CN106434777B - Microbial phospholipid type polyunsaturated fatty acid oil - Google Patents
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Abstract
The invention provides phospholipid type polyunsaturated fatty acid oil derived from microorganisms, wherein the content of phospholipid in the oil is not less than 38%, and the content of polyunsaturated fatty acid in the phospholipid is not less than 8%. The grease of the invention has the following beneficial effects: firstly, the polyunsaturated fatty acid in the grease is of microbial origin, so that the grease can meet the requirement of large-scale production and has lower cost; and secondly, the phospholipid content in the grease is higher, so that the grease is more beneficial to being absorbed by a human body.
Description
Technical Field
The invention relates to a phospholipid polyunsaturated fatty acid oil derived from microorganisms.
Background
Phospholipids are lipid compounds containing phosphoric acid, are commonly present in the cytoplasm and cell membrane of organisms, and play a key role in maintaining the function of the cell membrane and further maintaining the cell metabolism. Phospholipids can be classified into two major classes, glycerophospholipids and sphingophospholipids, according to their molecular structure. The glycerol phosphatide mainly comprises lecithin, cephalin, serine phosphatide, inositol phosphatide and the like. The lecithin is fully supplemented every day to achieve good curative effect on diseases caused by hypofunction of the nervous system, such as dysphoria, inappetence, tinnitus, impotence, neurogenic diarrhea, habitual constipation and dementia and memory loss caused by lack of nerve transmission substances. It was found that the offspring of pregnant rats raised with lecithin had significantly better memory in the intellectual testing (maze test) than the offspring of rats raised with no lecithin. In 1997 a seventh international meeting for lecithin made a conclusion: "it is recommended to pregnant women to take appropriate amount of lecithin, which is important for the development of children's intelligence. Lecithin was listed as one of five world nutritional foods by the Food and Agriculture Organization (FAO) of the United nations in 1998; the Food and Drug Administration (FDA) specified in 1999 that all infant formulas should be supplemented with appropriate amounts of lecithin.
Currently, phospholipids are mainly derived from plants, and commercially available phospholipids mainly contain saturated or monounsaturated fatty acids containing 16 or 18 carbon atoms such as palmitic acid, oleic acid, linoleic acid and stearic acid. However, in addition to these fatty acids, the human body needs to be supplemented with polyunsaturated fatty acids such as linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid. Docosahexaenoic acid is present in high concentrations in the brain and is believed to play a crucial role in the development of vision, cognitive function and other normal brain functions. Arachidonic acid is the most abundant and active polyunsaturated fatty acid in mammals, has great significance for the health of human bodies and is also important for the normal development of infants, and has the functions of preventing cardiovascular diseases, regulating immunity, assisting in inhibiting tumors, preventing canceration and regulating nerves, and can prevent and treat diseases such as hypertension, diabetes, obesity, virus infection and the like.
Compared with methyl ester/ethyl ester type or triglyceride type polyunsaturated fatty acid, the phospholipid type polyunsaturated fatty acid not only has the advantages of high bioavailability, high safety, good stability and the like, but also can provide rich phospholipid for organisms, and has the effect of 1+1>2 on the development of brains. The research shows that the human body has only 21 percent of digestibility to the ethyl ester type docosahexaenoic acid, and the digestibility to the phospholipid type docosahexaenoic acid exceeds 99 percent. The docosahexaenoic acid in the egg yolk is tightly combined with the lecithin in the egg yolk and is digested and absorbed by human bodies together with the lecithin, and the egg yolk is particularly suitable for pregnant and lying-in women and infants.
The invention patent with the Chinese patent publication number of CN103509047A discloses an extraction process of phosphatidylcholine from Antarctic krill and a preparation method of phosphatidylserine. The method obtains a phospholipid product with the content of the docosahexaenoic acid of 7.6-8.1 percent and the content of the eicosapentaenoic acid of 14.6-15.6 percent by collecting the Antarctic krill heads and a series of processes such as decoction, stirring and extraction. The disadvantages of this approach are: the source of raw materials is greatly limited, a large amount of polyunsaturated fatty acid is lost in the processes of decoction and the like, and the environment is damaged by the large fishing of the Antarctic krill.
