CN116458645A - Sn-2 long chain polyunsaturated fatty acid structural fat and preparation method and application thereof - Google Patents
Sn-2 long chain polyunsaturated fatty acid structural fat and preparation method and application thereof Download PDFInfo
- Publication number
- CN116458645A CN116458645A CN202310433524.9A CN202310433524A CN116458645A CN 116458645 A CN116458645 A CN 116458645A CN 202310433524 A CN202310433524 A CN 202310433524A CN 116458645 A CN116458645 A CN 116458645A
- Authority
- CN
- China
- Prior art keywords
- fatty acid
- mag
- tag
- fat
- long
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 235000020978 long-chain polyunsaturated fatty acids Nutrition 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 239000000194 fatty acid Substances 0.000 claims abstract description 41
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 39
- 229930195729 fatty acid Natural products 0.000 claims abstract description 39
- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 27
- -1 carbon chain fatty acid Chemical class 0.000 claims abstract description 19
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 8
- WBVHXPUFAVGIAT-UHFFFAOYSA-N [C].OCC(O)CO Chemical group [C].OCC(O)CO WBVHXPUFAVGIAT-UHFFFAOYSA-N 0.000 claims abstract description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 61
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 45
- 241000195493 Cryptophyta Species 0.000 claims description 33
- 108010048733 Lipozyme Proteins 0.000 claims description 26
- FCCDDURTIIUXBY-UHFFFAOYSA-N lipoamide Chemical group NC(=O)CCCCC1CCSS1 FCCDDURTIIUXBY-UHFFFAOYSA-N 0.000 claims description 25
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 claims description 23
- 108090001060 Lipase Proteins 0.000 claims description 22
- 102000004882 Lipase Human genes 0.000 claims description 22
- 239000004367 Lipase Substances 0.000 claims description 22
- 235000019441 ethanol Nutrition 0.000 claims description 22
- 235000019421 lipase Nutrition 0.000 claims description 22
- 238000000746 purification Methods 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 19
- 150000002632 lipids Chemical class 0.000 claims description 18
- 230000037396 body weight Effects 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 15
- 102000004157 Hydrolases Human genes 0.000 claims description 13
- 108090000604 Hydrolases Proteins 0.000 claims description 13
- 108010084311 Novozyme 435 Proteins 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 12
- LDVVTQMJQSCDMK-UHFFFAOYSA-N 1,3-dihydroxypropan-2-yl formate Chemical compound OCC(CO)OC=O LDVVTQMJQSCDMK-UHFFFAOYSA-N 0.000 claims description 11
- 210000000577 adipose tissue Anatomy 0.000 claims description 11
- 238000001704 evaporation Methods 0.000 claims description 11
- 150000002148 esters Chemical class 0.000 claims description 10
- 238000000926 separation method Methods 0.000 claims description 10
- 239000004519 grease Substances 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 9
- 239000012074 organic phase Substances 0.000 claims description 9
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 claims description 7
- 239000003960 organic solvent Substances 0.000 claims description 7
- 239000003480 eluent Substances 0.000 claims description 6
- 239000012071 phase Substances 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 3
- 230000008020 evaporation Effects 0.000 claims description 3
- 239000002808 molecular sieve Substances 0.000 claims description 3
- 239000000376 reactant Substances 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 239000002002 slurry Substances 0.000 claims description 3
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 3
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 2
- 238000011049 filling Methods 0.000 claims description 2
- 235000013305 food Nutrition 0.000 claims description 2
- 238000011068 loading method Methods 0.000 claims description 2
- 238000012544 monitoring process Methods 0.000 claims description 2
- 230000010355 oscillation Effects 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 15
- 125000002252 acyl group Chemical group 0.000 abstract description 8
- 230000008569 process Effects 0.000 abstract description 6
- 238000012546 transfer Methods 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 235000013376 functional food Nutrition 0.000 abstract description 2
- 238000009482 thermal adhesion granulation Methods 0.000 description 62
- 239000003925 fat Substances 0.000 description 55
- 241000699670 Mus sp. Species 0.000 description 54
- 235000019197 fats Nutrition 0.000 description 40
- 239000003921 oil Substances 0.000 description 32
- 235000019198 oils Nutrition 0.000 description 32
- 230000000694 effects Effects 0.000 description 29
- 230000015572 biosynthetic process Effects 0.000 description 22
- 150000004665 fatty acids Chemical class 0.000 description 20
- 210000004185 liver Anatomy 0.000 description 20
- 238000003786 synthesis reaction Methods 0.000 description 17
- 238000011740 C57BL/6 mouse Methods 0.000 description 15
- 230000001965 increasing effect Effects 0.000 description 15
- 210000003734 kidney Anatomy 0.000 description 15
- 239000002994 raw material Substances 0.000 description 15
- 229940088598 enzyme Drugs 0.000 description 14
- 229940040461 lipase Drugs 0.000 description 13
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 13
- NRYBAZVQPHGZNS-ZSOCWYAHSA-N leptin Chemical compound O=C([C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)CCSC)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(O)=O NRYBAZVQPHGZNS-ZSOCWYAHSA-N 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 11
- 102000016267 Leptin Human genes 0.000 description 11
- 108010092277 Leptin Proteins 0.000 description 11
- 229940039781 leptin Drugs 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 10
- 108010023302 HDL Cholesterol Proteins 0.000 description 10
- 210000004369 blood Anatomy 0.000 description 10
- 239000008280 blood Substances 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 10
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 10
- 229960002446 octanoic acid Drugs 0.000 description 10
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 210000005229 liver cell Anatomy 0.000 description 9
- 239000012188 paraffin wax Substances 0.000 description 9
- 238000004809 thin layer chromatography Methods 0.000 description 9
