CN110777171A - Method for preparing grease rich in saturated and unsaturated fatty acids simultaneously by using penicillium ecteinascidianum Asc2-4 - Google Patents
Method for preparing grease rich in saturated and unsaturated fatty acids simultaneously by using penicillium ecteinascidianum Asc2-4 Download PDFInfo
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Abstract
The invention discloses a method for preparing grease rich in saturated and unsaturated fatty acids by utilizing ascidian penicillium citrinum Asc 2-4. The method comprises the steps of inoculating Penicillium ascidianum Asc2-4 to a seed culture medium for shake cultivation; inoculating the cultured seeds to a fermentation culture medium, fermenting under a proper dissolved oxygen condition, and extracting; the fermentation medium comprises the following components: carbon source, potato, sodium citrate, sea salt, nitrogen source and KH
2PO
4、MgSO
4And FeCl
3. According to the invention, the oil rich in saturated and unsaturated fatty acids is prepared by optimizing the culture medium and culture conditions of Penicillium ecteinascidianum Asc2-4, the contents of oleic acid and linoleic acid account for 25-27% of the total oil mass and 46-54% of the unsaturated fatty acids, and the oil rich in oleic acid can be producedAnd linoleic acid.
Description
Technical Field
The invention belongs to the technical field of biological extraction. More particularly, relates to a method for preparing grease rich in saturated and unsaturated fatty acids by utilizing penicillium ecteinascidianum Asc 2-4.
Background
Microbial oils are oils synthesized by microorganisms such as yeast, mold, bacteria, and algae using exogenous nutrients and stored in cells. Compared with the land, the ocean has the extreme characteristics of high salt, high pressure and low oxygen, and forms the characteristic oil production property of the marine fungi.
The oil-producing fungi can generally utilize various carbon sources to grow thalli and accumulate oil and fat. The kind and concentration of the carbon source have an influence on the oil content and unsaturated components of the oleaginous fungi. hen, etc. the biomass, oil yield and oil content were 4.0g/L, 1.4g/L and 35.0% respectively, using pure glycerol for fermentation oil production. In addition, nitrogen sources have different effects on different fungi, and even for the same oleaginous fungi, the composition and concentration of nitrogen sources have different effects on biomass and grease yield. MalCan finds that the most suitable single nitrogen source for producing the fusarium oleophylum is potassium nitrate, the excessively high carbon-nitrogen ratio is not beneficial to the increase of biomass, and the excessively low carbon-nitrogen ratio is easy to prolong the fermentation days. Moreover, the metal ions can act on the growth of thalli, the intracellular and extracellular osmotic pressure and the enzyme activity of the oleaginous microorganisms, so that the oil synthesis capability of the oleaginous microorganisms is influenced, and therefore, even aiming at the same oleaginous fungi, the biomass and the oil yield of the oleaginous fungi are different due to different compositions and concentrations of different metal ions. The Zymond takes filamentous spore yeast as a research object, and 4.0g/L CaCl is added into a fermentation medium
2The oil yield is not obviously influenced, but the conversion rate of the substrate can be promoted. Xuehanming et al found Fe
3+Has effect in promoting the activity of desaturation enzyme of Phaeodactylum tricornutum. In addition, the rotation speed and the liquid loading amount determine the dissolved oxygen condition of the medium, and have an influence on the growth of the cells and the oil production. Qi et al found 70mL/250mL airThe amount of the strain has a larger influence on the biomass of the grease yeast EAM-AC16 than on the yield of the grease.
Sea squirt is a marine organism belonging to phylum chordata, subphylum urosum and class ascidiacea, and grows on natural sea area attached with pearl oyster cages. Research shows that the marine invertebrate symbiotic microorganisms reach hundreds of species in average, the density of the organisms is 40 percent of the total weight of host animals, and most of the organisms belong to the category of microorganisms difficult to culture. Sea squirts are animals living in the camps and the immortals, are sleeved with a layer of plant cellulose-like tunic outside like a sheath, and are unique to the animal kingdom. The unique ingestion and filter feeding system of the sea squirt enriches a large amount of microorganisms in the body and on the surface of the sea squirt. Compared with terrestrial microorganisms, the ascidian epiphyte can resist a plurality of extreme conditions such as high salt, high pressure, low oxygen, low light and the like which are peculiar to the ocean, thereby forming unique metabolic and physiological characteristics, generating metabolites with different chemical structures and providing active metabolites which can not be provided by the terrestrial microorganisms for human beings. The active substances have biological activities of antibiosis, antitumor, antivirus, blood pressure reduction, blood coagulation, hemolysis and the like, and become one of important contents for developing marine medicine resources.
