CN104862349B - It is a kind of improve rhodotorula glutinis grease yield culture medium and its application - Google Patents
It is a kind of improve rhodotorula glutinis grease yield culture medium and its application Download PDFInfo
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- CN104862349B CN104862349B CN201510210394.8A CN201510210394A CN104862349B CN 104862349 B CN104862349 B CN 104862349B CN 201510210394 A CN201510210394 A CN 201510210394A CN 104862349 B CN104862349 B CN 104862349B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
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Abstract
The present invention relates to it is a kind of improve rhodotorula glutinis grease yield culture medium, including glucose, yeast extract, dipotassium hydrogen phosphate, ammonium sulfate, sodium sulphate, epsom salt and spirulina protein zymolyte.Culture medium of the present invention, spirulina protein zymolyte can be can be used as to functional promotion object to be directly appended in dextrose culture-medium, also it can be used as a kind of organic nitrogen source and partly or entirely substitute organic nitrogen source yeast extract in dextrose culture-medium, addition manner can be adjusted according to different situations, obtain higher grease yield under the premise of lower cost to reach.
Description
Technical field
The present invention relates to microculture fields, and in particular to a kind of culture medium for improving rhodotorula glutinis grease yield.
Background technique
In recent years, as the aggravation of global energy crisis, green regenerative energy sources have become a hot topic of research, biodiesel contains
Sulfur content is low, has good burning performance, and is a kind of good renewable energy.The general raw materials for production of biodiesel are vegetable oil, however
Since the vegetable oil production period is long, mankind's eating requirements are big, are not enough to for producing biodiesel.Microbial oil is mainly C16
And C18Fatty acid composition, and microbial oil content is high, and the speed of growth is fast, and life cycle is short, compared with vegetable fat, micro- life
Object grease has certain advantage in terms of the raw material as biodiesel.Rhodotorula glutinis (Rhodotorula glutinis) is thin
Fat content in born of the same parents is more than the 20% of its dry weight, and rhodotorula glutinis is easy to cultivate, and is a kind of using several kinds of carbon source and nitrogen source
Good oleaginous microorganism.But in current research, since the grease yield of rhodotorula glutinis is low, rhodotorula glutinis grease does not have also
There are realization industrial applications.
Summary of the invention
(1) technical problems to be solved
The low defect of grease yield present in culture for rhodotorula glutinis in the prior art, the present invention provides one kind can
Improve the culture medium of rhodotorula glutinis grease yield.
(2) technical solution
Culture medium of the present invention, including glucose, dipotassium hydrogen phosphate, ammonium sulfate, sodium sulphate and epsom salt, with
And the spirulina protein zymolyte of addition.
Culture medium of the present invention further includes yeast extract.Spirulina protein zymolyte is added to dextrose culture-medium
In, it can both play the role of organic nitrogen source, functional materials therein may additionally facilitate the oil and fat accumulation of rhodotorula glutinis, and raising is glued red
The grease yield of yeast.
Culture medium of the present invention, preferably 40~100g/L of concentration of glucose, 0~1.5g/ of concentration of yeast extract
L, the concentration of dipotassium hydrogen phosphate are 1~7g/L, and 1~3g/L of concentration of ammonium sulfate, the concentration of sodium sulphate is 1~3g/L, seven water sulphur
The concentration of sour magnesium is 1~2g/L, and the concentration of spirulina protein zymolyte is 0.5-2g/L.
Spirulina protein zymolyte of the present invention is digested through alkali protease by spirulina protein and is made.Through basic protein
The spirulina protein of enzyme enzymatic hydrolysis is most obvious to the accumulation facilitation effect of rhodotorula glutinis grease.
In the present invention, the molecular weight of the spirulina protein zymolyte is less than 5KD.Molecular weight is selected to be less than the spirulina of 5K
Albumen is more advantageous to the absorption of rhodotorula glutinis compared with molecular weight more large protein, and the effect in terms of improving grease yield is more aobvious
It writes.
In the present invention, the additive amount of the spirulina protein zymolyte is 0.75~1.5g/L.Select the dense of this range
Degree, spirulina protein can both effectively facilitate the raising of rhodotorula glutinis grease yield, and be also unlikely to addition causes to waste too much.
In the present invention, the preparation method of the spirulina protein zymolyte is that spirulina protein and distilled water are pressed mass body
Product is made into mixed liquor than 1:10~1:14, and the basic protein of spirulina protein quality 4%~6% is added in the solution
Enzyme, the pH for adjusting the mixed liquor is 7.5, and 5~7h is digested under conditions of 55~65 DEG C, is divided step by step with ultrafiltration membrane enzymolysis liquid
From the molecular cut off of film is respectively 30KD and 5KD, and the enzymolysis liquid after separation is carried out vacuum freeze drying, it is small to obtain molecular weight
In the spirulina protein alkali protease zymolyte of 5KD.