- 108010028554 LDL Cholesterol Proteins 0.000 description 8
- 208000008589 Obesity Diseases 0.000 description 8
- 235000020824 obesity Nutrition 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 230000008859 change Effects 0.000 description 7
- 210000000056 organ Anatomy 0.000 description 7
- 230000003197 catalytic effect Effects 0.000 description 6
- 150000004667 medium chain fatty acids Chemical class 0.000 description 6
- 230000035484 reaction time Effects 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- 238000006136 alcoholysis reaction Methods 0.000 description 5
- 235000012000 cholesterol Nutrition 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- 238000005886 esterification reaction Methods 0.000 description 5
- 238000003780 insertion Methods 0.000 description 5
- 230000037431 insertion Effects 0.000 description 5
- 201000001320 Atherosclerosis Diseases 0.000 description 4
- 239000005635 Caprylic acid (CAS 124-07-2) Substances 0.000 description 4
- 208000031226 Hyperlipidaemia Diseases 0.000 description 4
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 4
- 241000700159 Rattus Species 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 210000003855 cell nucleus Anatomy 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 238000004043 dyeing Methods 0.000 description 4
- 210000003292 kidney cell Anatomy 0.000 description 4
- 210000005228 liver tissue Anatomy 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 210000005084 renal tissue Anatomy 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 125000005457 triglyceride group Chemical group 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 239000008096 xylene Substances 0.000 description 4
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 3
- 108010082126 Alanine transaminase Proteins 0.000 description 3
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 3
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 235000005911 diet Nutrition 0.000 description 3
- 230000037406 food intake Effects 0.000 description 3
- 235000021588 free fatty acids Nutrition 0.000 description 3
- 210000003494 hepatocyte Anatomy 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 230000037356 lipid metabolism Effects 0.000 description 3
- 150000004668 long chain fatty acids Chemical class 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- 230000004584 weight gain Effects 0.000 description 3
- 235000019786 weight gain Nutrition 0.000 description 3
- DUCVQOXJVCRDDH-UHFFFAOYSA-N (2-acetyloxy-3-hydroxypropyl) tetradecanoate Chemical compound CCCCCCCCCCCCCC(=O)OCC(CO)OC(C)=O DUCVQOXJVCRDDH-UHFFFAOYSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 108010010234 HDL Lipoproteins Proteins 0.000 description 2
- 102000015779 HDL Lipoproteins Human genes 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 206010020880 Hypertrophy Diseases 0.000 description 2
- 206010030113 Oedema Diseases 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 230000003044 adaptive effect Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- WTEOIRVLGSZEPR-UHFFFAOYSA-N boron trifluoride Chemical compound FB(F)F WTEOIRVLGSZEPR-UHFFFAOYSA-N 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 235000019577 caloric intake Nutrition 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 239000003651 drinking water Substances 0.000 description 2
- 235000020188 drinking water Nutrition 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 230000032050 esterification Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 2
- 235000012631 food intake Nutrition 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 230000003054 hormonal effect Effects 0.000 description 2
- 206010020718 hyperplasia Diseases 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 102000005861 leptin receptors Human genes 0.000 description 2
- 108010019813 leptin receptors Proteins 0.000 description 2
- 125000003473 lipid group Chemical group 0.000 description 2
- 230000003908 liver function Effects 0.000 description 2
- 210000004324 lymphatic system Anatomy 0.000 description 2
- 238000003760 magnetic stirring Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000006996 mental state Effects 0.000 description 2
- 210000000633 nuclear envelope Anatomy 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 230000008092 positive effect Effects 0.000 description 2
- 230000022558 protein metabolic process Effects 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 150000003626 triacylglycerols Chemical class 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000001993 wax Substances 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 235000019737 Animal fat Nutrition 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 241000003595 Aurantiochytrium limacinum Species 0.000 description 1
- 229910015900 BF3 Inorganic materials 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 206010019842 Hepatomegaly Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 238000008214 LDL Cholesterol Methods 0.000 description 1
- 108010007622 LDL Lipoproteins Proteins 0.000 description 1
- 102000007330 LDL Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 101001063890 Mus musculus Leptin Proteins 0.000 description 1
- 101000800132 Mus musculus Thyroglobulin Proteins 0.000 description 1
- 102000019280 Pancreatic lipases Human genes 0.000 description 1
- 108050006759 Pancreatic lipases Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- OGBUMNBNEWYMNJ-UHFFFAOYSA-N batilol Chemical class CCCCCCCCCCCCCCCCCCOCC(O)CO OGBUMNBNEWYMNJ-UHFFFAOYSA-N 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000007036 catalytic synthesis reaction Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
- 229960002327 chloral hydrate Drugs 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 208000027744 congestion Diseases 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000012303 cytoplasmic staining Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 230000032798 delamination Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000003028 elevating effect Effects 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000021323 fish oil Nutrition 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000000769 gas chromatography-flame ionisation detection Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000024924 glomerular filtration Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 231100000304 hepatotoxicity Toxicity 0.000 description 1
