CN104805160A - Method for synthesizing MELs (Mannosylerythrutol lipids) through bioconversion of kitchen waste oil - Google Patents
Method for synthesizing MELs (Mannosylerythrutol lipids) through bioconversion of kitchen waste oil Download PDFInfo
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- CN104805160A CN104805160A CN201510171790.4A CN201510171790A CN104805160A CN 104805160 A CN104805160 A CN 104805160A CN 201510171790 A CN201510171790 A CN 201510171790A CN 104805160 A CN104805160 A CN 104805160A
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Abstract
The invention discloses a method for synthesizing MELs (Mannosylerythrutol lipids) through bioconversion of kitchen waste oil. The method comprises steps as follows: Pseudozyma aphidis DSMZ70725 is inoculated into a fermentation medium and subjected to fermentation cultivation to obtain the MELs, and the fermentation medium contains the kitchen waste oil. According to the method, the kitchen waste oil is adopted to synthesize the MELs through bioconversion, the substrate is domestic waste, and the product is safe, non-toxic and environment-friendly; compared with edible vegetable oil, the kitchen waste oil has a higher grease conversion rate in the bioconversion synthesis process, so that the method effectively reduces the production cost, meets the environmental protection concept of waste material recycling, waste reduction and resource saving, has the characteristic of sustainable development and facilitates industrial production.
Description
Technical field
The present invention relates to biotechnology and field of microbial fermentation, particularly relate to a kind of method utilizing kitchen abendoned oil bio-transformation synthesis mannosylerythritol lipid.
Background technology
Bio-surfactant not only has the various surface propertys that chemical surfactant has, but also has following advantages: (1) selectivity is wide, environmentally friendly; (2) the huge and chemical structure of complexity make surfactivity and emulsifying capacity stronger; (3) molecular structure type is various, and have many special functional groups, specificity is strong; (4) raw material extensively to exist and inexpensive at nature; (5) fermentative production is typical " green " technique etc.
Mannosylerythritol lipid (Mannosylerythritollipids, MELs) is a kind of novel non-ionic type bio-surfactant, is the less class glycolipid of present studies in China.Mannosylerythritol lipid is applied to environmental protection, food, makeup, as emulsifying agent, tensio-active agent, flour quality improver, wetting Agent for Printing Inks, anti-agglomerating agent (suppressing the condensation of ice pellets in low-temperature storage ice slurry).The application of mannosylerythritol lipid in medicine industry is based on its distinctive physiologically active: as improved efficiency gene transfection, suppressing the variation of microorganism growth, inducing cell, can break up mankind's myelogenous leukemia cell line and melanoma cells, having stronger coordination ability etc. with glycoprotein.
At present, reported can produce MELs have yeast and mold, output is not quite similar, and foreign study production peak can reach 165g/L, and its industrialization prospect receives much concern.
Pseudozyma aphidis DSM 70725 (P.a.DSM70725) is found as the microorganism of a kind of production MELs newly.The people such as Rau.U reclaim the addition of culture medium carbon source and inorganic salt, fermentation condition, product and separation and purification etc. is studied the impact of product.And the carbon source that fermenting process utilizes is mainly soybean wet goods edible plants oil, supplementary interpolation seminose and erythritol are as carbon source in addition, and promote the synthesis of seminose erythritol, ferment after 10 days, production peak reaches 75g/L.
Publication number is the production method that CN103589764A patent discloses a kind of mannosylerythritol lipid, comprise: activated aphid pseudoyeast (Pseudozyma aphidis) DSMZ70725 is accessed in seed culture fluid and carries out seed culture, be separated after cultivating and obtain somatic cells; Somatic cells is accessed in fermention medium, cultivate 7 ~ 15d in 28 ~ 30 DEG C of condition bottom fermentations, obtain fermented liquid; From fermented liquid, separation and purification obtains mannosylerythritol lipid; Wherein, containing vegetables oil in described fermention medium.This inventive method substrate used is vegetables oil.
But use soybean wet goods edible plants oil as carbon source for industrial production, cause raw materials cost high, affect product competitive power in the market.
