CN117265027A - Method for preparing UPU structural fat rich in DHA through microbial fermentation - Google Patents

Method for preparing UPU structural fat rich in DHA through microbial fermentation Download PDF

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CN117265027A
CN117265027A CN202311406413.5A CN202311406413A CN117265027A CN 117265027 A CN117265027 A CN 117265027A CN 202311406413 A CN202311406413 A CN 202311406413A CN 117265027 A CN117265027 A CN 117265027A
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dha
upu
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张林尚
岳珂
毕艳兰
李军
陈小威
孙聪
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Henan University of Technology
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    • 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
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    • 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 invention belongs to the technical field of structural lipid preparation, and particularly relates to a method for preparing UPU structural lipid rich in DHA by microbial fermentation. According to the method for preparing the UPU structural fat rich in DHA through microbial fermentation, algae oil is used as a carbon source, rhodococcus turbidi is used as a fermentation strain, and the UPU structural fat rich in DHA is prepared through a microbial fermentation method. In addition, the fermentation method of the invention synthesizes grease and products naturally in cells, has high content of triglyceride, less byproducts and low cost, can prepare UPU structural grease by fermenting once, avoids the defect of complicated enzymatic synthesis process, has wide application prospect in the field of preparing UPU structural grease rich in DHA, and can also provide technical support for research, development and production of infant health food and dietary supplements.

Description

Method for preparing UPU structural fat rich in DHA through microbial fermentation
Technical Field
The invention belongs to the technical field of structural lipid preparation, and particularly relates to a method for preparing UPU structural lipid rich in DHA by microbial fermentation.
Background
UPU structured fat is a fat with special functions found in human milk fat, which refers to a class of triglycerides with unsaturated fatty acids attached to the sn-1,3 position and palmitic acid attached to the sn-2 position of the glycerol backbone. The existing research shows that UPU structured lipid can promote infants to absorb fat and mineral substances better, reduce the hardness of feces, and provide more and more complete fatty acid for the body.
Docosahexaenoic acid (DHA) is an omega-3 long chain polyunsaturated fatty acid, which is a fatty acid necessary for brain growth and maintenance function, and has important significance for human nerves, brain development, vision, etc. The infant body converts alpha-linolenic acid as a precursor substance to synthesize DHA, but the infant body can not meet the requirement of growth and development of the infant body, and the infant body has imperfect digestion and absorption system and low absorption and utilization rate of DHA, so DHA supplementation is needed through diet. DHAP DHA refers to UPU type structural grease with DHA connected with sn-1,3 position and palmitic acid (P) connected with sn-2 position of a glycerol skeleton, and is an important component of the UPU structural grease. DHA is applied to UPU structural fat, so that infant can absorb and utilize polyunsaturated fatty acid better, and the infant dietary supplement has the effects of improving gastrointestinal tract and promoting digestion, and has important application prospect in infant dietary supplements and health foods.
At present, methods for preparing UPU structural grease mainly comprise chemical synthesis and enzymatic synthesis, and researches on UPU type structural grease are focused on structural grease such as OPO, OPL and the like. Aiming at chemical synthesis, the invention patent with the application number of CN107473963A discloses a method for preparing OPO-type UPU structural grease by chemical synthesis, which comprises the steps of firstly reacting 1, 3-dichloro isopropanol with methyl hexadecanoate to obtain 1, 3-dichloro-2-hexadecanoate, then reacting 1, 3-dichloro-2-hexadecanoate with sodium oleate, and distilling to obtain 1, 3-dioleoyl-2-palmitic acid triglyceride (OPO). The UPU structured grease prepared by the chemical synthesis method can obtain OPO with a certain yield, but the reaction adopts a plurality of chemical intermediates and organic solvents, which is not in accordance with the development concept of green and health.