Chinese patent document CN 106047956A discloses a method for preparing grease rich in gamma-linolenic acid by fermenting ascidian-symbiotic penicillium citrinum Asc 2-4. The oil prepared by the method is only rich in unsaturated fatty acid oil, and the content of unsaturated fatty acid gamma-linolenic acid oil in the obtained oil is high.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for preparing grease rich in saturated and unsaturated fatty acids by utilizing penicillium ecteinascidianum Asc 2-4.
The invention aims to provide application of penicillium ecteinascidianum Asc2-4 in preparation of oil rich in saturated and unsaturated fatty acids.
The invention also aims to provide a method for preparing the grease rich in saturated fatty acid and unsaturated fatty acid by using the penicillium ecteinascidianum Asc 2-4.
The purpose of the invention is realized by the following technical scheme:
application of Penicillium ecteinascidianum Asc2-4 in preparing oil rich in saturated and unsaturated fatty acids. The saturated fatty acid grease refers to stearic acid and palmitic acid.
A method for preparing saturated and unsaturated fatty acid-rich oil by using Penicillium ascidianum Asc2-4 comprises the following steps:
s1, inoculating Penicillium ascidianum Asc2-4 to a seed culture medium for shake culture;
s2, inoculating the cultured seeds to a fermentation culture medium, fermenting under the conditions that the liquid loading amount is 40-80% of the volume of a culture container and the rotating speed is 120-280 rpm to obtain penicillium citrinum mycelia, and extracting oil rich in saturated and unsaturated fatty acids from the mycelia;
the fermentation medium comprises the following components: 50-150 g/L carbon source, 150-200 g/L potato, 0.1-0.5 g/L sodium citrate, 10-20 g/L sea salt and 0.1-1.5 g/L, KH nitrogen source
2PO
42～6g/L、MgSO
450～150mg/L、FeCl
32～6μg/L；
The carbon source is selected from one or more of glucose, maltose, glycerol, lactose and sucrose;
the nitrogen source is a mixture of yeast extract and peptone, potassium nitrate, ammonium sulfate, urea or beef extract;
the carbon-nitrogen ratio is 40-120;
the penicillium ecteinascidinium Asc2-4 was deposited at the Guangdong province culture Collection center at 2016, 7, 16, with the deposit numbers GDMCC NO: 60059.
in a preferred embodiment, the carbon source is glucose; the concentration of the carbon source is 100-125 g/L.
In a preferred embodiment, the nitrogen source is a mixture of yeast extract and peptone; the mass ratio of the yeast extract to the peptone is 1: 1.
in one preferred example, the carbon-nitrogen ratio is 100.
In one preferred embodiment, the composition of the fermentation medium is optimally: 100g/L glucose, 200g/L potato, 0.1g/L sodium citrate, 15g/L sea essence salt, 0.5g/L peptone,Yeast extract 0.5g/L, KH
2PO
44g/L、MgSO
4150mg/L、FeCl
32.0μg/L。
In one preferred example, the liquid content is 40% of the volume of the culture vessel, and the rotation speed is 220 rpm.
In one preferred example, the seed culture medium comprises the following components: 6-10 g/L of potato leaching powder, 18-22 g/L of glucose, 13-17 g/L of sea salt and 8-12 g/L of ammonium sulfate. In one preferred example, the composition of the seed culture medium is more preferably: 8g/L of potato extract powder, 20g/L of glucose, 15g/L of sea salt and 10g/L of ammonium sulfate.
In one preferred example, the culture conditions of S1 are 26-28 ℃ and 180-200 rpm for 2-3 days.
In one preferred example, the culture temperature of S2 is 26-28 deg.C, and the culture time is 5-7 days.
In one preferred example, the method for extracting the oil rich in saturated and unsaturated oil from the mycelium is an acid-thermal method, and the specific steps are as follows: after the mycelium culture is finished, carrying out solid-liquid separation, then drying the mycelium to obtain dry mycelium, and carrying out crushing and wall breaking treatment; then adding hydrochloric acid, standing at room temperature, heating in a boiling water bath, quickly cooling, and repeating for 2-3 times; and finally adding chloroform-methanol mixed solution, fully oscillating, centrifuging, and volatilizing to remove chloroform to obtain the grease.