The discarded grease of kitchen abendoned oil for producing in catering trade and food processing process, be commonly called as sewer oil, hogwash fat, swill oil, old oil, leftover bits and pieces wet goods, unit take-back cost is cheap.Kitchen abendoned oil, as a class of sewer oil, extensively exists in the life with people.In recent years, serve as the scandal of edible oil often by media report out about sewer oil, the public opinion causing society is paid close attention to.As everyone knows, sewer oil is very big to harm, and long-term eating can cause cancer.
At present, it is numerous that sewer oil serves as the reason that edible oil circulates in market, such as: Legal Regulation system is perfect not, utilize channel less to sewer oil.
Summary of the invention
The object of the present invention is to provide a kind of method utilizing kitchen abendoned oil to carry out bio-transformation synthesis seminose erythrose alcohol ester, not only effectively solve the problem that seminose erythrose alcohol ester raw materials for production cost is high, and provide a kind of method recycling sewer oil, effectively improve the industrial value of sewer oil.
For achieving the above object, the invention provides following technical scheme:
A kind of method utilizing kitchen abendoned oil bio-transformation synthesis mannosylerythritol lipid, comprise: aphid pseudoyeast (Pseudozyma aphidis) DSMZ70725 is accessed fermention medium, fermentation culture obtains mannosylerythritol lipid, and described fermention medium comprises kitchen abendoned oil.
Bacterial classification used herein is aphid pseudoyeast (Pseudozyma aphidis) DSMZ70725, this bacterial classification is purchased from German DSMZ, Deutsche Sammulung von Microorganismen und Zellkulturen (DSMZ).
Described kitchen abendoned oil is soybean oil, at least one in rapeseed oil, peanut oil, Fructus Maydis oil is produced.As preferably, described kitchen abendoned oil is produced by soybean oil.
Kitchen abendoned oil adopts prior art, by the swill reclaimed after filtration, obtain after dehydration.
Concrete fermentation culture method is as follows:
(1) activated aphid pseudoyeast (Pseudozyma aphidis) DSMZ70725 is accessed seed culture medium and carry out seed culture, obtain somatic cells;
(2) somatic cells is accessed fermention medium and carry out fermentation culture, obtain fermented liquid;
(3) from fermented liquid, separation and purification obtains mannosylerythritol lipid.
In step (1), described activation is: be inoculated in activation medium by aphid pseudoyeast DSMZ70725, and in 28 DEG C ~ 32 DEG C, shaking table shaking culture 1d ~ 2d, shaking speed is 150rpm ~ 200rpm.
Preferably, be inoculated in activation medium by aphid pseudoyeast (Pseudozyma aphidis) DSMZ70725, shaking table shaking culture 1.5d at 28 DEG C, shaking speed is 180rpm, is cultured to when bacterium liquid becomes muddy for subsequent use.
By weight percentage, described activation medium comprises following composition: yeast extract powder 0.05% ~ 0.15%, glucose 1.0% ~ 10.0%, soybean protein 0.1% ~ 1.0%.Concrete compound method is: by 3g yeast extract powder, 10g glucose, and 5g soybean protein adds in 1L water.
Be linked in seed culture medium by bacterium liquid after activation and cultivate, culture condition is shaking table shaking culture 1d ~ 3d at 28 ~ 32 DEG C, and shaking speed is 180rpm ~ 220rpm.As preferably, described seed culture condition is shaking table shaking culture 2d at 28 DEG C, and shaking speed is 180rpm.Seed culture fluid after cultivation is put into sterile centrifugation tube to carry out centrifugal, removes supernatant liquor, washs 3 times obtain somatic cells with the sodium chloride solution of concentration 0.9%.
By weight percentage, described seed culture medium comprises following component: glucose 2.0% ~ 6.0%, yeast extract powder 0.05% ~ 0.15%, SODIUMNITRATE 0.1% ~ 0.5%, magnesium sulfate 0.01% ~ 0.05%, potassium primary phosphate 0.01% ~ 0.05%.