In comparison, the enzymatic synthesis of UPU structural lipid has the advantages of mild reaction conditions, high selectivity, easy separation of lipase and the like. At present, the UPU structured grease is mainly produced by an enzyme method in industry. The enzyme method is a method for preparing UPU structured grease by acidolysis or transesterification with sn-1,3 specific lipase and palm stearin as a substrate and adding unsaturated fatty acid or unsaturated fatty acid ester. The invention patent with application publication number of CN102845537A discloses a method for preparing a human milk fat substitute rich in UPU structural fat by adopting a two-step enzymatic transesterification method, wherein the preparation process mainly comprises the steps of taking high-melting-point palm stearin as a substrate, carrying out transesterification reaction with formulated polyunsaturated fatty acid grease under the action of immobilized lipase (Lipozyme RM IM) after acidolysis, and obtaining a product with more than 61% of sn-2 palmitic acid, and higher sn-1,3 oleic acid and linoleic acid contents. Zhu Qisai (research on preparation of human milk fat substitute by acidolysis of lard oil) is carried out on lard oil serving as a substrate and vegetable fat fatty acid under the catalysis of Lipozyme RM IM to prepare the human milk fat substitute rich in UPU structural fat, wherein the sn-2 palmitic acid content of the obtained product is 63.08%, the content of the obtained product is 71.33% of total palmitic acid, and the fatty acid composition is similar to that of human milk fat. However, enzymatic preparation of UPU structured fats also has a technical bottleneck that has a high dependence on lipase (sn-1, 3 selectivity), which is expensive and the production technology is mastered in developed countries, resulting in a much higher selling price of UPU structured fats than ordinary vegetable fats. In addition, enzymatic processes also lead to the formation of various by-products (0-3% of monoglyceride, 0-10% of diglyceride, 60% -70% of fatty acids), limiting their further industrial application.
As is clear from the current state of research, most of the current methods for producing UPU structured lipid are directed to structured lipid containing monounsaturated fatty acid such as OPO and OPL, and few studies are conducted on DHAP DHA type UPU structured lipid containing polyunsaturated fatty acid. Therefore, the method for preparing the UPU structural fat rich in DHA economically and efficiently is explored, and has important significance for the research and development and production of infant health foods and dietary supplements.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a method for preparing the UPU structural fat rich in DHA by microbial fermentation, which is used for fermenting and culturing rhodococcus turbidi in a fermentation medium containing algae oil solid fat to obtain thalli, and then extracting grease to obtain the UPU structural fat rich in DHA, and has the advantages of low cost, natural products, high content of triglyceride and the like, and is suitable for preparing and applying the UPU structural fat rich in DHA.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a method for preparing a UPU structured lipid rich in DHA by microbial fermentation, comprising the steps of:
(1) Inoculating rhodococcus microorganisms into a basic culture medium for activation culture to obtain seed liquid; the rhodococcus microorganism is rhodococcus turbidi;
(2) Inoculating the seed liquid into a fermentation culture medium for fermentation culture, centrifuging after the culture is finished to remove fermentation liquid to obtain thalli, crushing the thalli, and extracting grease, wherein the obtained grease is UPU structural grease rich in DHA;
the fermentation medium is prepared by adding 8-16 g/L algae oil solid fat serving as a carbon source into an MSM (multi-stage fermentation) medium; in the Sn-2 fatty acid composition of the algae oil lipid-solidifying, the content of palmitic acid is 10% -30%, and the content of DHA is 40% -70%.
As a further improvement, in step (1), the basal medium is a nutrient broth medium.
Further preferably, in the step (1), the inoculation amount of the rhodococcus microorganism is 0.08 to 0.12v/v%; more preferably 0.1v/v%.
More preferably, the activation culture is performed at a temperature of 25 to 35℃and a rotation speed of 100 to 200rpm until the OD value of the seed solution reaches 0.8 to 1.5.OD value refers to the violet absorbance at 660 nm.
As a further improvement, in the step (2), the seed liquid is inoculated in an amount of 0.8 to 1.2v/v%, more preferably, 1.0v/v%.
MSM medium is a basic salt medium which is common in the art, and the composition of the present invention is not particularly limited. The MSM culture medium formula conventional in the art can be adopted, and the fermentation requirement of the rhodococcus microorganism can be met. As a further improvement, in the step (2), the composition of the MSM medium includes urea, magnesium sulfate, calcium chloride, phosphate, cobalt chloride, copper chloride, manganese chloride, nickel chloride, and water.
As a further improved scheme, in the step (2), the content of palmitic acid in the whole fatty acid composition of the algae oil solid fat is 30% -50%, and the content of DHA is 30% -50%.
As a further improvement, in the step (2), the fermentation culture is carried out at a temperature of 25-35 ℃ and a rotation speed of 100-200 rpm for 80-120 hours.
