CN109321608B - Application of aureobasidium pullulans in co-production of pullulan polysaccharide and phenethyl alcohol - Google Patents
Application of aureobasidium pullulans in co-production of pullulan polysaccharide and phenethyl alcohol Download PDFInfo
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
- C12P19/10—Pullulan
Abstract
The invention discloses an application of aureobasidium melanogenesis in co-production of pullulan polysaccharide and phenethyl alcohol, improves the tolerance of the aureobasidium melanogenesis to 2-PE by utilizing a fermentation product, and belongs to the technical field of bioengineering fermentation. The Aureobasidium melanogenesis classified name utilized by the invention is Aureobasidium melanogenesis A4, and the preservation number is CGMCC No. 13177. The strain can be applied to the fermentation production of 2-phenethyl alcohol, and simultaneously, another fermentation product pullulan polysaccharide is produced by fermentation, and the product can improve the tolerance of the aureobasidium melanogenesis to the 2-phenethyl alcohol. The fermentation process method also realizes the production of the pullulan polysaccharide and the 2-PE by one-time fermentation, so that the culture medium is fully utilized, the utilization rate of the raw materials is improved, and the fermentation cost is effectively reduced.
Description
Technical Field
The invention relates to an application of aureobasidium pullulans for producing melanin in co-production of pullulan polysaccharide and phenethyl alcohol, belonging to the technical field of bioengineering fermentation.
Background
Pullulan (pullulan), also called pullulan, pullulan or pullulan, is a tasteless and odorless white powder. The polysaccharide has a chemical structure of a straight-chain polysaccharide formed by polymerizing maltotriose repeating units connected by alpha-1, 4 glycosidic bonds through alpha-1, 6 glycosidic bonds, and has a molecular weight of 4.8 × 104-2.2 × 106 (the average molecular weight of commercial pullulan polysaccharide is 2 × 105, and the polysaccharide is composed of about 480 maltotriose). For the unique structural characteristics, the pullulan polysaccharide is endowed with a plurality of important physicochemical characteristics, namely, the pullulan polysaccharide is structurally rich in elasticity, and also has the excellent characteristics of strong film forming property, gas barrier property, plasticity and viscosity, easy water solubility, no toxicity, no harm, no color, no odor and the like. Therefore, the method has wide application in the aspects of food and food, chemical industry, medical treatment and medicine, tobacco, cosmetics, environmental protection, agricultural product preservation and the like.
Phenylethanol (also known as 2-phenylethanol, 2-phenylethanol or β -phenylethanol) is a colourless liquid with a rose fragrance. The rose aroma is widely applied to the food industry because of the elegant, fine and durable rose aroma, and is the second most spice in the current market. Meanwhile, due to the characteristics of water insolubility, stability under alkaline conditions and the like, the compound is also used for blending essence for soaps and cosmetics, and has wide application prospect in the industries of washing products and cosmetics; it can also be used for synthesizing important medical intermediates such as p-hydroxyphenylethanol and the like, and is a potential raw material of bulk chemical raw material styrene. Therefore, the research on the synthetic process of the phenethyl alcohol has important theoretical significance and application value.
Some strains for producing 2-phenethyl alcohol are reported in related researches at home and abroad, but it is known that 2-phenethyl alcohol has certain toxic effect on strain cells, the inhibition degree on the strain cells is increased along with the increase of the product concentration, and finally the amount of the product is reduced. In related researches at home and abroad, in-situ extraction is adopted to avoid toxic effects of products on bacterial strain cells, for example, organic solvents, oleyl alcohol, oleic acid, polypropylene glycol 1200, dibutyl sebacate and the like are additionally added while inoculation is carried out, and 2-PE in fermentation liquor is extracted by using macroporous resin, so that the fermentation cost is increased. The yeast strain (saccharomyces cerevisiae) reported at present is a main production strain of 2-phenethyl alcohol, and when the content of glucose is higher, a large amount of ethanol is produced in the production process, so that the absorption of yeast cells on amino acid and other nutrient substances is influenced. The reported strains only aim at 2-phenethyl alcohol for fermentation, and have the problems of single product, low utilization rate of fermentation raw materials, high energy consumption, high cost and the like. Thus, a method for producing 2-PE by using a fermentation medium and producing a product which improves the tolerance of the strain cell to 2-phenylethyl alcohol in the fermentation process is obtained, and the strain can greatly utilize glucose to be converted into another fermentation product in the fermentation process so as to reduce the production amount of the ethanol and reduce the influence on the permeability of the strain cell membrane. Therefore, the utilization rate of the raw materials is improved, which has extremely important significance for saving energy and reducing the cost of fermentation production.