As preferably, by weight percentage, described seed culture medium comprises following component: glucose 4.0%, yeast extract powder 0.1%, SODIUMNITRATE 0.3%, magnesium sulfate 0.3%, potassium primary phosphate 0.03%.
The compound method of concrete seed culture medium is: by 40g glucose, 1g yeast extract powder, 3gNaNO
3, 0.3g MgSO
47H
2o, 0.3g KH
2pO
4add in 1L water.
The somatic cells obtained access fermention medium is carried out fermentation culture, obtains fermented liquid.
The condition of described fermentation culture is shaking table shaking culture 7d ~ 15d at 28 DEG C ~ 32 DEG C, and shaking speed is 150rpm ~ 200rpm.Preferably, the condition of described fermentation culture is shaking table shaking culture 12d ~ 15d at 28 DEG C, and shaking speed is 180rpm.
By weight percentage, described fermention medium comprises following component: yeast extract powder 0 ~ 0.15%, magnesium sulfate 0.003% ~ 0.03%, potassium primary phosphate 0.003% ~ 0.03%, SODIUMNITRATE 0 ~ 0.4%, kitchen abendoned oil 2.0% ~ 15.0%; The pH value controlling fermention medium is 3.0 ~ 8.0.
As preferably, by weight percentage, described fermention medium comprises following component: yeast extract powder 0.1%, magnesium sulfate 0.03%, potassium primary phosphate 0.03%, SODIUMNITRATE 0.3%, kitchen abendoned oil 4.0% ~ 12.0%; The pH value controlling fermention medium is 3.0 ~ 8.0.
The compound method of concrete fermention medium is: by 1g yeast extract powder, 0.3gMgSO
47H
2o, 0.3g KH
2pO
4, 3g NaNO
3, 40mL ~ 120mL kitchen abendoned oil adds in 1L water, and pH value regulates.
In often liter of fermention medium, the access amount of somatic cells is 1.0g ~ 10.0g.As preferably, in often liter of fermention medium, the access amount of somatic cells is 2.4g.
Mannosylerythritol lipid is obtained through extracting and separating from the fermented liquid that fermentation culture obtains.Described separation and purification comprises: with an organic solvent extract fermented liquid, is separated and gets oil phase, and underpressure distillation removing organic solvent obtains the thick product of mannosylerythritol lipid; Thick for the mannosylerythritol lipid obtained product is dissolved in methyl alcohol, and with n-hexane extraction, be separated and get oil phase, underpressure distillation is removed methanol phase and is obtained mannosylerythritol lipid.
Compared with prior art, beneficial effect of the present invention is:
The present invention utilizes kitchen abendoned oil bio-transformation synthesis mannosylerythritol lipid, substrate is domestic refuse, product safety non-toxic, environmental protection, and compared with edible vegetable oil, the grease transformation efficiency of kitchen abendoned oil in bio-transformation building-up process is higher, and therefore method of the present invention not only effectively reduces production cost, and meets the environmental protection concept of " turn waste into wealth, cut the waste, economize on resources ", there is the feature of Sustainable development, be easy to realize industrialization and produce.
Accompanying drawing explanation
Fig. 1 is the schematic flow sheet that the present invention utilizes kitchen abendoned oil bio-transformation synthesis mannosylerythritol lipid.
Embodiment
In following examples, bacterial classification used is aphid pseudoyeast DSMZ70725.This bacterial classification is purchased from German DSMZ, Deutsche Sammulung von Microorganismen und Zellkulturen (DSMZ).
Embodiment 1-4
(1) actication of culture: the Erlenmeyer flask getting 250mL, often bottled enter the activation medium of 50mL, autoclave sterilization cools, by in the activation medium after aphid pseudoyeast (Pseudozyma aphidis) the DSMZ70725 inoculation to sterilizing of freezen protective, 28 DEG C, under rotary shaker rotating speed is the condition of 180rpm, shaking culture 1.5d, bacterium liquid is for subsequent use.
Wherein, activation medium comprises following composition: yeast extract powder 3g/L, glucose 10g/L, soybean protein 5.0g/L, water.