In one preferable example, the mass-volume ratio of the dry bacteria to the hydrochloric acid is 1-1.5 g: 5.5-6 mL, and the concentration of the hydrochloric acid is 4-4.5 mol/L.
In one preferable example, the standing time at room temperature is 30-35 min; the time for heating in the boiling water bath is 9-12 min.
In one preferable example, in the chloroform-methanol mixed solution, the volume ratio of chloroform to methanol is 1.5-2.5: 0.8 to 1.2.
In one preferred example, the saturated fatty acids include stearic acid and palmitic acid, and the unsaturated fatty acids include oleic acid and linoleic acid; the content of saturated fatty acid in the obtained grease is 46-50%, and the content of unsaturated fatty acid is 50-54%.
In one preferred example, the content of oleic acid accounts for 25-27% of the total oil mass, and accounts for 46-54% of the unsaturated fatty acid mass; the content of linoleic acid accounts for 25-27% of the total oil mass and 46-54% of the unsaturated fatty acid mass.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, the biomass and the grease concentration are respectively improved from 7.35g/L and 0.40g/L to 14.47g/L and 4.43g/L by optimizing the culture medium of Penicillium ecteinascidianum Asc2-4 by about 1 time and 10 times respectively.
(2) According to the invention, the proper rotating speed and liquid loading amount are determined by setting different dissolved oxygen conditions, and the optimized biomass and oil concentration are further increased to 19.90g/L and 8.76g/L which are respectively increased by about 0.3 time and 1 time.
(3) According to the invention, by optimizing the culture medium and culture conditions of Penicillium ascidianum Asc2-4, the grease rich in saturated and unsaturated fatty acids is prepared by fermenting Penicillium ascidianum Asc2-4, and the four fatty acids with the highest contents are as follows: linoleic acid, stearic acid, oleic acid and palmitic acid, wherein the content of oleic acid and linoleic acid with higher functional value accounts for 25-27% of the total oil mass and 46-54% of the unsaturated fatty acid mass. The invention can produce functional grease rich in oleic acid and linoleic acid by utilizing ascidian penicillium citrinum Asc 2-4.
Drawings
FIG. 1 shows the effect of five single carbon sources on biomass and oil concentration.
FIG. 2 is a graph showing the effect of different glucose concentrations on biomass and lipid concentrations.
FIG. 3 shows the effect of five nitrogen sources on biomass and oil and fat concentration.
FIG. 4 shows the effect of different carbon to nitrogen ratios on biomass and lipid concentrations.
FIG. 5 is a graph of mean principal effect.
FIG. 6 shows the effect of different dissolved oxygen conditions on the biomass and lipid concentration of Penicillium citrinum Asc 2-4; wherein A is the influence of different dissolved oxygen conditions on biomass; and B is the influence of different dissolved oxygen conditions on the oil concentration.
FIG. 7 is a gas chromatogram of Penicillium citrinum Asc2-4 oil.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. The present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are intended to be included in the scope of the present invention.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
(1) Apparatus and device
HZQ-F160 shaking incubator, Harbin east Union electronics technology development Co., Ltd; BMJ-25 type mold incubator, Shanghai Boxun industries Co., Ltd medical equipment factory; LS-B50L vertical pressure steam sterilization pot, Shanghai Hualin medical nuclear instruments, Inc.; GC-2010PLUS gas chromatograph, Shimadzu, Japan.
(2) Bacterial strains
The strain is as follows: penicillium echinocandioides Asc2-4(Penicillium citrinum Asc2-4), isolated from ascidians in the pond of quicksand shrimp, Renzhou, Guangdong province, and stored in the Guangdong province collection of microbial cultures with the accession number GDMCC NO: 60059.
example 1 method optimization
1. Method of producing a composite material
(1) Seed culture and fermentation
Seed culture: a single colony of Penicillium echinocandis Asc2-4 (isolated from ascidians in the pond of quicksand shrimp in Rezhou, Guangdong province, and stored in the Guangdong province center for culture of microbial strains with the accession number GDMCC NO: 60059) was inoculated into a 500mL Erlenmeyer flask containing 200mL seed medium and cultured for 2 days in a shaker at 28 ℃ and 180 rpm. The seed culture medium comprises the following components: 8g/L of potato extract powder, 20g/L of glucose, 15g/L of sea salt and 10g/L of ammonium sulfate.