As a further improved scheme, in the step (2), the rotating speed of the centrifugation is 3000-8000 rpm, and the centrifugation time is 3-10 min.
As a further improved scheme, in the step (2), the broken thalli is processed by an enzymolysis method or an acid heating method; the enzymolysis method is to add protease into thalli for enzymolysis to break thalli; the acid heating method is to put the cells in an acid solution and heat-treat them in a boiling water bath to crush them. More preferably, the acid heating method is a method in which the cells are placed in 2 to 4mol/L hydrochloric acid and treated in a boiling water bath for 3 to 6 minutes.
As a further improved scheme, in the step (2), the extraction is carried out by adopting a centrifugal separation method or a solvent extraction method; the centrifugal separation method is to centrifuge the crushed thalli and then separate to obtain grease; the solvent extraction is to extract the grease in the thalli by adopting the solvent, and combine the grease after 2 to 5 times of extraction. Further, the solvent is n-hexane.
Compared with the prior art, the invention has the outstanding advantages that:
according to the method for preparing the UPU structural fat rich in DHA through microbial fermentation, algae oil is used as a carbon source, and after rhodococcus turbidi is fermented, the UPU structural fat rich in DHA is obtained by extracting the oil, and the DHAP DHA content in the structural fat is most rich.
The algae oil fat fixing agent is a solid fat part obtained by separating algae oil, belongs to an intermediate byproduct of algae oil production, is low in cost and easy to obtain, has a market price far lower than carbon source raw materials such as DHA methyl ester and DHA ethyl ester, can greatly reduce the raw material cost, and realizes the reutilization of algae oil fat fixing agent. In addition, the invention adopts the rhodococcus turbidi as a fermentation strain, which not only has low price, but also can effectively promote the recombination of palmitic acid and DHA in the fixed fat of the algae oil in bacterial cells through strain fermentation, thus being combined into a new UPU type triglyceride.
Therefore, the UPU structural fat rich in DHA can break through the technical bottleneck of the traditional industrial enzyme method by adopting the microbial fermentation method, so that the UPU structural fat is not dependent on the sn-1,3 selective lipase with high price. The fermentation method also has the advantages of natural intracellular synthetic grease and products, high content of triglyceride, few byproducts and simple separation and purification, and can prepare the target UPU structural grease by fermenting once, thereby avoiding the production defect of complicated enzymatic synthesis process, having wide application prospect in the preparation field of the UPU structural grease rich in DHA and providing technical support for the research and development and production of infant health foods and dietary supplements.
Drawings
FIG. 1A shows the UPU structured lipid obtained by the method for preparing the UPU structured lipid rich in DHA by microbial fermentation of the invention and the raw material algae oil for fixing lipid 13 C-NMR spectrum comparison results.
Detailed Description
The technical scheme and technical effects of the present invention are further described below with reference to the detailed description and the accompanying drawings. The following description is merely illustrative of the invention and is not to be construed as limiting the scope of the invention. The following examples are all methods conventional in the art unless specifically indicated. The reagents used in the examples below, unless otherwise specified, are all conventional in the art and are commercially available.
The invention provides a method for preparing UPU structured lipid rich in DHA by microbial fermentation, which comprises the following steps:
(1) Inoculating rhodococcus microorganisms into a basic culture medium for activation culture to obtain seed liquid; the rhodococcus microorganism is rhodococcus turbidi;
(2) Inoculating the seed liquid into a fermentation culture medium for fermentation culture, centrifuging after the culture is finished to remove fermentation liquid to obtain thalli, crushing the thalli, and extracting grease, wherein the obtained grease is UPU structural grease rich in DHA;
the fermentation medium is prepared by adding 8-16 g/L algae oil solid fat serving as a carbon source into an MSM (multi-stage fermentation) medium; in the Sn-2 fatty acid composition of the algae oil lipid-solidifying, the content of palmitic acid is 10% -30%, and the content of DHA is 40% -70%.
As a further improvement, in step (1), the basal medium is a nutrient broth medium.
Further preferably, in the step (1), the inoculation amount of the rhodococcus microorganism is 0.08 to 0.12v/v%; more preferably 0.1v/v%.
More preferably, the activation culture is performed at a temperature of 25 to 35℃and a rotation speed of 100 to 200rpm until the OD value of the seed solution reaches 0.8 to 1.5.OD value refers to the violet absorbance at 660 nm.