- 235000009200 high fat diet Nutrition 0.000 description 1
- 230000003284 homeostatic effect Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000002267 hypothalamic effect Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 235000019626 lipase activity Nutrition 0.000 description 1
- 230000007056 liver toxicity Effects 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 235000021281 monounsaturated fatty acids Nutrition 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 230000003589 nefrotoxic effect Effects 0.000 description 1
- 231100000381 nephrotoxic Toxicity 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229940116369 pancreatic lipase Drugs 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 210000003240 portal vein Anatomy 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007127 saponification reaction Methods 0.000 description 1
- 230000036186 satiety Effects 0.000 description 1
- 235000019627 satiety Nutrition 0.000 description 1
- 150000004671 saturated fatty acids Chemical group 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 238000005809 transesterification reaction Methods 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 238000004260 weight control Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/115—Fatty acids or derivatives thereof; Fats or oils
- A23L33/12—Fatty acids or derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses Sn-2 long-chain polyunsaturated fatty acid structural fat and a preparation method and application thereof, belonging to the technical field of functional foods. The Sn-2 long chain polyunsaturated fatty acid structural fat adopting the structure is fat formed by connecting a long carbon chain fatty acid with a Sn-2 position of a glycerol carbon chain and connecting a medium carbon chain fatty acid with a Sn-1,3 position, wherein the carbon number of the long carbon chain fatty acid is 15-25, and the carbon number of the medium carbon chain fatty acid is 6-10. The invention adopts a two-step enzyme method to prepare the Sn-2 long chain polyunsaturated fatty acid structural fat, can greatly reduce the occurrence of acyl transfer, enhances the reaction specificity and simultaneously improves the process productivity, and is beneficial to the generation of special structural fat.
Description
Technical Field
The invention belongs to the technical field of functional foods, and particularly relates to Sn-2 long-chain polyunsaturated fatty acid structural fat, a preparation method and application thereof.
Background
Obesity can significantly increase the risk of developing diabetes, atherosclerosis, hyperlipidemia, hypertension, etc., and has become a major public health problem worldwide. The cause of obesity is mainly the ingestion of excessive dietary energy and the lack of calories in the body leading to the formation of systemic accumulation of fat in the body. How to optimize the type of fat in the diet and develop low calorie fat has become an effective way to prevent and control obesity. Currently, related studies for optimizing lipid species by using structural lipids to reduce the risk of illness have become a research hotspot in recent years. By utilizing the directional modification technology of the grease, specific fatty acid in the natural grease can be modified to exert corresponding nutrition characteristics, and the specific fatty acid can be used for preventing certain diseases or improving metabolic conditions, so that the field has become the consensus of the grease community and the scientific research workers in the nutrition community.
Among the structural lipids, the MLM type structural lipid is considered as the most ideal structural form of the structural lipid. The MLM structural grease is a special structural grease which is formed by connecting long carbon chain fatty acid (L) at the Sn-2 position of a glycerol carbon chain and connecting medium carbon chain fatty acid (M) at the Sn-1,3 positions. Studies have shown that MLM structural fat has the advantages and characteristics of good absorption and strong functions, has the greatest health-care and nutrition characteristics, and has the antibacterial and anti-inflammatory characteristics.
At present, the synthetic preparation method for MLM structural fat mainly adopts an enzymatic synthesis method. Compared with the chemical method, the enzymolysis method has the advantages of mild condition, high catalytic specificity, easy separation and the like, and is widely applied to the preparation of MLM structured lipid products. In the prior art, a one-step enzyme method and a three-step enzyme method are generally adopted to prepare the MLM structured lipid. The preparation of MLM structured lipid by one-step enzyme method has the defects of difficult removal of byproducts (such as diglyceride), complex purification and high acyl mobility; the three-step enzymatic process mainly comprises the steps of esterifying, hydrolyzing and acidolyzing the glycerol and the acyl donor by more than three different positioning lipases, so that the synthesis process has long time consumption, high cost and poor environmental protection benefit, and is difficult to realize large-scale industrial production.
Disclosure of Invention
Aiming at the prior art, the invention provides Sn-2 long-chain polyunsaturated fatty acid structural fat, a preparation method and application thereof, which opens up a new method for reducing weight and body fat rate and solves the technical problems of long time consumption, high cost and more byproducts in the synthesis of structural fat in the prior art.
In order to achieve the aim, the technical scheme adopted by the invention is to provide the application of the Sn-2 long-chain polyunsaturated fatty acid structured fat in preparing the food for reducing the weight and body fat rate; the Sn-2 long chain polyunsaturated fatty acid structural fat is formed by connecting a long carbon chain fatty acid with a Sn-2 position of a glycerol carbon chain and connecting a medium carbon chain fatty acid with a Sn-1,3 position, wherein the carbon number of the long carbon chain fatty acid is 15-25, and the carbon number of the medium carbon chain fatty acid is 6-10.