And (3) shaking table fermentation culture: the cultured seed solution was inoculated at 5% into a 250mL Erlenmeyer flask containing a fermentation medium and fermented for 7d in a shaker at 28 ℃. The composition of the initial fermentation medium was: 120g/L glucose, 200g/L potato, 0.1g/L sodium citrate, 0.2g/L dipotassium hydrogen phosphate, 2.0g/L potassium dihydrogen phosphate, 0.5g/L yeast extract, 2g/L ammonium sulfate and 15g/L sea salt.
(2) Carbon nitrogen source single factor optimization
Carbon source: single carbon source optimization includes glucose, maltose, glycerol, lactose and sucrose.
Carbon source concentration: the carbon source concentrations were set to 50g/L, 75g/L, 100g/L, 125g/L, and 150g/L for optimization.
Nitrogen source: mixing peptone, potassium nitrate, ammonium sulfate, urea and beef extract with yeast extract according to a mass ratio of 1: the ratio of 1 is added and optimized.
Carbon-nitrogen ratio: under the condition of a certain carbon source concentration, nitrogen sources with different concentrations are added, and the carbon-nitrogen ratio is optimized according to 40, 60, 80, 100 and 120.
Three replicates of each set of experiments were performed and the results averaged.
(3) Optimization of inorganic salts
For 8 kinds of inorganic salts, MgSO
4、CaCl
2、KH
2PO
4、NaH
2PO
4、K
2HPO
4、FeCl
3、ZnSO
4And CoCl
2. And comparing the addition of the inorganic salt with the non-addition of the inorganic salt according to three concentrations, and performing an orthogonal test on the three inorganic salts which obviously improve the oil yield to obtain the optimal combination. Three-factor three-level orthogonal test L9 (3) was performed on inorganic salts that significantly stimulate lipid accumulation
3) (Table 1). Three replicates of each set of experiments were performed and the results averaged.
TABLE 1 factor level table
(4) Setting of dissolved oxygen conditions
The rotation speed of a shaking table of 120rpm, 180rpm, 220rpm and 280rpm and the liquid loading amount of a 250mL conical bottle of 100mL, 150mL and 200mL are respectively combined to be set to 12 groups of different dissolved oxygen conditions, and the appropriate rotation speed and liquid loading amount are finally determined through the fermentation of the shaking table.
2. Evaluation index
(1) Biomass
The biomass is prepared by cell dry weight method, the fermented mycelium is obtained by vacuum filtration, the mycelium is dried in an oven with the temperature of 109 ℃, and the weight is constant and then the mycelium is weighed.
Biomass calculation:
(2) oil concentration and percentage oil content
Extracting grease by an acid-heat method: extracting total oil by adding 6.00mL of 4.00mol/L hydrochloric acid into 1.00g of dry thallus, standing at room temperature for 30min, heating in boiling water bath for 10min, quickly freezing at-20 deg.C, repeating for 2-3 times, adding 2 times volume of chloroform-methanol (2: 1, v/v) mixed solution, shaking thoroughly, centrifuging at 2000r/min for 15min, and volatilizing to remove chloroform-methanol to obtain oil.
Calculating the oil concentration:
calculating the percentage content of the grease:
(3) fatty acid component
And (4) carrying out methyl esterification on the sample, and analyzing the fatty acid component by using a gas chromatograph.
Gas chromatography conditions: SP-2560 gas phase capillary column (100X 0.25X 0.2); the detector is a hydrogen Flame Ion Detector (FID); the injection port temperature is 225 ℃; the temperature of the detector is 250 ℃; the split ratio is 10: 1.
temperature programming conditions: the column temperature is 100 deg.C, maintained for 13min, increased to 180 deg.C at 10 deg.C/min, maintained for 6min, increased to 200 deg.C at 1 deg.C/min, maintained for 20min, increased to 235 deg.C at 4 deg.C/min, and maintained for 20 min.