As a further improvement, in the step (2), the seed liquid is inoculated in an amount of 0.8 to 1.2v/v%, more preferably, 1.0v/v%.
MSM medium is a basic salt medium which is common in the art, and the composition of the present invention is not particularly limited. The fermentation requirements of microorganisms of the genus rhodococcus can be met using the MSM medium formulation conventional in the art. As a further improvement, in the step (2), the composition of the MSM medium includes urea, magnesium sulfate, calcium chloride, phosphate, cobalt chloride, copper chloride, manganese chloride, nickel chloride, and water. For example, the composition of MSM medium can be formulated as described in the reference (Goswami, L.; tejas Namboodiri, M.M.; vinoth Kumar, R.; renewable Energy 2017,105,400-406). Specifically, the composition of the MSM medium referred to in the following examples is: urea 0.6g/L, mgSO 4 ·7H 2 O 0.5g/L，CaCl 2 ·2H 2 O 0.02g/L，Na 2 HPO 4 ·12H 2 O 9g/L，KH 2 PO 4 1.5g/L，CoCl 2 ·6H 2 O 0.0002g/L，CuCl 2 ·2H 2 O 0.0010g/L，FeNaEDTA0.005g/L，H 3 BO 3 0.0003g/L，MnCl 2 ·H 2 O 0.002g/L，NiCl·6H 2 O 0.001g/L，ZnSO 4 ·7H 2 O 0.004g/L，NaMoO 4 ·H 2 O0.002 g/L, and water in balance. In other embodiments, other conventional MSM media formulations may be used for MSM media.
For natural algae oil, the fatty acid composition is in a reasonable range. As a further improved scheme, in the step (2), the content of palmitic acid in the whole fatty acid composition of the algae oil solid fat is 30% -50%, and the content of DHA is 30% -50%. In the following examples, the source manufacturer of algae oil for fixing lipid is Yi you kang biotechnology limited company. In the full-sample fatty acid composition of the algae oil for fixing fat, the content of palmitic acid is 42.96 percent, and the content of DHA is 35.47 percent; in the Sn-2 fatty acid composition of the algae oil for fixing fat, the content of palmitic acid is 14.07 percent, and the content of DHA is 57.69 percent. In other embodiments, the algae oil lipid-fixing agent can be natural algae oil lipid-fixing agent composed of other fatty acids in the field, the invention is not particularly limited, and the invention only needs to meet the conventional range of 10% -30% of palmitic acid and 40% -70% of DHA in the Sn-2 fatty acid composition. The algae oil lipid-fixing agent with the Sn-2 fatty acid composition can realize the redistribution of fatty acid on a glycerin skeleton through bacterial cell recombination by adopting the fermentation preparation method of the invention, so that the Sn-2 fatty acid composition is obviously changed, namely, the content of Sn-2 palmitic acid is effectively improved, and the content of Sn-2 polyunsaturated fatty acid DHA is effectively reduced.
As a further improvement, in the step (2), the fermentation culture is carried out at a temperature of 25-35 ℃ and a rotation speed of 100-200 rpm for 80-120 hours.
As a further improved scheme, in the step (2), the rotating speed of the centrifugation is 3000-8000 rpm, and the centrifugation time is 3-10 min.
The operation of crushing the cells is a relatively conventional operation in the art, and the present invention is not particularly limited thereto. As a further improved scheme, in the step (2), the broken thalli is processed by an enzymolysis method or an acid heating method; the enzymolysis method is to add protease into thalli for enzymolysis to break thalli; the acid heating method is to put the cells in an acid solution and heat-treat them in a boiling water bath to crush them. More preferably, the acid heating method is a method in which the cells are placed in 2 to 4mol/L hydrochloric acid and treated in a boiling water bath for 3 to 6 minutes. The acid heating method is an operation method for crushing thalli in a conventional laboratory, and the enzymolysis method is an operation method for crushing thalli adopted in factory production, and both can meet the requirements of crushing thalli required by the invention.