On the basis of the technical scheme, the invention can be improved as follows.
Further, the long carbon chain fatty acid has 22 carbon atoms, and the medium carbon chain fatty acid has 8 carbon atoms.
Further, the structural formula of the Sn-2 long-chain polyunsaturated fatty acid structural fat is shown as formula I:
wherein R is
The invention also discloses a preparation method of the Sn-2 long-chain polyunsaturated fatty acid structural ester, which comprises the following steps:
(1) Preparation of Sn-2 long chain monoglyceride 2D-MAG
The algae oil and Sn-1,3 specific hydrolase are dissolved in absolute ethyl alcohol according to the mass ratio of 8-10:2-5, and stirred and reacted for 4-16 h at 30-55 ℃; then adding a reactant after sn-1,3 specific hydrolase removal into a mixed solution formed by mixing n-hexane and KOH alcohol solution according to the volume ratio of 3:1, standing after violent oscillation, collecting ethanol phase and evaporating to remove organic solvent, thus obtaining 2D-MAG;
(2) Preparation of Sn-2 long chain polyunsaturated fatty acid 1,3C-2D-TAG
2D-MAG, octanoic acid, lipase and 4A molecular sieve are dissolved in an organic solvent together according to the mass ratio of 80-120:140-160:20-30:1-3, and stirred and reacted for 4-6 h at 35-45 ℃ to obtain 1,3C-2D-TAG.
Further, 2D-MAG was purified prior to use, the purification comprising the steps of:
dissolving 2D-MAG in n-hexane according to a feed liquid ratio of 1g to 15ml, then adding 85% ethanol water solution, standing and layering; taking the lower extraction layer, evaporating to remove the organic phase, dissolving the obtained material in a mixed solution formed by mixing ethanol and n-hexane according to the volume ratio of 9:1, centrifuging for 4-6 min at the speed of 3000-4000 rpm, taking the hexane phase of the upper 2D-MAG, and evaporating to remove the organic phase.
Further, the organic solvent is n-hexane.
Further, the Sn-1,3 specific hydrolase is Lipozyme RM IM or Novozym 435; the lipase is Lipozyme RMIM, lipozyme TL IM or Novozym 435.
Further, the stirring rate of the stirring reaction in the step (1) was 200rpm, the shaking time was 2 minutes, and the standing time was 5 mm.
Further, a purification step of 1,3C-2D-TAG is also included, and the purification method comprises the following steps:
s1: mixing silica gel and alumina with equal mass, dispersing the mixture in n-hexane according to the feed liquid ratio of 2g to 5mL, and filling the obtained slurry into a chromatographic column with the thickness of 300 multiplied by 30mm to obtain a separation and purification column;
s2: loading 1,3C-2D-TAG obtained by the reaction into a separation and purification column, eluting with eluent, monitoring the eluent flowing out of the separation and purification column by TLC and HPLC, collecting the eluent containing 1,3C-2D-TAG structural lipid, and removing the organic phase by evaporation.
The beneficial effects of the invention are as follows:
1. the invention adopts a two-step enzyme method to prepare the Sn-2 long chain polyunsaturated fatty acid structural fat, can greatly reduce the occurrence of acyl transfer, enhances the reaction specificity and simultaneously improves the process productivity, and is beneficial to the generation of special structural fat.
2. The Sn-2 long chain polyunsaturated fatty acid structured fat (MLM structured fat) prepared by the invention can reduce animal adipose tissues, reduce liver weight, increase fat quantity excreted in feces, remarkably increase HDL-cholesterol level in blood plasma, and remarkably reduce total cholesterol and LDL-cholesterol level, namely has positive effect on reducing body weight and body fat rate.
Drawings
FIG. 1 is a flow chart of the synthesis of structural esters of Sn-2 long chain polyunsaturated fatty acids;
FIG. 2 is a graph showing the effect of substrate ratio (algae oil/ethanol, mol/mol) on the synthesis rate of 2D-MAG;
FIG. 3 shows the effect of different species of Sn-1, 3-specific hydrolases on the 2D-MAG content of algae oil;
FIG. 4 shows the effect of Sn-1,3 specific hydrolase addition on 2D-MAG synthesis;
FIG. 5 is a graph showing the effect of reaction temperature on 2D-MAG synthesis;
FIG. 6 is the effect of reaction time on 2D-MAG synthesis;
FIG. 7 is a thin-layer chromatogram of different types of structural lipids;
FIG. 8 is a liquid chromatogram of 2D-MAG in the sample before and after purification;
FIG. 9 is a graph showing the effect of different lipase species on the insertion rate of caprylic acid at positions Sn-1,3 of monoglyceride;
FIG. 10 is the effect of different feed groups on weight gain in C57BL/6 mice; the left side of each group is the weight before feeding, and the right side is the weight after feeding;
FIG. 11 is a graph showing the effect of different feed groups on Blood Urea Nitrogen (BUN) concentration in C57BL/6 mice;
FIG. 12 is a graph showing the effect of different feed groups on C57BL/6 mice glutamic oxaloacetic transaminase (AST) concentration;
FIG. 13 is a graph showing the effect of different feed groups on the concentration of glutamic pyruvic transaminase (ALT) in C57BL/6 mice;
FIG. 14 is the effect of different feed groups on C57BL/6 mice cholesterol concentration;
FIG. 15 is a graph showing the effect of different feed groups on triglyceride concentration in C57BL/6 mice;
FIG. 16 is the effect of different feed groups on the high density lipoprotein (HDL-C) content of C57BL/6 mice;
FIG. 17 is a graph showing the effect of different feed groups on the low density lipoprotein (LDL-C) content of C57BL/6 mice;
FIGS. 18 and 19 are the effect of different feed groups on liver and kidney tissue sections of C57BL/6 mice;
FIG. 20 is a graph showing the effect of different feed groups on leptin concentration in C57BL/6 mice.