At present, no article reports that another strain capable of improving the tolerance of strain cells to 2-phenethyl alcohol is produced in the process of producing 2-PE in the prior art, and a fermentation process method that the 2-phenethyl alcohol is produced by using one aureobasidium melanophorum and the tolerance of the strain cells to 2-phenethyl alcohol is improved by producing another product pullulan is produced in the process of producing 2-PE.
Disclosure of Invention
In order to solve the problems that the 2-phenylethyl alcohol produced by fermentation has high cost and low utilization rate of raw materials, and the 2-PE produced by fermentation has toxic and side effects on cells, the invention provides the application of the aureobasidium melanophore in the co-production of pullulan and phenylethyl alcohol;
the Aureobasidium pullulans used in the invention is classified as Aureobasidium melanogenesis A4, the preservation date is 2016, 10 and 24 days, the preservation unit is the common microorganism center of China microorganism culture preservation management committee, the preservation address is No. 3 of No.1 Hospital of Western Lu of Beijing city, North Chengyang, and the preservation number is CGMCC No. 13177.
The invention provides an application of the aureobasidium pullulans strain for producing melanin in the co-production of pullulan polysaccharide and phenethyl alcohol, which comprises the following steps:
the method comprises the following steps: inoculating seeds of Aureobasidium melanosporum A4 to an YPD culture medium for culture, culturing for 24-36 hours at 30 ℃ in a constant-temperature incubator to obtain first-stage seeds, inoculating the obtained first-stage seeds to a shake flask containing a second-stage seed culture medium for culture, and culturing for 12-24 hours at 30 ℃ to obtain second-stage seeds;
step two: and (3) inoculating the secondary seeds obtained in the step one into a fermentation culture medium, adjusting the pH value, continuously supplementing biomass sugar into the culture medium after fermenting for 24-36 h, continuously fermenting for 24-48 h, and separating and purifying two products, namely pullulan polysaccharide and 2-phenylethyl alcohol.
The method for preparing the first-level seed culture medium comprises the following steps: namely YPD culture medium, which comprises the following components: 0.5 (wt)% to 1.5 (wt)% glucose, 1 (wt)% to 1.5 (wt)% yeast extract, 1.5 (wt)% to 2.5 (wt)% peptone, natural pH; the sterilization condition of the culture medium is sterilization at 115 ℃ for 25-35 minutes. .
The method for preparing the secondary seed culture medium comprises the following steps: the composition of the culture medium is: 3 (wt)% -4 (wt)% glucose, 1.5 (wt)% -2.0 (wt)% yeast powder, 2 (wt)% -3 (wt)% peptone, 0.5 (wt)% -1 (wt)% K%2HPO4,0.04(wt)％～0.08(wt)％MgSO4·7H2O,0.5(wt)％～1.2(wt)％NaCl,0.06(wt)％～1.0(wt)％(NH4)2SO4Natural pH. The sterilization condition of the culture medium is sterilization at 115 ℃ for 25-35 minutes.
The method for preparing the fermentation medium comprises the following steps: the aqueous solution comprises the following components: 2.5 (wt)% to 3 (wt)% biomass sugar, 1 (wt)% to 1.5 (wt)% yeast extract, 0.4 (wt)% to 0.6 (wt)% K2HPO4，0.3(wt)％～0.5(wt)％KH2PO4，0.03(wt)％～0.05(wt)％MgSO4·7H20, the pH value is 5.5-6.5, and the sterilization condition is that the sterilization is carried out for 25-35 minutes at 115 ℃.