As a further improved scheme, in the step (2), the extraction is carried out by adopting a centrifugal separation method or a solvent extraction method; the centrifugal separation method is to centrifuge the crushed thalli and then separate to obtain grease; the solvent extraction is to extract the grease in the thalli by adopting the solvent, and combine the grease after 2 to 5 times of extraction. Further, the solvent is n-hexane. The centrifugal separation method is a method for extracting grease which is convenient for factory production and application, and the solvent extraction method is an extraction method which meets the operation requirement of a laboratory, and both are suitable for the requirement of grease extraction in the invention.
Specifically, in the following examples, nutrient broth media was purchased from Guangdong Crypton microorganism technologies Inc. Rhodococcus turbidi (Rhodococcus opacus) was purchased from North Nature Biotechnology institute under the number BNCC337018.
Example 1
The embodiment provides a method for preparing DHA-rich UPU structural fat by microbial fermentation, which comprises the following specific steps:
(1) And (3) activating and culturing: rhodococcus microorganisms (specifically, rhodococcus turbidi) were inoculated in an inoculum size of 0.1% (v/v) into 100mL of a nutrient broth, and cultured in a shaker at 30℃and a rotation speed of 160r/min until the OD (660 nm) was 1.0, to obtain a seed solution.
(2) Fermentation culture: inoculating the seed solution into 100mL of fermentation medium according to the inoculum size of 1% (v/v), and fermenting at 30 ℃ at 160r/min for 96h. After the culture is finished, centrifuging (5000 rpm,10 min) to remove fermentation liquor to obtain thalli, then placing the thalli in 4mL of 3mol/L hydrochloric acid, treating in a boiling water bath for 5min to crush the thalli, then cooling to room temperature, extracting grease in the thalli by adopting 2mL of solvent n-hexane for 3 times, combining three n-hexane layers, drying n-hexane for 2h at 70 ℃, and obtaining the grease which is the UPU structural grease rich in DHA.
Wherein, the fermentation medium is prepared by adding 8g/L algae oil solid fat into MSM medium as carbon source; in the Sn-2 fatty acid composition of the algae oil for fixing fat, the content of palmitic acid is 14.07 percent, and the content of DHA is 57.69 percent.
Example 2
The embodiment provides a method for preparing DHA-rich UPU structural fat by microbial fermentation, which comprises the following specific steps:
(1) And (3) activating and culturing: rhodococcus microorganisms (specifically, rhodococcus turbidi) were inoculated in an inoculum size of 0.1% (v/v) into 100mL of a nutrient broth, and cultured in a shaker at 30℃and a rotation speed of 160r/min for 36 hours to obtain a seed solution.
(2) Fermentation culture: inoculating the seed solution into 100mL of fermentation medium according to the inoculum size of 1% (v/v), and fermenting at 30 ℃ at 160r/min for 96h. After the culture is finished, centrifuging (6000 rpm,8 min) to remove fermentation liquor to obtain thalli, then placing the thalli in 4mL of 2mol/L hydrochloric acid, treating in a boiling water bath for 6min to break the thalli, then cooling to room temperature, extracting grease in the thalli by adopting 2mL of solvent n-hexane for 3 times, combining three n-hexane layers, drying n-hexane for 2h at 70 ℃, and obtaining the grease which is the UPU structural grease rich in DHA.
Wherein, the fermentation medium is prepared by adding 12g/L algae oil solid fat into MSM medium as carbon source; in the Sn-2 fatty acid composition of the algae oil for fixing fat, the content of palmitic acid is 14.07 percent, and the content of DHA is 57.69 percent.
Example 3
The embodiment provides a method for preparing DHA-rich UPU structural fat by microbial fermentation, which comprises the following specific steps:
(1) And (3) activating and culturing: rhodococcus microorganisms (specifically, rhodococcus turbidi) were inoculated in an inoculum size of 0.1% (v/v) into 100mL of a nutrient broth, and cultured in a shaker at 30℃and a rotation speed of 160r/min for 36 hours to obtain a seed solution.
(2) Fermentation culture: inoculating the seed solution into 100mL of fermentation medium according to the inoculum size of 1% (v/v), and fermenting at 30 ℃ at 160r/min for 96h. After the culture is finished, centrifuging (7000 rpm,5 min) to remove fermentation liquor to obtain thalli, then placing the thalli in 4mL of 4mol/L hydrochloric acid, treating in a boiling water bath for 3min to crush the thalli, then cooling to room temperature, extracting grease in the thalli by adopting 2mL of solvent n-hexane for 3 times, combining three n-hexane layers, drying n-hexane for 2h at 70 ℃, and obtaining the grease which is the UPU structural grease rich in DHA.