In the present invention, spirulina protein and distilled water are preferably pressed matter by the preparation method of the spirulina protein zymolyte
Amount volume ratio 1:12 is made into mixed liquor, and the alkali protease of spirulina protein quality 5% is added in the solution, adjusts institute
The pH for stating mixed liquor is 7.5, and 5~7h is digested under conditions of 60 DEG C, is separated step by step with ultrafiltration membrane to enzymolysis liquid, the retention point of film
Son amount is respectively 30KD and 5KD, and the enzymolysis liquid after separation is carried out vacuum freeze drying, obtains the spirulina that molecular weight is less than 5KD
Albumen alkali protease zymolyte.The protein zymolyte digested according to this method is in terms of improving rhodotorula glutinis grease yield
Effect it is most obvious.
In the present invention, the pH of the culture medium is 5~5.5.
Culture medium of the present invention is preferably 75~85g/L of concentration of glucose, 1~1.5g/ of concentration of yeast extract
L, dense 2.5~3.5g/L of dipotassium hydrogen phosphate, 1.5~2.5g/L of concentration of ammonium sulfate, 1.5~2.5g/L of concentration of sodium sulphate,
The concentration 1.5g/L of epsom salt, 1~1.5g/L of concentration of spirulina protein zymolyte.
Culture medium of the present invention is also preferably the concentration 40g/L of glucose, the concentration 0 of yeast extract~
0.75g/L, the concentration 7g/L of dipotassium hydrogen phosphate, the concentration 2g/L of ammonium sulfate, the concentration 2g/L of sodium sulphate, epsom salt it is dense
1.5g/L is spent, the concentration of the spirulina protein zymolyte is 0.75~1.5g/L, and yeast extract and spirulina protein digest
The concentration summation of object is 1.5g/L.This preferred embodiment completely or partially replaces the organic of dextrose culture-medium using spirulina protein
Nitrogen source yeast extract, can effectively improve the grease yield of rhodotorula glutinis, and can also use up a step reduces production cost.
Culture medium of the present invention can be used for cultivating the rhodotorula glutinis bacterial strain of all kinds, and its grease yield also can be corresponding
It improves, the significant effect in terms of improving 2.704 grease yield of rhodotorula glutinis.
(3) beneficial effect
The culture medium of the present invention for improving rhodotorula glutinis grease yield, in the glucose culture of traditional rhodotorula glutinis
Spirulina protein zymolyte is added in base.On the one hand, zymolyte is a kind of organic nitrogen source in spirulina protein, can be improved and glues red ferment
Female biomass, on the other hand, functional materials therein may additionally facilitate the oil and fat accumulation of rhodotorula glutinis, improve rhodotorula glutinis
Fat content, two all combine, and the grease yield of rhodotorula glutinis is improved.
Spirulina protein zymolyte of the present invention can be used as functional promotion object and be directly appended to dextrose culture-medium
In, it also can be used as a kind of organic nitrogen source and partly or entirely substitute organic nitrogen source yeast extract in dextrose culture-medium, applying
During, method is flexible, addition manner is adjusted according to different situation, with reach obtained under the premise of lower cost it is higher
Grease yield.
Spirulina protein of the present invention is easily obtained, and preparation method is simple for the zymolyte of spirulina protein,
There is higher practical application value, is suitable for large-scale promotion.
Specific embodiment
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case where spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
In embodiment Rhodotorula glutinis fungus purchase in China General Microbiological culture presevation administrative center (number:
CGMCC2.704)
Spirulina used in embodiment is blunt top spirulina, is taken from spirulina research institute, Beijing Forestry University.
The various reagents used in embodiment are purchased from biotechnology difficult to understand Co., Ltd, Beijing section hundred.
1 molecular weight of embodiment is less than the preparation of 5KD spirulina protein alkali protease zymolyte
The extraction of spirulina protein
1) it is that 1:10 dissolves by mass volume ratio by blunt top spirulina dry powder and distilled water, spirulina suspension is made
Liquid, ultrasound 15min under conditions of ultrasonic power is 700w are broken to spirulina then with liquid nitrogen to its multigelation 3 times
Wall, the spirulina liquid after obtaining broken wall.
