucose, 1g/L ammonium chloride, 0.5g/L KCl, KH2PO40.1g/L，MgSO4·7H2O 0.1g/L，ZnSO4·7H2O 0.05g/L，CaCO320g/L。
(3) Inoculating the cultured seed solution into a 250mL triangular flask filled with 40mL liquid fermentation medium according to the inoculation amount of 10% (v/v), and culturing for 8 days at the conditions of the rotating speed of a shaking table of 180rpm and the temperature of 30 ℃ to obtain a fermentation liquid. The liquid fermentation medium is starch liquefied liquid 160g/L, ammonium chloride 3g/L, KCl 0.5g/L, KH2PO40.1g/L，MgSO4·7H2O 0.1g/L，ZnSO4·7H2O 0.2g/L，CaCO330g/L。
(4) Extracting polymalic acid: centrifuging the fermentation liquor to remove thalli and residual calcium carbonate, collecting supernatant, adding equal volume of absolute ethyl alcohol, slightly stirring, standing for 1h, centrifuging to remove polysaccharide precipitate, collecting supernatant, continuously adding equal volume of absolute ethyl alcohol, standing overnight at 4 ℃, centrifuging, collecting precipitate, washing the precipitate with 80% ethanol, and freeze-drying to obtain the polymalic acid.
The corn starch liquefied liquid is prepared by gelatinizing corn starch and liquefying the corn starch by α -amylase, wherein the specific process conditions are that the corn starch and water are prepared according to the mass-volume ratio of 1: 5, the pH value is adjusted to 5.5 by hydrochloric acid, the mixture is heated and continuously stirred to be pasty, when the temperature is reduced to 60 ℃, the commercially available α -amylase (more than or equal to 3700 activity units/g) is added according to the mass ratio of the enzyme to the corn starch of 1: 400, and the mixture is stirred and enzymolyzed for 60min at 60 ℃ to obtain the corn starch liquefied liquid.
The polymalic acids obtained in examples 1 to 6 were respectively subjected to yield detection by the following methods: adding 2M sulfuric acid with the same volume into the fermentation supernatant, and hydrolyzing at constant temperature of 90 ℃ overnight. Then taking malic acid in the hydrolyzed solution to be analyzed by high performance liquid chromatography; the chromatographic column adopts C18 chromatographic column, and the mobile phase is 50mM KH2PO4The solution was injected at a flow rate of 0.7mL/min and a sample size of 5. mu.L at a temperature of 25 ℃. The detection results are as follows:
sequence listing
<110> Guangxi university
<120> aureobasidium pullulans for producing polymalic acid by using starch and application thereof
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<213> Aureobasidium pullulans (Aureobasidium pullulans)
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Claims (5)
1. An aureobasidium pullulans for producing polymalic acid by using starch is characterized in that: the Classification of said Aureobasidium pullulans is named as Aureobasidium pullulans (A)Aureobasidium pullulans) GXL-1 with a preservation number of CCTCC NO: m2018519, preservation date of 2018, 08 and 01 months, preservation unit: china center for type culture Collection, collection address: wuhan, Wuhan university.
2. The aureobasidium pullulans for producing polymalic acid by using starch according to claim 1, wherein: said Aureobasidium pullulans (A), (B), (C)Aureobasidium pullulans) GXL-1 can be used for producing polymalic acid by fermenting with one or more of tapioca starch, wheat starch, corn starch and starch liquefied liquid as carbon source.
3. The aureobasidium pullulans for producing polymalic acid by using starch as claimed in claim 2, wherein the starch liquefaction liquid is prepared by gelatinizing tapioca starch, wheat starch or corn starch and then carrying out α -amylase liquefaction.
4. The aureobasidium pullulans for producing polymalic acid by using starch according to claim 2, wherein: said Aureobasidium pullulans (A), (B), (C)Aureobasidium pullulans) GXL-1 does not produce melanin in the process of producing polymalic acid by fermentation using starch as a carbon source.
5. Use of the aureobasidium pullulans of any one of claims 1 to 4 (A), (B)Aureobasidium pullulans) Application of GXL-1 in production of polymalic acid.
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| CN112094752B (en) * | 2020-09-16 | 2022-02-15 | 广西大学 | Method for producing ultra-low molecular weight pullulan by fermenting aureobasidium pullulans |
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