Wherein, the fermentation medium is prepared by adding 16g/L algae oil solid fat into MSM medium as carbon source; in the Sn-2 fatty acid composition of the algae oil for fixing fat, the content of palmitic acid is 14.07 percent, and the content of DHA is 57.69 percent.
Comparative example 1
This comparative example provides a microbial fermentation process which differs from example 1 in that the fermentation medium of step (2) is not supplemented with algae oil for lipid fixation, but with glucose 12g/L as a carbon source in MSM medium; the remaining parameters and process operations were the same as in example 1.
Comparative example 2
This comparative example provides a microbial fermentation process which differs from example 1 in that the fermentation medium of step (2) is not supplemented with algae oil fixed fat, but with 8g/L arachidonic acid oil fat (20:4) as carbon source in MSM medium; the remaining parameters and process operations were the same as in example 1. The fatty acid composition of the arachidonic acid oil (ARA oil as a raw material) had a palmitic acid content of 7.47% and an arachidonic acid content of 46.58%, and the specific composition is shown in Table 2.
Comparative example 3
This comparative example provides a microbial fermentation method which is different from example 1 in that the fermentation medium of step (2) is not added with algae oil fixed fat, but 10g/L of algae oil fixed fat fatty acid (obtained after hydrolyzing algae oil fixed fat, fatty acid composition and algae oil fixed fat) is added as a carbon source in the MSM medium; the remaining parameters and process operations were the same as in example 1.
Test examples
1. Determination of biomass and microbial cell oil content
Biomass measurement: the cells of example 1 and comparative examples 1 to 3 were fermented for 96 hours, and the fermentation broth was removed by centrifugation to obtain wet cells, which were then washed with water 2 times and then with n-hexane 6 times, and then freeze-dried to obtain dry cells, which were weighed to calculate biomass.
Measurement of the oil content of the cells: taking 0.04-0.10g of freeze-dried dry bacterial powder, adding the dry bacterial powder into a headspace bottle, then adding 4mL of 3mol/L hydrochloric acid, then adding 2mL of n-hexane, then screwing a cover, and carrying out boiling water bath for 5min. Then cooling to room temperature, centrifuging at 6000r/min, taking the upper layer of normal hexane into a centrifuge tube, and extracting for three times. And combining the n-hexane layers for three times, drying n-hexane, and drying at 70 ℃ for 2 hours to obtain the grease. And calculating the oil content according to the quality of the grease and the quality of the fungus powder.
The measurement results of the biomass and the microbial cell oil content are shown in Table 1.
TABLE 1 biological and oil content results for example 1 and comparative examples 1-3
Example 1 Comparative example 1 Comparative example 2 Comparative example 3
Biomass (g/L) 4.21 2.82 2.51 No bacteria growth
Oil content (%) 47.91 58.02 40.5 -
As is clear from Table 1, the biomass obtained by the fermentation in example 1 of the present invention was 4.21g/L, the oil content was 47.91% (DCW), and the UPU yield was 0.35g/L. From the biomass point of view, the biomass of the example 1 is improved by 1.39-1.70g/L compared with that of the comparative example 1 and the comparative example 2, which shows that the algae oil fixed fat is beneficial to improving the biomass of bacteria. In addition, strain fermentation was performed with algal oil lipid-fixing fatty acids (comparative example 3), and it was found that strain could not grow, indicating that the mixed fatty acids had inhibitory effect on rhodococcus turbidi.
2. Determination of fatty acid composition of full sample and Sn-2-position fatty acid composition
Full-sample fatty acid composition determination: the fatty acid composition of the oil was analyzed by gas chromatography using boron trifluoride or simple methyl esterification (GB 5009.168-2016).
Determination of Sn-2 fatty acid composition: and (3) determining the composition of the Sn-2 fatty acid by referring to a determination method of the 2-fatty acid component of the triglyceride molecule of the GB/T24894-2010 animal and vegetable oil.
The measurement results of the total fatty acid composition and the Sn-2 fatty acid composition of the raw materials and the fats and oils of example 1, comparative example 1 and comparative example 2 are shown in Table 2.