2) by the spirulina liquid after broken wall at -4 DEG C, it is centrifuged 20min under conditions of 8000r/min, collects supernatant, and will
Supernatant (NH4)2SO4Crystal is saltoutd.
3) by the mixture after saltouing at -4 DEG C, it is centrifuged 20min under the conditions of 8000r/min, collects precipitating, is with concentration
The phosphate buffer that 0.005M, pH are 6.86 dissolves precipitating, dialyses, the albumen after obtaining desalination, after the desalination
Albumen carry out vacuum freeze drying, obtain spirulina protein dry product.
The enzymatic hydrolysis of spirulina protein
1) it is that 1:12 dissolves by mass volume ratio by spirulina protein dry product and distilled water, obtains lysate, dissolving
5% alkali protease of the quality of spirulina protein dry product is added in liquid, adjusting and keep the pH of lysate is 7.5,
6min is digested under conditions of 60 DEG C of bath temperature；After enzymatic hydrolysis in 85 DEG C of water-baths enzyme deactivation 15~20min, 8000r/min from
Heart 20min, takes supernatant, obtains spirulina protein alkali protease enzymolysis liquid.
2) enzymolysis liquid is separated step by step with ultrafiltration membrane, the molecular cut off of film is respectively 30KD and 5KD, and control peristaltic pump turns
Speed is 28-34r/min, and ultrafiltration diaphragm plate outlet pressure is 2.0 × 105Pa, collects molecular weight and is less than the part 5KD, it is cold to carry out vacuum
Be lyophilized it is dry, obtain molecular weight be less than 5KD spirulina protein alkali protease zymolyte.
The culture of 2 rhodotorula glutinis of embodiment
1) activation of rhodotorula glutinis bacterial strain
The Rhodotorula glutinis fungus (number: CGMCC2.704) bought from China General Microbiological culture presevation administrative center is protected
It ensconces in ampoul tube, is stored in after being activated according to explanation to it in 4 DEG C of environment.One is taken from the slant medium of preservation of bacteria strain
Ring bacterium is inoculated in brewer's wort slant medium, cultivates 72h in 30 DEG C of constant incubator, obtains the strain of primary activation.
The strain of a ring primary activation is taken to be inoculated in malt juice liquid medium, constant temperature oscillation is trained under the conditions of 30 DEG C, 140r/min
It supports for 24 hours, obtains the rhodotorula glutinis of activation.
2) shaking flask culture
The rhodotorula glutinis 5mL for taking activation, is inoculated into the triangular flask of the 250mL equipped with 45mL dextrose culture-medium, is training
The spirulina alkali protease zymolyte 1g/L that addition molecular weight in base is less than 5KD is supported, at 30 DEG C of temperature, the condition of 140r/min
Lower culture 120h must cultivate mature rhodotorula glutinis.
3) extraction of grease
The mature rhodotorula glutinis of culture is centrifuged, is dried, is calculated after carrying out grease extraction with chloroform methanol method
Grease yield is 1.38g/L.
Malt extract medium preparation process described in the present embodiment is that 100g fructus hordei germinatus leaching powder is taken to be dissolved in 1L distilled water, is dissolved
Afterwards in 1 × 105Pa high pressure sterilization 10min, the preparation method of the brewer's wort slant medium is to add in malt extract medium
Add 2% agar.
The composition of dextrose culture-medium described in the present embodiment and the preparation method is as follows: glucose 40g/L, ammonium sulfate 2g/
L, yeast powder 1.5g/L, sodium sulphate 2g/L, potassium dihydrogen phosphate 7g/L, epsom salt 1.5g/L.By the above substance and spiral
Algae protein zymolyte is uniformly mixed, in 1 × 105Pa high pressure sterilization 10min after being completely dissolved.
Embodiment 3
Compared with embodiment 2, difference is only that, by KH in dextrose culture-medium2PO4Concentration be adjusted to 3g/L, grease
Yield is 1.49g/L.
Embodiment 4
Compared with embodiment 2, difference is, by KH in dextrose culture-medium2PO4Concentration be adjusted to 3g/L, glucose
Concentration be adjusted to 60g/L, the additive amount of spirulina protein zymolyte is 2g/L, grease yield 2.30g/L.
Embodiment 5
Compared with embodiment 2, difference is, by KH in dextrose culture-medium2PO4Concentration be adjusted to 3g/L, glucose
Concentration section be 80g/L, grease yield 3.39g/L.
Embodiment 6
Compared with embodiment 2, difference is, spirulina alkali protease of the culture medium middle-molecular-weihydroxyethyl less than 5KD digests
The additive amount of object is 1.5g/L, and the additive amount of yeast extract is 0g/L, grease yield 1.54g/L.