Detailed Description
The following describes the present invention in detail with reference to examples.
Examples
The synthesis flow of the Sn-2 long-chain polyunsaturated fatty acid structural ester is shown in figure 1, and the specific synthesis steps are as follows:
1. preparation of Sn-2 long chain monoglyceride 2D-MAG
0.9g of algae oil and a proper amount of absolute ethyl alcohol (based on the complete reaction of triglyceride in the algae oil) are weighed and added into a 50ml conical flask with a plug, 0.4g of Sn-1,3 specific hydrolase (4% -16% and substrate mass fraction) is added, and magnetic stirring is carried out for 4-16 h under the reaction conditions of the rotating speed of 200rpm and the temperature of 30-55 ℃. The reaction mixture was then centrifuged and the lipase was removed by filtration. Taking a centrifugal sample, adding 30mL of normal hexane and 10mL of 0.8mol/L KOH alcohol solution (30% ethanol), shaking vigorously for 2min, standing for 5min, adding 15mL of normal hexane into the lower alcohol-water solution for secondary extraction, shaking vigorously for 2min, standing and layering. After formation of the two layers, the 2D-MAG-rich ethanol phase was collected and washed twice with the same volume of n-hexane. Mixing the supernatants obtained by the two extractions, and removing the organic solvent by rotary evaporation at the water area temperature of 40 ℃.
Analysis of the fatty acid composition of algae oil (Table 1) revealed that C22:6 (44.37%) and C16:0 (27.5%) are the main fatty acid components of algae oil, and that C22:5, C14:0 and C18:1 were higher in content, reaching 7.42%,6.21% and 5.37%, respectively. Among the total fatty acids of the algae oil, the polyunsaturated fatty acids are mostly n-3PUFA with physiological activity, and the content of the polyunsaturated fatty acids accounts for about 85.38% of the content of PUFA (polyunsaturated fatty acids) of the algae oil. DHA content was highest at Sn-2 position of algae oil triglyceride (47.38%), followed by C16:0 (18.85%) and C22:5 (11.34%). The proportion of PUFA at Sn-2 position of the algae oil is up to 64.29%, which shows that the algae oil is an ideal preparation raw material of Sn-2 long-chain polyunsaturated fatty acid structural fat. At present, a plurality of commercial DHA algae oils are marketed, wherein the species of schizochytrium limacinum and Wu Kenshi algae are mainly used, wu Kenshi algae oil is selected as a donor source of DHA, and although different algae fatty acid compositions are different, the DHA content of most algae oil is higher than other sources, so that the algae oil is an ideal source for providing rich DHA next to fish oil.
TABLE 1 algal oil fatty acid and sn-2 fatty acid content
(1) In the reaction process of enzyme catalysis synthesis of 2D-MAG, the composition of the product after the reaction reaches equilibrium is closely related to the quantity ratio of substrate substances. As can be seen from FIG. 2, the production rate of 2D-MAG increased significantly (p < 0.05) with increasing algae oil/ethanol molar ratio in the substrate, and the production rate of 2D-MAG was 37.2% when algae oil/ethanol molar ratio was 1:80, which was higher than that of the other experimental groups (p < 0.05).
(2) To screen out Sn-1,3 specific hydrolases most suitable for catalyzing 2D-MAG, 6 different lipases Novozym 435, lipozyme RM IM, lipozyme TL IM, newase F, lipase AY and Lipase AK were screened and tested. As can be seen from FIG. 3, in the alcoholysis reaction of the enzyme-catalyzed algae oil, the effects of the different lipases for catalyzing and synthesizing 2D-MAG are greatly different, and the activities of the six lipases for catalyzing and generating 2D-MAG are as follows from high to low: lipozyme RM IM > Novozym 435>Lipozyme TL IM>Newlase F>Lipase AK>Lipase AY. Among them, lipozyme RM IM and Novozyme 435 have relatively high content of catalyzing 2D-MAGs, 34.7% and 28.1% respectively, which reflects that Lipozyme RM IM has higher catalytic ability than other lipases. The contents of Lipozyme TL IM and Lipozyme AK and Newlase F catalyzing the formation of 2D-MAG by algae oil were 13.3%, 9.6% and 11.3%, respectively, significantly lower than Lipozyme RM IM and Novozym 435 (p < 0.05). The Sn-2 acyl position on the triglyceride has larger steric hindrance, and the Sn-2 position is more difficult to be acylated than the Sn-1,3 position. Therefore, the catalytic effect of the lipase with the same specificity is greatly influenced by steric hindrance and fatty acid structure, and compared with the lipase Novozyme 435 and Lipozyme TL IM, the Lipozyme RM IM energy region and the stereoselectivity catalyze hydrolysis, esterification and transesterification reactions of the Sn-1,3 positions of the triglyceride, and the 2D-MAG catalytic capability is better, so that the invention preferentially adopts the Lipozyme RM IM lipase as Sn-1,3 specific hydrolase to prepare the 2D-MAG.