3. Results
(1) Carbon nitrogen source optimization of fermentation medium
1) Carbon source optimization
As shown in FIG. 1, Penicillium citrinum Asc2-4 can synthesize fats and oils using these five carbon sources. The biomass and the grease concentration of the sucrose, the maltose and the glycerol are similar and are respectively 5.65g/L and 0.32 g/L; 5.00g/L and 0.35 g/L; 5.78g/L and 0.25g/L, it was found that the three carbon sources were similarly utilized by the microorganism. The utilization efficiency of the penicillium citrinum Asc2-4 on lactose is the lowest, and lactose is not suitable as a carbon source. Glucose is used as a carbon source, the biomass and the grease concentration of the strain are highest and respectively reach 7.35g/L and 0.40g/L, and the glucose is determined to be the most suitable single carbon source.
2) Carbon source concentration optimization
As shown in FIG. 2, in the Penicillium citrinum Asc2-4, the biomass gradually increases in the glucose concentration range of 50-100 g/L, while the oil concentration first decreases and then increases. From 100-150 g/L of glucose concentration, the decrease of biomass and oil concentration is probably caused by the inhibition of the bacterial growth and the oil accumulation by the overhigh glucose concentration. The glucose concentration was 125g/L relative to the biomass at 100 g/L. The glucose concentration of 100g/L, biomass and lipid concentration both reached the highest, 8.33g/L and 0.56g/L, respectively (FIG. 2), and thus the optimum glucose concentration was 100 g/L.
3) Nitrogen source optimization
As can be seen from FIG. 3, the five nitrogen sources were optimized by adding them to the yeast extract in a ratio of 1:1, respectively. As can be seen from FIG. 3, ammonium sulfate is more favorable for growth of cells and accumulation of oil and fat than potassium nitrate as the inorganic nitrogen source, and it is found that the efficiency of the cells using the amino nitrogen source is higher than that using the nitro nitrogen source. When the organic nitrogen source is peptone, the biomass and the oil concentration of the Penicillium citrinum Asc2-4 reach the highest values, namely 8.78g/L and 2.71g/L respectively, and the peptone is the most suitable nitrogen source of the Penicillium citrinum Asc2-4 (figure 3). The biomass and the oil content of the two organic nitrogen sources, namely the beef extract and the urea, are lower than those of the peptone. Ammonium sulfate and peptone were used as nitrogen sources, the biomass of the strain was 8.25g/L and 8.58g/L, respectively, and the oil concentration was 0.46g/L and 0.75g/L, respectively, and therefore peptone + yeast extract (mass ratio 1: 1) was the most suitable nitrogen source.
4) Carbon to nitrogen ratio optimization
As can be seen from FIG. 4, when Penicillium citrinum Asc2-4 is cultured under the condition of carbon-nitrogen ratio (40-120), the biomass is in a descending trend and the oil concentration is gradually increased within the range of 40-80, which may be because the lack of nitrogen source affects the growth of the bacteria, and the bacteria enter the oil accumulation stage more quickly due to the gradual decrease of the nitrogen source content, thereby increasing the oil accumulation. The biomass and the grease concentration reach the highest values respectively at 11.59g/L and 2.71g/L (figure 4) when the carbon-nitrogen ratio is 100, which shows that the bacteria is more favorable for grease synthesis under the condition of high carbon-nitrogen ratio. The carbon to nitrogen ratio of 120 caused a decrease in lipid concentration, probably because insufficient nitrogen source restricted hyphal growth and lipid synthesis was inhibited.
(2) Medium inorganic salt optimization
1) Influence of different inorganic salts on growth and oil production of Penicillium citrinum Asc2-4
TABLE 2 Effect of different inorganic salts on Biomass and oil concentration
As can be seen from Table 2, appropriate amount of CaCl was added
2Can increase biomass, but has inhibition effect on oil production of thalli, and the oil concentration of the oil is reduced by 5 percent. K
2HPO
4Neither promotion nor inhibition of the growth of the bacteria nor oil production. ZnSO of suitable concentration
4Can increase biomass, but does not promote grease accumulation. Is suitable for adding NaH
2PO
4And CoCl
2Can promote the growth of thallus, and the oil concentration is respectively increased by 2% and 3%. Wherein the bacterium is MgSO
4And KH
2PO
4The amplitude of stimulating and increasing the grease concentration is large, and is respectively increased by about 30% and 28%. Adding proper amount of Penicillium citrinum Asc2-4Fe of suitable concentration
3+The oil concentration can be greatly improved by 33 percent. In summary, the inorganic salt having an obvious oil concentration promoting effect is MgSO
4、KH
2PO
4And FeCl
3Therefore, the three inorganic salts are selected for further orthogonal optimization.