In the second step: and inoculating the secondary seed culture medium into a fermentation culture medium of fermentation liquor according to the inoculation amount of 4-6% (v/v), wherein the fermentation culture temperature is 30 ℃, the stirring speed during fermentation is 180-250 rpm/min, supplementing 70% (g/mL) biomass sugar into the culture medium after fermenting for 24-36 h, continuing to ferment for 24-48 h, and separating and purifying the products of pullulan and 2-phenylethyl alcohol.
And the pH value is adjusted by hydrochloric acid solution or sodium hydroxide solution.
And the biomass sugar in the second step is one or more than two of cane sugar, maltose or corn hydrolysate.
After the fermentation in the second step is finished, pullulan polysaccharide and 2-phenethyl alcohol can be obtained by separation and purification, and the separation and purification steps are as follows:
centrifuging 5-10 mL of fermentation liquor at 10000rpm for 10min, adding an extracting agent into supernatant, reversing and uniformly mixing for several times, centrifuging at 10000rpm for 10min, and taking supernatant for gas phase detection; adding anhydrous ethanol with the volume twice that of the lower layer;
and step two, after fermentation is carried out for 24-36 h, 70% (g/mL) of biomass sugar is added, 10-20% (v/v) of an extracting agent is added, then the mixture is placed in a constant-temperature incubator at 28 ℃ for continuous culture for 24-48 h, centrifugation is carried out for 10min at 10000 revolutions, the extracting agent is added into supernatant, the mixture is inverted and mixed uniformly for several times, centrifugation is carried out for 10min at 9000 revolutions, and the supernatant is taken for gas phase detection.
The extractant is trialkyl phosphorus oxide (TRPO).
The biomass sugar is glucose, sucrose or maltose, or corn hydrolysate.
The invention has the beneficial effects that:
1. the strain provided by the invention utilizes an optimized fermentation medium, 2-PE is produced by controlling a fermentation process through fermentation, and simultaneously another product pullulan is produced, so that the tolerance of strain cells to 2-phenethyl alcohol can be improved, the full utilization of the fermentation medium is realized, the utilization rate of raw materials is further improved, and the fermentation cost is reduced.
2. The production method for producing pullulan polysaccharide and 2-PE by using the strain provided by the invention has the advantages of mild conditions, environmental friendliness, green and natural products and the like.
3. The method realizes the simultaneous fermentation production of pullulan and 2-PE by utilizing aureobasidium melanogenesis, so that the yield of 2-PE is further improved in the presence of pullulan, thereby improving the utilization rate of raw materials, saving energy and reducing the cost of fermentation production.
Drawings
The Aureobasidium melanogenesis strains utilized by the invention are classified as Aureobasidium melanogenesis A4, and the preservation date is as follows: 24 days 10 months 2016, China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, No. 3 Hospital No.1 Sail No. 3, having a collection number of CGMCC No.13177, in the Beijing area, on the sunward area.
FIG. 1 is a photograph showing the results of qualitative chromatography of pullulan.
FIG. 2 is a qualitative chromatogram of 2-phenylethyl alcohol.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.