(3) In general, enzymes are positively correlated with the reaction rate and the amount of product produced as catalysts for synthetic reactions. The invention examines the influence of the addition amount of 4-16% Sn-1,3 specific hydrolase (accounting for the total mass of the reaction substrate) on the 2D-MAG synthesis rate. As can be seen from FIG. 4, when the content of Lipozyme RM IM was increased from 4% to 10%, the 2D-MAG production was increased from 22.4% to 39.1% (p < 0.05), which indicates that the enzyme addition amount had a significant effect on the enzymatic hydrolysis effect, and the esterification reaction was mainly represented as an enzyme-controlled reaction. When the amount of enzyme added exceeded 10%, the 2D-MAG production gradually decreased from 39.1% to 33.5% (p < 0.05), indicating that increasing the amount of enzyme did not further increase the rate of product production, which was then manifested as a substrate-controlled reaction.
(4) Generally, an increase in the reaction temperature accelerates the reaction rate to shorten the reaction time, but an excessively high temperature decreases the lipase activity and the stability of the reactants. As shown in FIG. 5, the synthesis rate of 2D-MAG was changed into two stages, and when the reaction temperature was increased from 30℃to 35℃the synthesis rate of 2D-MAG was increased from 33.5% to 35.8%, and when the reaction system was further increased from 35℃to 55℃the synthesis rate was decreased. This shows that 35℃is the optimum temperature for the catalytic synthesis of 2D-MAG by Lipozyme RM IM, at which the yield of 2D-MAG is highest, and therefore 35℃is the preferred temperature level for the synthesis of 2D-MAGs.
(5) Most enzymes are not completely immobilized at their optimum temperature, which is related to the duration of the reaction, and the optimum temperature of the enzyme moves in a direction in which the value of the temperature required for the reaction is low when the reaction time is prolonged. As can be seen from fig. 6, the TAG (triglyceride) gradually decreased from the highest value of 91.3% to 3.4% (p < 0.05) after 2.5 hours as the reaction continued, indicating that the enzyme-catalyzed reaction had a higher reaction rate within 0 to 3 hours. During this time, 2D-MAG and 1,2-DAG gradually increased, with 2D-MAG reaching a maximum of 41.2% (p < 0.05) after 3h, indicating a faster rate of TAG alcoholysis reaction in the range of 0-3 hours, starting to decrease 2D-MAG content as the reaction continued, and decreasing to 19% (p < 0.05) after 6h. Too long an alcoholysis reaction time may lead to the formation of large amounts of free fatty acids and even further alcoholysis to glycerol and EEs (ethyl ester) after the transfer of the acyl group at the Sn-2 position to 1-MAG/1, 3-DAG. And the reaction time and the factors of accumulation of the synthesized products are combined, and the esterification reaction efficiency is highest and the time cost is lower when the reaction time is 3 hours.
2. Purification of Sn-2 long chain monoglyceride 2D-MAG
1g of the product obtained in the step (1) was dissolved in 15ml of n-hexane, then 10ml of an aqueous 85% ethanol solution was added, and the mixed solution was transferred to a separation funnel and allowed to stand for delamination. After formation of the two layers, the upper layer containing the ester and unreacted triglyceride residues was removed, leaving the lower extract layer containing 2D-MAG. And (3) evaporating the lower extraction layer to dry other organic phases by rotating under the water bath condition of 40 ℃, adding a sample into an ethanol/n-hexane (90:10v/v) solution, centrifuging for 5 minutes at the speed of 3500rpm, sucking the hexane phase of the upper 2D-MAG, and evaporating to remove the organic phase to obtain the purified 2D-MAG.
(1) Thin Layer Chromatography (TLC) is a simple reaction product characterization method that makes use of the different simple, rapid characterization of the product by the difference in separation of the components in the stationary phase. The invention uses TLC technique to analyze algae oil, 2D-MAG prepared in step (1) and purified 2D-MAG, and makes preliminary judgment on each component according to the specific shift value (Rf) of spots in thin layer chromatography (figure 7). The results show that the algae oil contains monoglyceride (rf≡0.05), free fatty acid (rf≡0.1-0.4), diglyceride (rf=0.48) and triglyceride (rf≡0.70), respectively. Only one spot (Rf approximately 0.06) appears in the 2D-MAG obtained by enzymatic reaction of algae oil and ethanol in the TLC plate, and the content of monoglyceride in the prepared final product is preliminarily judged to be high. The product was not purified, so the spot of TLC was tailing. After the 2D-MAG is further extracted and purified, the spot tailing phenomenon of the 2D-MAG disappears and spots are more concentrated, which shows that the monoglyceride is formed by catalyzing the alcoholysis of the algae oil by using Sn-1,3 specific hydrolase, and the purity of the monoglyceride is improved by the purification method.