(2) seed culture: the Erlenmeyer flask getting 250mL, often bottled enter the seed culture medium of 50mL, autoclave sterilization cools, and the bacterium liquid got after 1mL activation is inoculated in the Erlenmeyer flask that 50mL seed culture medium is housed, 28 DEG C, under rotary shaker rotating speed is the condition of 180rmp, cultivate 2d; Then seed culture fluid is centrifugal, remove supernatant liquor, the sodium chloride solution with 0.9% washs 3 times, then according to thalline weight in wet base, and the sodium chloride solution adding appropriate 0.9% dissolves, and makes somatic cells weight in wet base concentration be 0.12g/mL.
Wherein seed culture medium is made up of following component: glucose 40g/L, yeast extract powder 1g/L, NaNO
33g/L, MgSO
47H
2o 0.3g/L, KH
2pO
40.3g/L, water.
(3) fermentation culture: the Erlenmeyer flask getting 250mL, often bottled enter the fermention medium of 50mL, getting above-mentioned concentration is 0.12g/mL bacterium liquid, is inoculated in fermention medium according to 1mL bacterium liquid/50mL fermention medium, 28 DEG C, rotary shaker rotating speed be 180rpm condition bottom fermentation cultivate 8d.
Wherein fermention medium ferment substratum is made up of following component: yeast extract powder 1g/L, MgSO
47H
2o 0.3g/L, KH
2pO
40.3g/L, NaNO
33g/L, water, pH value regulates.
Wherein, embodiment 1 adds soybean oil, and addition is 80mL/L fermention medium; Embodiment 2-4 adds kitchen abendoned oil, and addition is respectively 40mL/L, 80mL/L, 120mL/L fermention medium, as shown in table 1.
(4) extracting and separating: after fermentation culture, gets isopyknic ethyl acetate with fermented liquid, carries out extraction 2 ~ 3 times to fermented liquid, merges organic phase, and vacuum rotary steam obtains the thick product of mannosylerythritol lipid (MELs); Again thick for mannosylerythritol lipid product is dissolved in 25mL methyl alcohol, add 35mL n-hexane extraction, reclaim methanol phase, vacuum rotary steam obtains purer mannosylerythritol lipid, the output of embodiment 1-4 is respectively 31.11g/L, 6.67g/L, 39.71g/L, 63.77g/L, as shown in table 1.
In table 1 embodiment 1-4 fermention medium, grease adds situation, MELs output and grease transformation efficiency
Note: grease transformation efficiency (g/mL)=MELs output (g)/grease addition (mL)
As can be seen from Table 1: under above-mentioned fermentation condition, along with the increase of kitchen abendoned oil addition, MELs output and grease transformation efficiency also increase; When kitchen abendoned oil is the same with soybean oil addition, when adding kitchen abendoned oil, MELs output is higher, and namely grease transformation efficiency is higher, and therefore adding kitchen abendoned oil can refuse reclamation, reduces production cost, can increase again the output of mannosylerythritol lipid.
Embodiment 5-25
Following examples are probed into the liquid amount of fermention medium, pH value, kitchen abendoned oil addition and inoculum size.
(1) actication of culture: the Erlenmeyer flask getting 250mL, often bottled enter the activation medium of 50mL, autoclave sterilization cools, by aphid pseudoyeast (Pseudozyma aphidis) the DSMZ70725 bacterial strain of freezen protective, be seeded in the activation medium after sterilizing, 28 DEG C, under rotary shaker rotating speed is the condition of 180rpm, cultivate 1.5d, bacterium liquid is for subsequent use.
Wherein, activation medium comprises following composition: yeast extract powder 3g/L, glucose 10g/L, soybean protein 5.0g/L, water.
(2) seed culture: the Erlenmeyer flask getting 250mL, often bottled enter the seed culture medium of 50mL, autoclave sterilization cools, and the bacterium liquid after every bottle of seed culture fluid gets 1mL activation is inoculated, 28 DEG C, under rotary shaker 180rpm condition, cultivate 2d; After seed culture, seed liquor is centrifugal, remove supernatant liquor, the sodium chloride solution with 0.9% washs 3 times, then according to thalline weight in wet base, and the sodium chloride solution adding appropriate 0.9% dissolves, and makes somatic cells weight in wet base concentration be 0.12g/mL.