The invention patent with Chinese patent publication No. CN103815434A discloses a process for preparing yolk powder rich in yolk lecithin DHA, which comprises the steps of firstly selecting eukaryotic microorganisms and marine microalgae for proliferation and fermentation in a nutritional agent, then preparing DHA nutritional essence powder as an additive component of laying hen feed according to a certain proportion together with deep sea fish oil, fishbone powder and natural plant mixed powder, collecting eggs laid by laying hens after feeding for 10 days, taking yolk, adding soybean lecithin homogenate, and homogenizing into liposome by a high-pressure homogenizer; the yolk lecithin DHA liposome is taken as a core material, the monoglyceride sodium caseinate is taken as an emulsifier, and the dextrin is taken as a wall material, and the finished product is prepared by mixing, emulsifying, drying and screening the materials through a high-pressure homogenizer. The method has low conversion rate, the DHA content is only 6-7%, and the cost is high.
Therefore, it is necessary to provide a phospholipid-type polyunsaturated fatty acid oil which is environmentally friendly and less expensive.
Disclosure of Invention
The purpose of the present invention is to provide a phospholipid-type polyunsaturated fatty acid oil containing polyunsaturated fatty acids derived from microorganisms.
In order to achieve the above object, the present invention provides a phospholipid-type polyunsaturated fatty acid oil derived from microorganisms, wherein the phospholipid content in the oil is not less than 38%, and the polyunsaturated fatty acid content in the phospholipid is not less than 8%.
The grease of the invention has the following beneficial effects: firstly, the polyunsaturated fatty acid in the grease is of microbial origin, so that the grease can meet the requirement of large-scale production and has lower cost; and secondly, the phospholipid content in the grease is higher, so that the grease is more beneficial to being absorbed by a human body.
Further, in the fat or oil of the present invention, the microbial source is Mortierella alpina, yeast, Schizochytrium, Dunaliella salina, or dinoflagellate.
In the fat or oil of the present invention, the polyunsaturated fatty acid includes linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
Example 1
The method takes microspherical algae as a fermentation strain and comprises the following steps in sequence:
step 1, fermenting to obtain fermentation liquor of microbial thallus rich in eicosapentaenoic acid, and processing the fermentation liquor to obtain dry thallus rich in eicosapentaenoic acid.
Step 2, 500g of the dry cell and about 2.5L of n-hexane were put in a 5L three-necked flask and extracted in a water bath at 50 ℃ with continuous stirring. Extracting for 4 hours, carrying out suction filtration, collecting filtrate, repeating for several times until the oil in the thallus is almost completely extracted, obtaining 7.5L of an organic phase rich in eicosapentaenoic acid, and measuring the actual weight of the oil in the organic phase to be 193 g;
and 3, concentrating the 7.5L of the organic phase rich in the eicosapentaenoic acid to 1.1L by reduced pressure evaporation.
And 4, taking 320g of commercial powder phospholipid and the 1.1L of organic phase rich in eicosapentaenoic acid, putting into a 3L three-neck flask, and uniformly shearing by using a shearing machine. 48g of lipase and 9.6g of water were added to the mixture, and the mixture was stirred with electric power at a rotational speed of 200rpm, and the temperature of the water bath was set at 50 ℃ and kept constant, and the reaction was stopped after the reaction was started for 4 hours when the temperature reached 50 ℃.
And 5, centrifuging the reacted mixture, separating out lipase, collecting supernatant, cooling to normal temperature after desolventizing, and adding 3L of acetone to remove grease. The obtained solid was freeze-dried to remove acetone, whereby 297g of powdered phospholipid was obtained. The powdered phospholipid of this example is a specific form of phospholipid type polyunsaturated fatty acid oil. The step is a purification step, and mainly aims to remove impurities such as lipase, solvent, grease and the like and obtain high-purity powder phospholipid. The detection shows that the phospholipid content of the powder phospholipid is 96%, and the total content of polyunsaturated fatty acids (the sum of eicosatetraenoic acid and eicosapentaenoic acid) in the phospholipid is 8%.
Example 2
The method comprises the following steps of taking yeast producing linolenic acid as a fermentation strain:
step 1, fermenting to obtain a fermentation liquor of microbial thallus rich in linolenic acid, and processing the fermentation liquor to obtain the microbial thallus rich in linolenic acid
Drying the thalli;
and 2, taking 3kg of the dry bacteria and 15L of n-hexane in a 25L reaction kettle, and stirring and refluxing in a water bath at 65 ℃ for extraction. Extracting for 4 hr, centrifuging, collecting supernatant, extracting thallus with 12L n-hexane, repeating several times until the oil in thallus is almost completely extracted to obtain linolenic acid-rich organic phase 37.8L, and measuring to obtain actual oil 1113 g.