2) Quadrature test
TABLE 3 Effect of inorganic salt Quadrature test on oil and fat concentration
Results of the pole difference analysis of the orthogonal test are shown in Table 3, KH
2PO
4、MgSO
4And FeCl
3The data of the three highest levels of the three inorganic salts have the average values of A2, B3 and C1, so that the optimal concentration of the inorganic salt is A2B3C1, namely KH
2PO
44.0g/L、MgSO
4150.0mg/L、FeCl
32.0 mu g/L, and the influencing factor C is more than A and more than B, FeCl
3The effect on the strain fermentation for oil production was the greatest (fig. 5). The inorganic salt concentration ratio is as follows: KH (Perkin Elmer)
2PO
44g/L、MgSO
4150mg/L、FeCl
32.0 mu g/L, the oil concentration reaches 4.63g/L after the optimization of inorganic salt, and is improved by 41 percent.
(3) Optimization of dissolved oxygen conditions
As can be seen from FIG. 6, the liquid loading of 100 to 200mL showed a tendency to decrease under each rotation speed condition, indicating that the bacteria is favorable for biomass growth and oil accumulation at a low liquid loading (100 mL). When the dissolved oxygen conditions are 180rpm, 100mL, 180rpm and 150mL, the biomass is higher and reaches 20.39g/L and 20.62g/L respectively, probably because the liquid shearing force under the conditions is more suitable for the growth of the thallus. Under the condition of high rotating speed of 280rpm, the biomass is 11.79-17.44 g/L, which shows that the Penicillium citrinum Asc2-4 has higher liquid shear force resistance, and the characteristic has important significance for industrial oil production. Under the condition of 100mL of 220rpm, the biomass is 19.90g/L, and the oil concentration and the percentage content of the oil are 8.76g/L and 44% (FIG. 6), and it is known that the 100mL liquid loading amount and the 220rpm rotating speed are suitable dissolved oxygen conditions.
(4) Analysis of fatty acid component of Penicillium citrinum Asc2-4
And (3) analyzing the optimized penicillium citrinum Asc2-4 grease by using fatty acid gas chromatography. The results are shown in FIG. 7 and Table 4.
TABLE 4 The percentage contents of fatty acid components in Penicillium citrinum Asc2-4 oil
As shown in fig. 7 and table 4, the penicillium citrinum Asc2-4 oil is mainly C16 and C18 fatty acids, and the four highest-content fatty acids are: linoleic acid > stearic acid > oleic acid > palmitic acid, wherein the fatty acids with higher functional value are oleic acid (C18: 1) and linoleic acid (C18: 2), the contents are 26.05% and 26.67%, respectively, and the total percentage of the two fatty acids reaches 52.72%. Therefore, the penicillium citrinum Asc2-4 can be used for producing the oleic acid and linoleic acid functional oil.
According to the results, the optimal fermentation medium formula suitable for growth and oil production of penicillium citrinum Asc2-4 comprises the following components: 100g/L glucose, 200g/L potato, 0.1g/L sodium citrate, 15g/L sea essence salt, 100g/L glucose, 0.5g/L peptone, 0.5g/L yeast extract, KH
2PO
44g/L，MgSO
4150mg/L，FeCl
32.0μg/L。
The biomass and the oil concentration are respectively increased from 7.35g/L and 0.40g/L to 14.47g/L and 4.43g/L which are respectively increased by about 1 time and 10 times through the culture medium optimization of the Penicillium ascidianum Asc 2-4.
The proper rotating speed and liquid loading amount are determined by setting different dissolved oxygen conditions and are respectively 220rpm and 100mL, and the optimized biomass and the optimized grease concentration are further improved to 19.90g/L and 8.76g/L which are respectively improved by about 0.3 time and 1 time.
Analysis on fatty acid components shows that the content of unsaturated fatty acid reaches 53.37%, wherein the content of oleic acid and linoleic acid reaches more than 26%, and thus, penicillium citrinum Asc2-4 can be used as a strain for producing functional oil and fat of oleic acid and linoleic acid.
