阅读说明：本技术 利用海鞘桔青霉Asc2-4制备同时富含饱和与不饱和脂肪酸油脂的方法 (Method for preparing grease rich in saturated and unsaturated fatty acids simultaneously by using penicillium ecteinascidianum Asc2-4 ) 是由 雷晓凌 罗正东 聂芳红 钟敏 刘唤明 于 2019-11-26 设计创作，主要内容包括：本发明公开了一种利用海鞘桔青霉Asc2-4制备同时富含饱和与不饱和脂肪酸油脂的方法。该方法将海鞘桔青霉Asc2-4接种于种子培养基进行摇床培养；将培养后的种子接种于发酵培养基,在合适的溶氧条件下进行发酵后进行提取；发酵培养基的组成为：碳源、土豆、柠檬酸钠、海精盐、氮源、KH<Sub>2</Sub>PO<Sub>4</Sub>、MgSO<Sub>4</Sub>和FeCl<Sub>3</Sub>。本发明通过海鞘桔青霉Asc2-4的培养基和培养条件优化,制备出同时富含饱和与不饱和脂肪酸的油脂,油酸和亚油酸含量占总油脂质量的25％～27％,占不饱和脂肪酸质量的46％～54％,能够生产出富含油酸和亚油酸的功能性油脂。(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 2 PO 4 、MgSO 4 And 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.)

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.

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.