(2) FIG. 8 shows the HPLC results of 2D-MAG samples before and after purification. The maximum sample peaks appear at 4.9min and 5.5min before 2D-MAG purification, respectively, which indicates that two types of monoglyceride exist simultaneously or the phenomenon of acyl transfer occurs in the molecule. Monoglycerides are typically mixtures of 1-MAG and 2-MAG due to the relatively low activation energy of intramolecular acyl transfer. After purification treatment, 2D-MAG only has the maximum sample peak at 5.5min and has no sample peak at 4.9min, which shows that the solvent extraction method adopted in the research can be used for crystallization and separation under the low-temperature condition to obtain 2D-MAG with the purity of 88.5 percent, and the preparation method and the purification method are proved to be reliable and effective by TLC and HPLC of structural fat.
3. Preparation of Sn-2 long chain polyunsaturated fatty acid structured ester 1,3C-2D-TAG
100g of purified 2D-MAG, 150g of octanoic acid, 25g of lipase and 2g of 4A molecular sieve are added into 1000mL of normal hexane, magnetic stirring is carried out for 5h under the reaction condition of 200rpm and 40 ℃, after the reaction is finished, the mixture is centrifuged for 10min at 5000rpm, so as to remove the lipase, and 1,3C-2D-TAG is obtained.
To determine the appropriate lipases in the esterification reaction, three lipases Lipozyme RM IM, lipozyme TL IM and Novozyme 435 were compared and screened. As can be seen from FIG. 9, the three lipases catalyze the insertion of octanoic acid into Sn-1,3 to form MLM structural lipid with a significantly different synthesis rate, wherein Lipozyme RM IM has the highest catalytic activity, and the insertion rate of octanoic acid reaches 34.6%. The insertion rates of octanoic acid after Lipozyme TL IM and Novozyme 435 catalysis are 25.1% and 32.2%, respectively, and the catalytic activities of the three lipases are as follows from high to low: lipozyme RM IM > Lipozyme TL IM > Novozym 435.
4. Purification of Sn-2 long chain polyunsaturated fatty acid structural ester 1,3C-2D-TAG
After removal of the lipase by centrifugation, the organic phase was dried over anhydrous sodium sulfate and the excess solvent was removed by evaporation in vacuo. The reaction mixture was purified using column chromatography: 10g of silica gel and 10g of alumina were added to 50mL of n-hexane to prepare a slurry, which was then poured into a 300X 30mm column. The 1,3C-2D-TAG (containing MAG, DAGs, TAGs) prepared in step 3 was loaded onto a column and eluted with n-hexane/diethyl ether (95/5, v/v) solution. Recovering the eluted fraction, analyzing the eluted fraction by TLC and HPLC, and collecting the purified 1,3C-2D-TAG structural lipid according to the analysis result.
Experimental example
1. Determination of Sn-2-fatty acid composition of Sn-2-long chain polyunsaturated fatty acid structural ester by GC-FID method
Fatty acid methyl esterification was analyzed by the following method: with 2mL of 0.5M NaOH-CH 3 OH was saponified with 3g of the sample at 60℃for 30 minutes and reacted with 14% boron trifluoride at 60℃for 5 minutes. After the completion of the reaction, fatty acid methyl esters were extracted with about 2mL of hexane, and then the mole percentage of the fatty acid composition at the Sn-2 position of the resulting monoglyceride was calculated.
Gas chromatography conditions: the chromatographic column is FFAP capillary column (Agilent, 30m×0.25mm×0.5 μm; detector is FID. Carrier gas is N) 2 The flow rate was set at 1.0mL/min, the inlet pressure was 25psi, and the split ratio was set at 30:1. The initial oven temperature was set to 140 ℃ for 1min, then increased to 230 ℃ at a rate of 10 ℃/min and maintained for 8min. The temperature of the detector was maintained at 280 ℃. The peak time and relative peak area will be used for quantitative and qualitative analysis of fatty acid methyl esters, according to standard analysis of fatty acids. The composition and content of fatty acids before and after modification of structural fats of Sn-2 long chain polyunsaturated fatty acids are shown in Table 2.
TABLE 2 fatty acid composition and content before and after modification of algae oil
Note that: NF represents not found; SFA represents saturated fatty acid; MUFA represents monounsaturated fatty acids; PUFA represents polyunsaturated fatty acids
The fatty acids of the algae oil are mainly C22:6 (44.37%) and C16:0 (27.5%), and secondarily C14:0, C18:1 and C22:5, with contents of 6.21%, 5.37% and 7.42%, respectively (Table 2). Wherein the proportion of PUFA at the Sn-2 position of the algae oil is up to 64.29 percent, which is significantly higher than that of SFA (28.16 percent) and MUFA (5.01 percent), and especially 47.38 percent of DHA is mainly concentrated at the Sn-2 position of triglyceride in the natural algae oil and is far higher than the content of other fatty acids, which indicates that the natural algae oil is one of ideal modified raw materials of the MLM structural fat of the Sn-2 polyunsaturated fatty acid. The modified MLM structural grease mainly comprises C22:6, C16:0 and C8:0, and the content is 38.14%, 24.39% and 20.35% respectively. Compared with natural algae oil, the insertion rate of caprylic acid in the structural fat is 33.16 percent and is mainly positioned at the Sn-1,3 positions of the glycerol skeleton, 93.82 percent of the caprylic acid inserted into the structural fat is distributed at the Sn-1,3 positions of the glycerol skeleton, and 7.48 percent of the caprylic acid is distributed at the Sn-2 positions. The DHA at the Sn-1,3 positions in the modified structural fat is reduced from 45.87% to 37.52% (p < 0.05), while the octanoic acid at the Sn-1,3 positions is increased to 29.33%. In addition, the modified structural grease Sn-2 DHA content does not change significantly (39.38%).