Example 2
A method for preparing saturated and unsaturated fatty acid-rich oil by using Penicillium ascidianum Asc2-4 comprises the following steps:
s1, inoculating Penicillium ascidianum Asc2-4 to a seed culture medium, and culturing for 3d in a shaker at 26 ℃ and 200 rpm; the seed culture medium comprises the following components: 8g/L of potato extract powder, 20g/L of glucose, 15g/L of sea salt and 10g/L of ammonium sulfate;
s2, inoculating the cultured seeds to a fermentation medium, and fermenting for 5d in a shaking table at 26 ℃ under the conditions that the liquid loading amount is 40% of the volume of the culture container and the rotating speed is 220rpm to obtain the penicillium citrinum mycelium, wherein the fermentation medium comprises the following components: 100g/L glucose, 200g/L potato, 0.1g/L sodium citrate, 15g/L sea essence salt, 0.5g/L peptone and 0.5g/L, KH yeast extract
2PO
44g/L、MgSO
4150mg/L、FeCl
32.0 mu g/L; the mass ratio of the yeast extract to the peptone is 1: 1;
s3, extracting grease by an acid-thermal method: extracting total oil by adding 6.00mL of 4.00mol/L hydrochloric acid into 1.00g of dry thallus, standing at room temperature for 30min, heating in boiling water bath for 10min, quickly freezing at-20 deg.C, repeating for 2-3 times, adding 2 times volume of chloroform-methanol (2: 1, v/v) mixed solution, fully oscillating, centrifuging at 2000r/min for 15min, and volatilizing to remove chloroform-methanol to obtain oil rich in saturated and unsaturated fatty acids.
Example 3
A method for preparing saturated and unsaturated fatty acid-rich oil by using Penicillium ascidianum Asc2-4 comprises the following steps:
s1, inoculating Penicillium ascidianum Asc2-4 to a seed culture medium, and culturing for 2d in a shaking table at 28 ℃ and 180 rpm; the seed culture medium comprises the following components: 6g/L of potato extract powder, 18g/L of glucose, 13g/L of sea salt and 8g/L of ammonium sulfate;
s2, inoculating the cultured seeds to a fermentation medium, wherein the liquid loading amount is the culture volumeFermenting for 5d in a shaker at 28 ℃ under the conditions of 40% of the volume of the device and 180rpm to obtain the penicillium citrinum mycelium, wherein the fermentation medium comprises the following components: 50g/L glucose, 150g/L potato, 0.1g/L sodium citrate, 10g/L sea essence salt, 0.1g/L peptone and 0.1g/L, KH yeast extract
2PO
42g/L、MgSO
450mg/L、FeCl
32.0 mu g/L; the mass ratio of the yeast extract to the peptone is 1: 1;
s3, extracting grease by an acid-thermal method: extracting total oil by adding 6.00mL of 4.00mol/L hydrochloric acid into 1.00g of dry thallus, standing at room temperature for 30min, heating in boiling water bath for 10min, quickly freezing at-20 deg.C, repeating for 2-3 times, adding 2 times volume of chloroform-methanol (2: 1, v/v) mixed solution, fully oscillating, centrifuging at 2000r/min for 15min, and volatilizing to remove chloroform-methanol to obtain oil rich in saturated and unsaturated fatty acids.
Example 4
A method for preparing saturated and unsaturated fatty acid-rich oil by using Penicillium ascidianum Asc2-4 comprises the following steps:
s1, inoculating Penicillium ascidianum Asc2-4 to a seed culture medium, and culturing for 2d in a shaking table at 28 ℃ and 180 rpm; the seed culture medium comprises the following components: 6g/L of potato extract powder, 18g/L of glucose, 13g/L of sea salt and 8g/L of ammonium sulfate;
s2, inoculating the cultured seeds to a fermentation medium, and fermenting for 3d in a shaking table at 26 ℃ under the conditions that the liquid loading amount is 80% of the volume of the culture container and the rotating speed is 180rpm to obtain the penicillium citrinum mycelium, wherein the fermentation medium comprises the following components: 150g/L glucose, 200g/L potato, 0.5g/L sodium citrate, 20g/L sea essence salt, 1.5g/L peptone and 1.5g/L, KH yeast extract
2PO
46g/L、MgSO
4150mg/L、FeCl
36 mu g/L; the mass ratio of the yeast extract to the peptone is 1: 1;
s3, extracting grease by an acid-thermal method: extracting total oil by adding 6.00mL of 4.00mol/L hydrochloric acid into 1.00g of dry thallus, standing at room temperature for 30min, heating in boiling water bath for 10min, quickly freezing at-20 deg.C, repeating for 2-3 times, adding 2 times volume of chloroform-methanol (2: 1, v/v) mixed solution, fully oscillating, centrifuging at 2000r/min for 15min, and volatilizing to remove chloroform-methanol to obtain oil rich in saturated and unsaturated fatty acids.