The first embodiment is as follows:
the method comprises the following steps: taking a ring of aureobasidium pullulans strain from an YPD plate by using an inoculating ring, and inoculating the ring of aureobasidium pullulans strain into a primary seed culture medium, wherein the aqueous solution of the primary seed culture medium (YPD culture medium) comprises the following components: 1.1 wt% glucose, 1.5 wt% yeast extract, 2.0 wt% peptone, natural pH; the medium was sterilized at 115 ℃ for 25 minutes. The tube was placed in a shaker at 180rpm/min at 30 ℃ for 30 hours while being tilted. The culture solution is used as a first-class seed;
inoculating the primary seeds cultured for 30 hours into a secondary seed culture medium, wherein the inoculation amount is 10% (v/v) of the culture medium, and the aqueous solution of the secondary seed culture medium comprises the following components: 1.5 wt% glucose, 1.8 wt% yeast powder, 2.5 wt% peptone, 0.6 wt% K2HPO4,0.06(wt)％MgSO4·7H2O,0.5(wt)％NaCl,1.0(wt)％(NH4)2SO4Natural pH; the medium was sterilized at 115 ℃ for 25 minutes. Adding according to the inoculation amount of 5% (v/v); then placing the shake flask in a shaking table with the temperature of 30 ℃ and the rpm of 200/min for culturing for 20 hours, and obtaining a secondary seed solution;
step two: inoculating the secondary seed solution into 50mL of fermentation medium according to the inoculation amount of 5% (v/v), wherein the fermentation medium comprises the following components: 2.5 wt% glucose, 1.6 wt% yeast extract, 0.6 wt% K2HPO4，0.5(wt)％KH2PO4，0.05(wt)％MgSO4·7H2O, pH 6, and medium sterilization at 115 ℃ for 30 minutes. The inoculated fermentation medium was placed on a shaker at 30 ℃ and 220rpm/min for 10 hours, then 0mL and 6mL of 70% (w/v, g/mL) glucose was added, and the mixture was further placed on a shaker at 28 ℃ and 180rpm/min for 36 hours.
(4) After fermentation is finished, respectively measuring 25mL of fermentation liquor, then adding 5mL of extraction agent TRPO, centrifuging at 10000rpm/min for 10min, dividing the fermentation liquor into 3 layers, taking an upper layer organic solvent to measure that the content of 2-PE is 0.42g/L and 0.52g/L respectively, pouring a second layer into a new centrifugal tube and adding 2 times of volume of absolute ethyl alcohol to obtain extracellular polysaccharide, measuring the content of the extracellular polysaccharide to be 28.62 g/L and 39.7g/L, improving the yield of pullulan polysaccharide in the fermentation liquor, and increasing the tolerance of strain cells to 2-phenethyl alcohol, thereby increasing the yield.
Example two:
the method comprises the following steps: taking an aureobasidium pullulans strain for producing melanin, inoculating the aureobasidium pullulans strain into a primary seed culture medium (YPD culture medium), wherein the primary seed culture medium (YPD culture medium) is weighed as (wt): 1.1% glucose, 1.5% yeast extract, 2.0% peptone, natural pH; the medium was sterilized at 115 ℃ for 25 minutes. The tube was placed in a shaker at 180rpm/min at 30 ℃ for 30 hours while being tilted. The culture solution is used as a first-class seed;
inoculating the primary seeds cultured for 30 hours into a secondary seed culture medium, wherein the inoculation amount is 10% (v/v) of the culture medium, and the aqueous solution of the secondary seed culture medium comprises the following components: 1.5 wt% glucose, 1.8 wt% yeast powder, 2.5 wt% peptone, 0.6 wt% K2HPO4,0.06(wt)％MgSO4·7H2O,0.5(wt)％NaCl,1.0(wt)％(NH4)2SO4Natural pH; the medium was sterilized at 115 ℃ for 25 minutes. Adding according to the inoculation amount of 5% (v/v); then placing the shake flask in a shaking table with the temperature of 30 ℃ and the rpm of 200/min for culturing for 20 hours, and obtaining a secondary seed solution;
step two: inoculating the secondary seed solution into 100mL of hair in an amount of 7% (v/v)In the fermentation medium, the fermentation medium comprises the following components (wt): 2.5% maltose, 1.5% yeast extract, 0.8% K2HPO4，0.5％KH2PO4，0.07％MgSO4·7H2O, pH 6, and medium sterilization at 115 ℃ for 30 minutes. The inoculated fermentation medium was incubated for 8 hours at 30 ℃ on a 220rpm/min shaker, then 0mL, 6mL 70% (w/v, g/mL) maltose was added and the incubation was continued for 36 hours at 28 ℃ on a 180rpm/min shaker.