And 3, concentrating the 37.8L of organic phase rich in linolenic acid to 3.3L by reduced pressure evaporation.
And 4, putting 500g of commercially available powdered phospholipid and 1.7L of the organic phase rich in linolenic acid into a 3L three-neck flask, fully mixing the materials by using a shearing machine to obtain a uniform phase, and stopping shearing. 100g of lipase and 20g of water are added into the mixture, electric stirring is carried out, the rotating speed is set to be 300rpm, the water bath temperature is set to be constant at 48 ℃, the reaction is stopped after the reaction is started for 6 hours when the temperature reaches 48 ℃.
And 5, centrifuging the reacted mixture, separating out lipase, collecting supernatant, and desolventizing to obtain 989.3g of microbial oil. 500g of the microbial oil is taken and introduced with steam of 110 ℃, hydration reaction is carried out for 5 minutes, then cooling and centrifugation are carried out to obtain crude phospholipid, and the crude phospholipid is subjected to vacuum dehydration and drying to obtain 389g of the linolenic acid-rich phospholipid type polyunsaturated fatty acid oil. Through detection, the content of phospholipid in the microbial oil is 59.2%, and the total content of polyunsaturated fatty acids (linolenic acid) in the phospholipid is 9.7%.
Example 3
The dinoflagellate is taken as a fermentation strain, and the method comprises the following steps in sequence:
step 1, fermentation is carried out to obtain fermentation liquor of microbial thalli rich in docosahexaenoic acid, and the fermentation liquor is processed to obtain dry thalli rich in docosahexaenoic acid.
And 2, taking 27kg of the dried thallus and about 100L of n-hexane in a 200L reaction kettle, and continuously stirring and extracting in a water bath at 50 ℃. And (3) after 4 hours of extraction, settling and separating, filtering the supernatant by a 0.5um titanium rod, repeating for a plurality of times until the grease in the thalli is almost completely extracted, obtaining an organic phase 293L rich in docosahexaenoic acid, and measuring the actual oil content to be 12.9 kg.
Step 3, 293L of organic phase rich in docosahexaenoic acid is concentrated to 19.3L by thin film evaporation.
And 4, putting 5kg of commercially available powdered phospholipid and the 12L of docosahexaenoic acid-rich organic phase into a 25L reaction kettle, and fully and uniformly grinding by using a colloid mill. 1kg of lipase and 200g of water were added to the mixture, the reaction was stirred at 200rpm and 50 ℃ and stopped after the reaction was started for 8 hours at 50 ℃.
And 5, filtering the reacted mixture, separating out lipase, collecting filtrate, and evaporating the filtrate by using a thin film to obtain 12.2kg of microbial oil rich in phospholipid docosahexaenoic acid. This step is a process for purifying phospholipids. Through detection, the content of phospholipid in the microbial oil is 38%, and the total content of polyunsaturated fatty acids (the sum of docosahexaenoic acid and eicosapentaenoic acid) in the phospholipid is 15.7%.
Example 4
Taking mortierella alpina as a fermentation strain, and sequentially operating according to the following steps:
step 1, fermenting to obtain fermentation liquor of microbial thalli containing arachidonic acid, and processing the fermentation liquor to obtain dry thalli rich in arachidonic acid;
step 2, taking 100kg of dry bacteria of eicosatetraenoic acid and 300L of n-hexane in 1m3The reaction vessel (2) was stirred at 55 ℃ to extract. Extracting for 4 hoursFiltering, fine-filtering the filtrate with 0.5um titanium rod, and collecting the filtrate. Repeating for several times until the oil in the thallus is almost completely extracted to obtain an organic phase 1242L containing eicosatetraenoic acid oil, and measuring the actual oil content in the organic phase to be 51.7 kg.
And 3, concentrating the 1242L of the eicosatetraenoic acid-rich organic phase to 101.7L by reduced pressure evaporation.
And 4, putting 17kg of commercial powder phospholipid and the 101.7L of organic phase containing the eicosatetraenoic acid grease into a 200L reaction kettle, and fully and uniformly mixing by using a shearing machine. 3kg of lipase and 100g of water are added into the mixture, the mixture is stirred and reacted, the rotating speed is set to be 1500rpm, the temperature is set to be constant at 55 ℃, the reaction is stopped after the reaction is started for 8 hours when the temperature reaches 55 ℃.