Wherein seed culture medium is made up of following component: glucose 40g/L, yeast extract powder 1g/L, NaNO
33g/L, MgSO
47H
2o0.3g/L, KH
2pO
40.3g/L, water.
(3) fermentation culture: the Erlenmeyer flask getting 250mL, every bottle of a certain amount of fermention medium, getting above-mentioned concentration is that 0.12g/mL bacterium liquid is inoculated, 28 DEG C, rotary shaker rotating speed is that the condition bottom fermentation of 180rpm is cultivated after 10d and obtained fermented liquid.
Wherein fermention medium ferment substratum is made up of following component: yeast extract powder 1g/L, MgSO
47H
2o 0.3g/L, KH
2pO
40.3g/L, NaNO
33g/L, a certain amount of kitchen abendoned oil, water, adjust ph is to certain value.
(4) extracting and separating: after fermentation culture, gets isopyknic ethyl acetate with fermented liquid, carries out extraction 2 ~ 3 times to fermented liquid, merges organic phase, and vacuum rotary steam obtains the thick product of mannosylerythritol lipid (MELs); Again thick for mannosylerythritol lipid product is dissolved in 25mL methyl alcohol, adds 35mL n-hexane extraction, reclaim methanol phase, vacuum rotary steam obtains purer mannosylerythritol lipid, and the output of product is shown in Table 2.
Wherein the step (1) of embodiment 5-25, (2), (4) are all identical, and in step (3), the kitchen abendoned oil addition (mL/L fermention medium) of fermention medium, inoculum size (mL/L fermention medium), fermention medium liquid amount (mL) and initial pH value are as shown in table 2.
The condition of table 2 embodiment 5-25 fermention medium, MELs output and grease transformation efficiency
Note: grease transformation efficiency (g/mL)=MELs output (g)/grease addition (mL)
As can be seen from the embodiment 5-25 fermentation results of table 2, when kitchen abendoned oil addition is 105.2mL/L fermention medium, inoculum size is 20mL/L fermention medium, fermention medium liquid amount 50mL (250mL Erlenmeyer flask), initial pH value are 5.93, leavening temperature be 28 DEG C, under shaking speed is the condition of 180rpm, MELs output after 10 days of fermenting is the highest, and production peak is 60g/L; When kitchen abendoned oil addition be 95mL/L fermention medium, inoculum size 25mL/L fermention medium, fermention medium liquid amount 65mL (250mL Erlenmeyer flask), initial pH value be 4.43 time, grease transformation efficiency is the highest, is 0.583g/L.
Claims (9)
1. one kind utilizes the method for kitchen abendoned oil bio-transformation synthesis mannosylerythritol lipid, comprise: aphid pseudoyeast (Pseudozyma aphidis) DSMZ70725 is accessed fermention medium, fermentation culture obtains mannosylerythritol lipid, it is characterized in that, described fermention medium comprises kitchen abendoned oil.
2. the method utilizing kitchen abendoned oil bio-transformation synthesis mannosylerythritol lipid according to claim 1, is characterized in that, described kitchen abendoned oil is soybean oil, at least one in rapeseed oil, peanut oil, Fructus Maydis oil produced.
3. the method utilizing kitchen abendoned oil bio-transformation synthesis mannosylerythritol lipid according to claim 2, is characterized in that, described kitchen abendoned oil is produced by soybean oil.
4. the method utilizing kitchen abendoned oil bio-transformation synthesis mannosylerythritol lipid according to claim 1, it is characterized in that, by weight percentage, described fermention medium comprises following component: yeast extract powder 0 ~ 0.15%, magnesium sulfate 0.003% ~ 0.03%, potassium primary phosphate 0.003% ~ 0.03%, SODIUMNITRATE 0 ~ 0.4%, kitchen abendoned oil 2.0% ~ 15.0%; The pH value controlling fermention medium is 3.0 ~ 8.0.