(4) After fermentation is finished, 50mL of fermentation liquor is measured respectively, 10mL of extraction agent TRPO is added, centrifugation is carried out at 12000rpm/min for 10min, the fermentation liquor is divided into 3 layers, the upper layer organic solvent is taken to measure the content of 2-PE to be 0.6g/L and 0.68g/L respectively, the second layer is poured into a new centrifugal tube, 2 times of volume of absolute ethyl alcohol is added, extracellular polysaccharide is obtained, the content of the extracellular polysaccharide is measured to be 30.1g/L and 40.8g/L respectively, the yield of pullulan in the fermentation liquor is improved, the tolerance of strain cells to 2-phenethyl alcohol is improved, and therefore the yield is increased.
Example three:
the method comprises the following steps: taking an aureobasidium pullulans strain for producing melanin, inoculating the aureobasidium pullulans strain into a primary seed culture medium, wherein the primary seed culture medium (YPD culture medium) is weighed as (wt): 1.1% glucose, 1.5% yeast extract, 2.0% peptone, natural pH; the medium was sterilized at 115 ℃ for 25 minutes. The tube was placed in a shaker at 180rpm/min at 28 ℃ for 30 hours. The culture solution is used as a first-class seed;
inoculating the primary seeds cultured for 30 hours into a secondary seed culture medium, wherein the inoculation amount is 10% (v/v) of the culture medium, and the aqueous solution of the secondary seed culture medium comprises the following components: 1.5 wt% glucose, 1.8 wt% yeast powder, 2.5 wt% peptone, 0.6 wt% K2HPO4,0.06(wt)％MgSO4·7H2O,0.5(wt)％NaCl,1.0(wt)％(NH4)2SO4Natural pH; the medium was sterilized at 115 ℃ for 25 minutes. Adding according to the inoculation amount of 5% (v/v); then placing the shake flask in a shaking table at 28 ℃ and 200rpm/min for culturing for 20 hours, and obtaining a secondary seed solution;
step two: inoculating the secondary seed solution into 100mL of fermentation medium according to the inoculation amount of 7% (v/v), wherein the fermentation medium comprises the following components (wt): 2.5% of sucrose, 1.6% of yeast extract and 0.8% of K2HPO4，0.5％KH2PO4，0.07％MgSO4·7H2O, pH 6, and medium sterilization at 115 ℃ for 30 minutes. The inoculated fermentation medium was placed in a shaker at 30 ℃ and 220rpm/min for 6 hours, then 0mL, 6mL 70% (w/v, g/mL) sucrose was added and the incubation continued in a shaker at 27 ℃ and 180rpm/min for 36 hours.
(4) After fermentation is finished, 50mL of fermentation liquor is measured respectively, 10mL of extraction agent TRPO is added, centrifugation is carried out at 12000rpm/min for 10min, the fermentation liquor is divided into 3 layers, the upper layer organic solvent is taken to measure the content of 2-PE to be 0.8g/L and 0.94g/L respectively, the second layer is poured into a new centrifugal tube, 2 times of volume of absolute ethyl alcohol is added, extracellular polysaccharide is obtained, the content of the extracellular polysaccharide is measured to be 33.32g/L and 41.45g/L respectively, the yield of pullulan in the fermentation liquor is improved, the tolerance of strain cells to 2-phenethyl alcohol is improved, and therefore the yield is increased.
Example four: identification of pullulan
(1) Dissolving the fermentation product (exopolysaccharide) and the pullulan into deionized water according to the proportion of 1g/10mL to obtain a sample solution and a pullulan standard solution; dissolving maltotriose and glucose in deionized water according to the proportion of 10 mg/mL; a display system: acetonitrile and distilled water according to volume ratio n-butanol: acetic acid: mixing distilled water at a ratio of 8:3:2, and placing in a glass bottle with a plug for later use. Color developing agent: concentrated sulfuric acid and absolute ethyl alcohol are mixed according to the volume ratio of concentrated sulfuric acid: and (3) uniformly mixing the absolute ethyl alcohol V in a ratio of 1:6, and filling the mixture into a spray can for later use.