And 5, centrifuging the reacted mixture, separating out lipase, collecting supernatant, and desolventizing to obtain 65.1kg of microbial oil. Taking 15kg of the microbial oil, heating to 80 ℃, adding 1.5kg of pure water for hydration reaction for 30min, and then centrifuging to obtain 7.47kg of crude concentrated phospholipid. This step is the process for enriching the phospholipids in the microbial oil. Removing water from 7.47kg of crude concentrated phospholipid at 85-90 deg.C under-0.095 Mpa. Cooling to below 50 deg.C after the water content in the crude phospholipid is less than 0.5%, to obtain 5.58kg microbial oil rich in phospholipid type eicosatetraenoic acid. Through detection, the content of phospholipid in the microbial oil is 63.2%, and the total content of polyunsaturated fatty acid (the sum of linolenic acid and eicosatetraenoic acid) in the phospholipid is 26.7%.
Example 5
The method takes Dunaliella salina as a fermentation strain and comprises the following steps in sequence:
step 1, fermentation is carried out to obtain fermentation liquor of microorganism thallus rich in linolenic acid, and the fermentation liquor is processed to obtain dry thallus rich in linolenic acid.
Step 2, putting 500kg of the dry thalli into a workshop extraction tank, adding 1500L of normal hexane, controlling the temperature to be 55 ℃, starting a colloid mill to perform circular extraction for 3 hours, performing sedimentation separation, fine-filtering supernatant by using a 0.5um titanium rod, collecting supernatant, repeating the steps for a plurality of times until the grease in the thalli is almost completely extracted, obtaining 3870L of an organic phase rich in linolenic acid, and measuring the actual grease in the organic phase to be 259 kg;
and 3, concentrating the 3870L organic phase rich in the linolenic acid to 513L by decompression and evaporation.
And 4, putting 27kg of commercially available powdered phospholipid and the 110L of docosahexaenoic acid-rich organic phase into a 200L reaction kettle, uniformly mixing by shearing, adding 5kg of lipase and 200g of water into the mixture, stirring for reaction, setting the rotation speed to be 180rpm, setting the temperature to be 40 ℃, keeping the temperature, starting timing when the temperature reaches 40 ℃, and stopping the reaction after 12 hours of reaction.
Step 5, filtering the reacted mixture, separating out lipase, collecting filtrate, and continuously adding 100L of n-hexyl into the filtrate
Adding 2.5kg pure water into alkane, microfiltering with 0.2um pore size ceramic membrane at 35-40 deg.C under 0.15MPa, and evaporating the semi-solid obtained by membrane interception with thin film to obtain 40.7kg microbial oil rich in phospholipid type linolenic acid. This step is a process of simultaneously performing enrichment and purification treatment of phospholipids. Through detection, the content of phospholipid in the microbial oil is 63.3%, and the total content of polyunsaturated fatty acids (linolenic acid) in the phospholipid is 21.3%.
Example 6
Using schizochytrium limacinum as a fermentation strain, and sequentially operating according to the following steps:
step 1, fermenting to obtain fermentation liquor of microbial thalli rich in docosahexaenoic acid, and centrifuging the fermentation liquor to obtain concentrated solution with the water content of 75%;
and 2, putting 2000kg of the concentrated solution into a workshop extraction tank, adding 8L of alkaline protease, continuously stirring in a water bath at 50 ℃ for breaking the wall for 4 hours, and then adding 2000L of hexane and 1000L of absolute ethyl alcohol for extraction. Centrifuging, collecting upper layer solvent oil liquid to obtain docosahexenoic acid-rich organic phase 2207L, and measuring the actual oil content to 217.8 Kg.
Step 3, concentrating 2207L of docosahexaenoic acid organic phase to 427L by thin film evaporation.
And 4, putting 51kg of commercially available powdered phospholipid and 100L of docosahexaenoic acid-rich organic phase into a 250L reaction kettle, fully and uniformly mixing by using a colloid mill, adding 10kg of lipase and 2.5kg of water into the mixture, stirring for reaction, setting the rotation speed at 200rpm, setting the temperature at 45 ℃ for constant temperature, starting timing when the temperature reaches 45 ℃, and stopping the reaction after 8 hours of reaction.