5. the method utilizing kitchen abendoned oil bio-transformation synthesis mannosylerythritol lipid according to claim 4, it is characterized in that, by weight percentage, described fermention medium comprises following component: yeast extract powder 0.1%, magnesium sulfate 0.03%, potassium primary phosphate 0.03%, SODIUMNITRATE 0.3%, kitchen abendoned oil 4.0% ~ 12.0%; The pH value controlling fermention medium is 3.0 ~ 8.0.
6. the method utilizing kitchen abendoned oil bio-transformation synthesis mannosylerythritol lipid according to claim 1, it is characterized in that, in often liter of fermention medium, the access amount of somatic cells is 1.0g ~ 10.0g.
7. the method utilizing kitchen abendoned oil bio-transformation synthesis mannosylerythritol lipid according to claim 6, it is characterized in that, in often liter of fermention medium, the access amount of somatic cells is 2.4g.
8. the method utilizing kitchen abendoned oil bio-transformation synthesis mannosylerythritol lipid according to claim 1, it is characterized in that, the temperature of fermentation culture is 28 DEG C ~ 32 DEG C, and shaking table concussion is cultivated, and shaking speed is 150rpm ~ 200rpm.
9. the method utilizing kitchen abendoned oil bio-transformation synthesis mannosylerythritol lipid according to claim 1, it is characterized in that, also comprise purification procedures: with an organic solvent the fermented liquid that fermentation culture obtains is extracted, oil phase is got in separation, and underpressure distillation removing organic solvent obtains the thick product of mannosylerythritol lipid; Thick for the mannosylerythritol lipid obtained product is dissolved in methyl alcohol, and with n-hexane extraction, be separated and get oil phase, underpressure distillation is removed methanol phase and is obtained mannosylerythritol lipid.
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| CN106174662A (en) * | 2016-08-25 | 2016-12-07 | 浙江大学 | A kind of heat stability mannosylerythritol lipid carrier and its preparation method and application |
| CN108619996A (en) * | 2018-04-26 | 2018-10-09 | 南京理工大学 | A kind of isolation and purification method of mannose erythrose alcohol ester |
| CN108653186A (en) * | 2018-08-20 | 2018-10-16 | 广州无添加主义化妆品有限公司 | A kind of skin care articles for washing and preparation method thereof |
| CN109046192A (en) * | 2018-08-28 | 2018-12-21 | 浙江大学 | A kind of plants essential oil chitosan nano mcirocapsule and its preparation method and application |
| CN112280806A (en) * | 2019-07-23 | 2021-01-29 | 广东现代汉方科技有限公司 | Method for preparing ceramide by emulsification fermentation and application thereof |
| CN113924884A (en) * | 2021-11-09 | 2022-01-14 | 广西壮族自治区农业科学院 | Fruit retention treatment method for improving storage and transportation resistance of sunshine rose |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106174662A (en) * | 2016-08-25 | 2016-12-07 | 浙江大学 | A kind of heat stability mannosylerythritol lipid carrier and its preparation method and application |
| CN106174662B (en) * | 2016-08-25 | 2019-02-19 | 浙江大学 | A kind of thermal stability mannosylerythritol lipid carrier and its preparation method and application |
| CN108619996A (en) * | 2018-04-26 | 2018-10-09 | 南京理工大学 | A kind of isolation and purification method of mannose erythrose alcohol ester |
| CN108653186A (en) * | 2018-08-20 | 2018-10-16 | 广州无添加主义化妆品有限公司 | A kind of skin care articles for washing and preparation method thereof |
| CN109046192A (en) * | 2018-08-28 | 2018-12-21 | 浙江大学 | A kind of plants essential oil chitosan nano mcirocapsule and its preparation method and application |
| CN112280806A (en) * | 2019-07-23 | 2021-01-29 | 广东现代汉方科技有限公司 | Method for preparing ceramide by emulsification fermentation and application thereof |
| CN113924884A (en) * | 2021-11-09 | 2022-01-14 | 广西壮族自治区农业科学院 | Fruit retention treatment method for improving storage and transportation resistance of sunshine rose |
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