Embodiment 7
Compared with embodiment 2, difference is, spirulina alkali protease of the culture medium middle-molecular-weihydroxyethyl less than 5KD digests
The addition concentration of object is 0.75g/L, and the additive amount of yeast extract is 0.75g/L, grease yield 1.96g/L.
Embodiment 8
Compared with embodiment 2, difference be, in culture medium the concentration of glucose be 100g/L, dipotassium hydrogen phosphate it is dense
Degree is 1g/L, and the concentration of ammonium sulfate is 1g/L, and the concentration of sodium sulphate is 1g/L, and the concentration of epsom salt is 2g/L, spirulina
The addition concentration of protein zymolyte is 0.5g/L, grease yield 1.78g/L.
Embodiment 9
Compared with embodiment 2, difference is, the concentration of glucose is 80g/L, the concentration of dipotassium hydrogen phosphate in culture medium
For 3g/L, the concentration of ammonium sulfate is 3g/L, and the concentration of sodium sulphate is 3g/L, and the concentration of epsom salt is 1g/L, spirulina egg
The addition concentration of white zymolyte is 2g/L, grease yield 2.05g/L.
Comparative example 1
Compared with embodiment 2, difference is, the spiral that molecular weight is less than 5K is added not in the dextrose culture-medium
The zymolyte of alginine protease, grease yield 1.25g/L.
Comparative example 2
Compared with embodiment 2, difference is, the spirulina protein alkali protease that addition molecular weight is 5KD~30KD
Zymolyte, grease yield 1.35g/L.
Comparative example 3
Compared with embodiment 5, difference is, does not add the spirulina protein that molecular weight is less than 5K in the medium
Zymolyte, grease yield 1.86g/L.
As seen from the above embodiment, spirulina protein zymolyte is added in Portugal's culture medium, it can be to the grease of rhodotorula glutinis
Facilitation is played in accumulation, and grease yield is improved；Moreover, rhodotorula glutinis can also completely or partially substitute glucose culture
Organic nitrogen source yeast extract in base, uses spirulina protein zymolyte as the organic nitrogen source of rhodotorula glutinis, can both meet glue it is red
The growth of yeast, moreover it is possible to improve its grease yield.Therefore, the culture medium of the present invention for improving rhodotorula glutinis grease yield,
Preparation method is simple, easy to operate, has biggish practical application value.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvement and replacement can also be made, these are improved and replacement
Also it should be regarded as protection scope of the present invention.
Claims (5)
1. a kind of culture medium for improving 2.704 grease yield of rhodotorula glutinis CGMCC, which is characterized in that the concentration of glucose is
80g/L, the concentration of yeast extract are 1.5g/L, and the concentration of potassium dihydrogen phosphate is 3g/L, and the concentration of ammonium sulfate is 2g/L, sodium sulphate
Concentration be 2 g/L, the concentration of epsom salt is 1.5g/L, and the concentration of spirulina protein zymolyte is 1g/L；The spiral
Algae protein zymolyte is digested through alkali protease by spirulina protein and is made.
2. culture medium according to claim 1, which is characterized in that the molecular weight of the spirulina protein zymolyte is less than
5KD。
3. culture medium according to claim 2, which is characterized in that the pH of the culture medium is 5.0~5.5.
4. culture medium according to claim 1 or 3, which is characterized in that the preparation method of the spirulina protein zymolyte
For spirulina protein and distilled water are made into mixed liquor by mass volume ratio 1:10~1:14, spiral is added in the solution
The alkali protease of algae albumen quality 4%~6%, the pH for adjusting the mixed liquor is 7.5, digests 5 under conditions of 55~65 DEG C
~7h.
5. application of any one of Claims 1 to 4 culture medium in terms of cultivating rhodotorula glutinis.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559788A (en) * | 2011-12-31 | 2012-07-11 | 北京林业大学 | Culture medium for improving content of oil in Rhodotorula glutinis and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559788A (en) * | 2011-12-31 | 2012-07-11 | 北京林业大学 | Culture medium for improving content of oil in Rhodotorula glutinis and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 利用粘红酵母生产微生物油脂研究进展;乔凤杰等;《食品工业科技》;20141231;第35卷(第11期);第391页右栏第3段至第392页左栏第1段及第393页右栏第3段至第394页左栏第1段 |
| 粘红酵母在味精废水中发酵生产油脂;邢旭等;《生物加工过程》;20100131;第8卷(第1期);第6-10页 |
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