(2) Respectively diluting the pullulan standard solution, the pullulan hydrolysate, the sample solution and the sample enzymolysis solution by 100 times, carrying out sample application on a silica gel chromatography plate according to the sequence of the pullulan standard solution, the pullulan hydrolysate, maltotriose, the sample enzymolysis solution, the sample solution and glucose, carrying out chromatography in a developing solution, taking out and drying after the liquid is chromatographed to the top of the silica gel plate, and repeating the chromatography for 2-3 times. And spraying the color developing agent onto the silica gel plate after chromatography, drying, heating and developing on an alcohol lamp, and observing experimental phenomena. The results indicated that the product obtained was pullulan (fig. 1).
Example five: qualitative detection of 2-phenylethyl alcohol
(1) And (3) adding 10% (v/v) n-hexane into the fermentation liquor after the fermentation is finished, placing the fermentation liquor in a shaking table at 25 ℃ for overnight extraction for 12 hours, centrifuging the fermentation liquor for 10min at 1000 revolutions, and detecting the 2-phenethyl alcohol by a gas chromatography-mass spectrometer through a filtering membrane of supernate.
(2) The detected chromatogram is shown in FIG. 2, and it is found that the strain can produce 2-PE.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (7)
1. The application of the aureobasidium melanogenesis in the coproduction of pullulan and phenethyl alcohol is characterized in that the aureobasidium melanogenesis is classified as aureobasidium melanogenesis (Aureobasidium melanogenesis:)Aureobasidium melanogenum) A4, date of deposit: 24 days at 10 months in 2016, the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, No. 3 Hospital No.1 Sail Luo of North Chen of the Chaoyang region in Beijing, with the collection number of CGMCC No. 13177;
the application comprises the following steps: the method comprises the following steps: aureobasidium pullulans (A) for producing melaninAureobasidium melanogenum) Inoculating the seeds of A4 into YPD culture medium to obtain primary seeds, inoculating the obtained primary seeds into a shake flask containing secondary seed culture medium to culture to obtain secondary seeds;
step two: inoculating the secondary seeds obtained in the step one into a fermentation culture medium, adjusting the pH value, continuously supplementing biomass sugar into the culture medium after fermenting for 24-36 h, continuously fermenting for 24-48 h, and measuring the concentrations of 2-phenethyl alcohol and extracellular polysaccharide;
step three: and (3) adding an extractant TRPO into the fermentation liquor obtained after the fermentation in the step two, centrifuging for 20min at 10000rpm, dividing the fermentation liquor into 3 layers, taking the supernatant, performing gas phase detection on 2-phenethyl alcohol, and adding 2 times of volume of absolute ethyl alcohol for separation and purification to obtain pullulan.
2. The use according to claim 1, wherein the YPD seed medium of step one comprises the following components: 0.5 (wt)% to 1.5 (wt)% glucose, 1 (wt)% to 1.5 (wt)% yeast extract, 1.5 (wt)% to 2.5 (wt)% peptone, natural pH.
3. The use of claim 1, wherein the secondary seed medium of step one comprises the following: 3 to 4 wt% glucose, 1.5 to 2.0 wt% yeast powder, 2 to 3 wt% peptone, 0.5 to 1 wt% K2HPO4, 0.04（wt）%~0.08（wt）% MgSO4·7H2O, 0.5（wt）%~1.2（wt）% NaCl, 0.06（wt）%~1.0（wt）% (NH4)2SO4Natural pH.
4. The use of claim 1, wherein the fermentation medium of step two comprises the following components in percentage by mass: 2.5 wt% -3 wt% biomass sugar, 1 wt% -1.5 wt% yeast extract, 0.4 wt% -0.6 wt% K2HPO4，0.3（wt）%~0.5（wt）% KH2PO4，0.03（wt）%~0.05（wt）% MgSO4·7H2O, the pH value is 5.5-6.5.
5. The use of claim 1, wherein the secondary seed obtained in step two is inoculated into the fermentation medium in an amount of 4 (v/v)% -7 (v/v)%.
6. The use according to claim 1, wherein the pH adjustment in step two is carried out with a hydrochloric acid solution or a sodium hydroxide solution.
7. The use of claim 1, wherein the biomass sugar in step two is one or more of sucrose, maltose or corn hydrolysate.
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