When the test is carried out under the same conditions, compared with examples 2, 3 and 4, the content of the saturated fatty acid oil and the unsaturated fatty acid oil obtained in example 2 is the highest. The content of saturated fatty acid in the obtained oil was 46%, and the content of unsaturated fatty acid was 54%. The oleic acid content accounts for 27 percent of the total oil mass and 50 percent of the unsaturated fatty acid mass; the content of linoleic acid accounts for 27 percent of the total oil mass and 50 percent of the unsaturated fatty acid mass.
The applicant declares that the above detailed description is a preferred embodiment described for the convenience of understanding the present invention, but the present invention is not limited to the above embodiment, i.e. it does not mean that the present invention must be implemented by means of the above embodiment. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (10)
1. Application of Penicillium ecteinascidianum Asc2-4 in preparing oil rich in saturated and unsaturated fatty acids.
2. A method for preparing grease rich in saturated and unsaturated fatty acids simultaneously by utilizing penicillium ecteinascidianum Asc2-4 is characterized by comprising the following steps:
s1, inoculating Penicillium ascidianum Asc2-4 to a seed culture medium for shake culture;
s2, inoculating the cultured seeds to a fermentation culture medium, fermenting under the conditions that the liquid loading amount is 40-80% of the volume of a culture container and the rotating speed is 120-280 rpm to obtain penicillium citrinum mycelia, and extracting oil rich in saturated and unsaturated fatty acids from the mycelia;
the fermentation medium comprises the following components: 50-150 g/L carbon source, 150-200 g/L potato, 0.1-0.5 g/L sodium citrate, 10-20 g/L sea salt and 0.1-1.5 g/L, KH nitrogen source
2PO
42～6g/L、MgSO
450～150mg/L、FeCl
32～6μg/L；
The carbon source is selected from one or more of glucose, maltose, glycerol, lactose and sucrose;
the nitrogen source is a mixture of yeast extract and peptone, potassium nitrate, ammonium sulfate, urea or beef extract;
the carbon-nitrogen ratio is 40-120;
the penicillium ecteinascidinium Asc2-4 was deposited at the Guangdong province culture Collection center at 2016, 7, 16, with the deposit numbers GDMCC NO: 60059.
3. the method of claim 2, wherein the carbon source is glucose; the concentration of the carbon source is 100 g/L.
4. The method of claim 3, wherein the nitrogen source is a mixture of yeast extract and peptone; the mass ratio of the yeast extract to the peptone is 1: 1.
5. the method of claim 4, wherein the carbon to nitrogen ratio is 100.
6. The method according to claim 5, wherein the liquid content is 40% by volume of the culture vessel, and the rotation speed is 220 rpm.
7. The method of claim 6, wherein the composition of the seed medium is: 6-10 g/L of potato leaching powder, 18-22 g/L of glucose, 13-17 g/L of sea salt and 8-12 g/L of ammonium sulfate.
8. The method of claim 7, wherein the composition of the seed medium is: 8g/L of potato extract powder, 20g/L of glucose, 15g/L of sea salt and 10g/L of ammonium sulfate.
9. The method according to any one of claims 2 to 8, wherein the fermentation medium has a composition of: 100g/L glucose and 20g/L potato0g/L, 0.1g/L sodium citrate, 15g/L sea essence salt, 0.5g/L peptone and 0.5g/L, KH yeast extract
2PO
44g/L、MgSO
4150mg/L、FeCl
32.0μg/L。
10. The method according to claim 2, wherein the culture conditions of S1 are 26-28 ℃ and 180-200 rpm for 2-3 days; the culture temperature of S2 is 26-28 ℃, and the culture time is 5-7 d;
the saturated fatty acids include stearic acid and palmitic acid, and the unsaturated fatty acids include oleic acid and linoleic acid;
the content of saturated fatty acid in the obtained grease is 46-50%, and the content of unsaturated fatty acid is 50-54%; the oleic acid content accounts for 25-27% of the total oil mass and 46-54% of the unsaturated fatty acid mass; the content of linoleic acid accounts for 25-27% of the total oil mass and 46-54% of the unsaturated fatty acid mass.
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