TABLE 2 data table of main fatty acid composition of oil samples (%)
As can be seen from the analysis of Table 2, the invention prepares the UPU structural fat rich in DHA by fermenting rhodococcus turbidimetric with algae oil fixed fat as the only carbon source. The fatty acid composition of the UPU prepared in examples 1-3 is mainly 37.61% -38.55% DHA, 26.34% -27.5% palmitic acid and 15.56% -16.55% docosapentaenoic acid (DPA), and sn-2 position is mainly palmitic acid (51.12% -53.61%). Compared with the solid fat of the raw material algal oil, the full-sample fatty acid composition of the oil obtained by fermentation and culture in examples 1-3 is similar, but the sn-2 fatty acid composition is obviously changed, the palmitic acid content is improved from 14.07% of the solid fat of the raw material algal oil to 51.12% -53.61% after fermentation, and the unsaturated fatty acid content 22:5 and 22:6 in the full-sample fatty acid of the oil after fermentation are obviously reduced, which indicates that the fatty acid is subjected to recombination distribution on a glycerol skeleton in the strain fermentation process. Meanwhile, the fatty acid composition of example 1 was also significantly different from that of comparative example 1. In addition, the composition of the full-sample fatty acid of comparative example 2 was very significantly changed from that of the ARA oil, and the arachidonic acid content was undetectable from 46.58% in the starting material, indicating that arachidonic acid (20:4) was not recombined in the bacterial cell, but converted, and it was concluded that not all polyunsaturated fatty acids were able to be recombined in the bacterial cell. From the full-scale fatty acid composition, it was determined that arachidonic acid could not be used to prepare UPU structured fats, and thus the sn-2 fatty acid composition was not measured. As can be seen from comparative examples 1, 2 and 3, the whole samples of the three examples and sn-2 fatty acids were similar in composition, and the triglyceride structure was presumed to be similar, and were all UPU type structural lipids rich in DHA. In conclusion, the experiment effectively proves that DHA (22:6) can be recombined in bacterial cells and combined into new triglyceride.
3. Composition of triglyceride 13 C-NMR analysis
Determination of the composition of the triglyceride: about 10mg of algae oil lipid-fixing raw material and about 10mg of DHA-rich UPO structural lipid (i.e. bacterial oil) prepared in example 1 were weighed respectively, and triglyceride composition measurement was carried out by UPLC-MS under specific test conditions and parameter references (Lin-Shang Zhang; mei-Yun Chu; facile and green production of human milk fat substitute through Rhodococcus opacus fermentation, journal of Agricultural and Food Chemistry,2020,68 (35): 9368-9376).
13 C-NMR test: about 20mg of oil was taken, and the mixture was placed in a nuclear magnetic resonance tube, and 0.6mL of deuterated chloroform was added for dissolution, followed by nuclear magnetic resonance scanning.
TABLE 3UPLC-MS identification of the major triglyceride composition of DHA-enriched UPU germ oil
Table 3 shows the triglyceride composition (content > 1%) of the UPU type structural lipid obtained in the present invention, and it can be seen from Table 3 that DHAP DHA (6.66%) and DPA P DHA (6.62%) are the most abundant triacylglycerol species. The result shows that the DHA-rich UPU structured lipid product is successfully prepared.
To further confirm the triglyceride structure of UPU, a method of using 13 C-nuclear magnetic resonance analysis is carried out on the algae oil lipid-fixing raw material and the UPU structural lipid prepared by the invention, 13 the results of the C-NMR test are shown in FIG. 1. As can be seen from FIG. 1, sn-1,3 in the algae oil fat fixing is mainly saturated fatty acid-palmitic acid, and DHA is mainly distributed at sn-2 position. The sn-2 palmitic acid peak in the UPU structural fat rich in DHA prepared by the invention is obviously higher than that of raw material fat, which indicates that the palmitic acid is concentrated at the sn-2 position. Sn-1,3 is that the peak height of DHA is obviously higher than that of raw material grease, which indicates that the Sn-1,3 position mainly distributes DHA. The results of the nuclear magnetic resonance again prove that the structural ester obtained by fermentation of the invention is UPU structural ester.