And 5, filtering the reacted mixture, separating out lipase, collecting supernatant, and desolventizing to obtain 96.9kg of microbial oil. Taking 20kg of the microbial oil, adding 60L of acetone, stirring at 20-30 ℃ for reaction for 0.5h, and repeatedly treating for 2 times. The resulting wet solid was vacuum dried to remove acetone to give 8.71kg of powdered phospholipid.
And 6, putting 35L of the organic phase rich in docosahexaenoic acid concentrated in the step 3 and 8.71kg of the powdered phospholipid in the step 5 into a 70L reaction kettle, uniformly shearing by using a shearing machine, adding 1kg of lipase and 160g of water into the mixture, stirring for reaction, setting the rotation speed at 170rpm, setting the temperature at 58 ℃ for constant temperature, starting timing to react for 5 hours when the temperature reaches 58 ℃, and stopping the reaction.
And 7, filtering the mixture reacted in the step 6, separating out lipase, collecting supernatant, and desolventizing to obtain 25.1kg of microbial oil. Adding 70L acetone into the microbial oil, stirring at 20-30 deg.C for reaction for 0.5h, and repeating the treatment for 2 times. The resulting wet solid was vacuum dried to remove acetone to obtain 8.03kg of powdered phospholipid. The powdered phospholipid of the embodiment is a special form of microbial oil rich in phospholipid type polyunsaturated fatty acids. The step is a purification step and mainly aims at removing impurities such as grease, solvent and the like. The detection shows that the content of phospholipid in the microbial oil is 97.9%, and the total content of polyunsaturated fatty acids (the sum of arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid) in the phospholipid is 35%.
Claims (1)
1. A phospholipid-type polyunsaturated fatty acid oil derived from a microorganism, characterized in that: the content of phospholipid in the oil is 97.9%, and the polyunsaturated fatty acid in the phospholipid is arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid, and the total content is 35%;
the preparation method of the phospholipid polyunsaturated fatty acid oil comprises the following steps:
using schizochytrium limacinum as a fermentation strain, and sequentially operating according to the following steps:
step 1, fermenting to obtain fermentation liquor of microbial thalli rich in docosahexaenoic acid, and centrifuging the fermentation liquor to obtain concentrated solution with the water content of 75%;
step 2, putting 2000kg of the concentrated solution into a workshop extraction tank, adding 8L of alkaline protease, continuously stirring in a water bath at 50 ℃ for breaking the wall for 4 hours, and then adding 2000L of hexane and 1000L of absolute ethyl alcohol for extraction; centrifuging, and collecting upper layer solvent oil liquid to obtain docosahexaenoic acid-rich organic phase 2207L;
step 3, concentrating the organic phase to 427L by thin film evaporation;
step 4, putting 51kg of commercially available powdered phospholipid and 100L of docosahexaenoic acid-rich organic phase into a 250L reaction kettle, fully and uniformly mixing by using a colloid mill shearing machine, adding 10kg of lipase and 2.5kg of water into the mixture, stirring for reaction, setting the rotating speed to be 200rpm, setting the temperature to be 45 ℃, keeping the temperature constant, starting timing when the temperature reaches 45 ℃, and stopping the reaction after 8 hours of reaction;
step 5, filtering the reacted mixture, separating out lipase, collecting supernatant, and desolventizing to obtain 96.9kg of microbial oil; adding 60L acetone into 20kg of the microbial oil, stirring at 20-30 deg.C for reaction for 0.5 hr, and repeating the treatment for 2 times; the obtained wet solid was vacuum dried to remove acetone to obtain 8.71kg of powdered phospholipid;
step 6, putting 35L of the organic phase rich in docosahexaenoic acid concentrated in the step 3 and 8.71kg of the powdered phospholipid in the step 5 into a 70L reaction kettle, shearing and uniformly mixing the organic phase and the powdered phospholipid by a shearing machine, adding 1kg of lipase and 160g of water into the mixture, stirring the mixture for reaction, setting the rotating speed to be 170rpm, setting the temperature to be 58 ℃, keeping the temperature constant, starting timing the reaction for 5 hours when the temperature reaches 58 ℃, and stopping the reaction;
step 7, filtering the mixture reacted in the step 6, separating out lipase, collecting supernatant, and desolventizing to obtain 25.1kg of microbial oil; adding 70L acetone into the microbial oil, stirring at 20-30 deg.C for reaction for 0.5h, and repeating for 2 times; the obtained wet solid was vacuum-dried to remove acetone, whereby 8.03kg of the phospholipid-type polyunsaturated fatty acid oil derived from the microorganism was obtained.
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