In summary, according to the method for preparing the UPU structural fat rich in DHA by microbial fermentation, which is provided by the invention, algae oil is used as a carbon source, rhodococcus turbidi is used as a fermentation strain, and the UPU structural fat rich in DHA is prepared by a microbial fermentation method. In addition, the fermentation method of the invention synthesizes grease and products in cells naturally, has high content of triglyceride, few byproducts and simple separation and purification, can prepare the target UPU structural grease by fermenting once, avoids the production defect of complicated enzymatic synthesis process, has wide application prospect in the preparation field of the UPU structural grease rich in DHA, and can also provide technical support for research, development and production of infant health foods and dietary supplements.
The above examples are only preferred embodiments of the present invention, and are merely for illustrating the present invention, not for limiting the present invention. Alterations, substitutions, modifications and the like will be apparent to those skilled in the art without departing from the spirit of the invention.

Claims (10)

1. A method for preparing a UPU structured lipid rich in DHA by microbial fermentation, comprising the steps of:
(1) Inoculating rhodococcus microorganisms into a basic culture medium for activation culture to obtain seed liquid; the rhodococcus microorganism is rhodococcus turbidi;
(2) Inoculating the seed liquid into a fermentation culture medium for fermentation culture, centrifuging after the culture is finished to remove fermentation liquid to obtain thalli, crushing the thalli, and extracting grease, wherein the obtained grease is UPU structural grease rich in DHA;
the fermentation medium is prepared by adding 8-16 g/L algae oil solid fat serving as a carbon source into an MSM (multi-stage fermentation) medium; in the Sn-2 fatty acid composition of the algae oil lipid-solidifying, the content of palmitic acid is 10% -30%, and the content of DHA is 40% -70%.
2. The method for preparing a UPU structured lipid rich in DHA by microbial fermentation according to claim 1, wherein in step (1), the basal medium is a nutrient broth medium.
3. The method for preparing the UPU structured fat rich in DHA by microbial fermentation according to claim 1, wherein the inoculation amount of the rhodococcus microorganism in the step (1) is 0.08-0.12 v/v%; the activation culture is carried out at the temperature of 25-35 ℃ and the rotating speed of 100-200 rpm until the OD value of the seed solution reaches 0.8-1.5.
4. The method for producing a UPU structured fat rich in DHA by microbial fermentation according to claim 1, wherein the seed liquid is inoculated in an amount of 0.8-1.2 v/v% in step (2).
5. The method for preparing the UPU structured fat rich in DHA by microbial fermentation according to claim 1, wherein in step (2), the composition of the MSM medium comprises urea, magnesium sulfate, calcium chloride, phosphate, cobalt chloride, copper chloride, manganese chloride, nickel chloride, and water.
6. The method for preparing the UPU structural fat rich in DHA by microbial fermentation according to claim 1, wherein in the step (2), the content of palmitic acid in the full-scale fatty acid composition of the algae oil fixed fat is 30% -50% and the content of DHA is 30% -50%.
7. The method for preparing a UPU structured lipid rich in DHA by microbial fermentation according to any one of claims 1 to 6, wherein in the step (2), the fermentation culture is carried out at a temperature of 25 to 35 ℃ and a rotation speed of 100 to 200rpm for 80 to 120 hours.
8. The method for preparing a UPU structured lipid rich in DHA by microbial fermentation according to any one of claims 1 to 6, wherein in the step (2), the rotational speed of centrifugation is 3000 to 8000rpm and the centrifugation time is 3 to 10min.
9. The method for preparing the UPU structural fat rich in DHA by microbial fermentation according to any one of claims 1 to 6, wherein in the step (2), the broken thalli are subjected to an enzymolysis method or an acid heating method; the enzymolysis method is to add protease into thalli for enzymolysis to break thalli; the acid heating method is to put the cells in an acid solution and heat-treat them in a boiling water bath to crush them.
10. The method for preparing the UPU structural fat rich in DHA by microbial fermentation according to any one of claims 1 to 6, wherein in the step (2), the extraction is performed by a centrifugal separation method or a solvent extraction method; the centrifugal separation method is to centrifuge the crushed thalli and then separate to obtain grease; the solvent extraction is to extract the grease in the thalli by adopting the solvent, and combine the grease after 2 to 5 times of extraction.
CN202311406413.5A 2023-10-27 2023-10-27 Method for preparing UPU structural fat rich in DHA through microbial fermentation Pending CN117265027A (en)

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