2. Animal experiment
(1) Experimental animal
C57BL/6 mice (about 6 weeks old, male and female halves), average body weight (25+ -2) g, provided by the experimental animal center of the army specialty medical center, experimental animal eligibility number: SCXK (Beijing) 2019-0010. The environment temperature of the feeding room is kept to be (24+/-1) DEGC, the relative humidity is kept to be (50+/-5)%, and the illumination time is 12 hours. Sn-2 long chain polyunsaturated fatty acid 1,3C-2D-TAG was prepared by the method of the examples. The mixed feed for mice is provided by experimental animal center of army special medical center.
(2) Raising and sampling mice
70C 57BL/6 mice were randomly divided into 6 groups after 7 days of laboratory adaptive feeding: normal group, high fat group, 1,2,3C-TAG group, 1,3C-2D-TAG low dose group, 1,3C-2D-TAG medium dose group, 1,3C-2D-TAG high dose group (20, 50, 100 g/kg.d), after 5D of adaptive feeding, the mice were fed with free-feeding drinking water for 6 weeks according to the corresponding feed formula (Table 3) in groups of 2 cages each. Daily weighing the food intake of the mice, replacing feed and drinking water, weighing the weight of the mice every week, replacing 2 times of cage padding, regularly removing mildew feed and excrement, and timely sterilizing and cleaning the feeding environment.
After the end of the feeding period, the mice were fasted for 24 hours, weighed and recorded, and anesthetized and sacrificed by intraperitoneal injection of 3% chloral hydrate. Collecting whole blood from eyeballs, separating serum, and freezing for detection; then dissecting and separating liver, kidney, testis periphery and kidney periphery fat from the mice, weighing, and fixing in Ma Fuer forest solution for subsequent pathological staining analysis. After the materials are obtained, all the mouse carcasses are subjected to centralized harmless treatment.
Table 3 Experimental mice feed formulation Table (%)
(3) Influence of structural fat on mouse body weight
The experimental mice were weighed 1 time a week during the feeding period and their body weights were recorded, and the mice body weights were finally measured after the end of the feeding period. Dissecting kidney, liver, kidney Zhou Zhi, etc., washing off floating blood with PBS solution, drying with filter paper, weighing, and calculating organ index: organ index (%) =wet organ weight (g)/body weight of mice (g) ×100.
Obesity is closely related to hyperlipidemia and atherosclerosis, and body weight is the most visual reflection index of the obesity degree of rats. After 6 weeks of feeding of C57BL/6 mice, all experimental groups had higher body weight than before (fig. 10), with a 6.1g increase in body weight for the mice in the blank group, significantly higher than before (p < 0.05). The weight of the mice in the high-fat group is increased by 13.7g compared with that before feeding, and the weight of the mice in the high-fat group is obviously higher than that of the mice in a blank group (p < 0.05) before feeding and after feeding, which indicates that the animal modeling is successful. The body weight of the mice added with 10% of 1,2,3C-TAG group has no obvious change compared with the mice before feeding, which shows that 1,2,3C-TAG structural fat can effectively prevent and control obesity of the mice. The body weight of the 1,3C-2D-TAG experimental groups added with the low dose, the medium dose and the high dose is respectively increased by 11.3 percent (p < 0.05), 8.9 percent (p < 0.05) and 1.3 percent (p < 0.05) compared with that before feeding. The medium and high dose 1,3C-2D-TAG experimental groups were significantly lower in weight than the high-fat model group except the low dose group, indicating that 1,3C-2D-TAG was able to reduce the increase in weight of C57BL/6 mice. In addition, in the whole feeding experiment process, normal mice have no abnormal mental state, are active and active, and high-fat mice have slightly larger body states, relatively lower spirit and less activity. The mice in the 1,3C-2D-TAG experimental group had good mental state, lively and agile response, and no death phenomenon, which indicated that 1,3C-2D-TAG inhibited weight gain in rats not by reducing food intake.
Liver and kidney are important tissues and organs of the mice for lipid metabolism, the liver index change can better judge the damage condition of the liver, the liver index increase indicates that the liver has the changes of congestion, edema, hyperplasia, hypertrophy and the like, and the decrease indicates organ atrophy, growth blockage or degenerative disease. As can be seen from table 4, the liver index (LW/BW) of the blank group was lower than that of the model group and each experimental group, wherein the model group was 4.86%, which is significantly higher than that of the high, medium and low dose 1,3C-2D-TAG experimental groups (p < 0.05), indicating that 1,3C-2D-TAG can have a certain inhibitory effect on liver hyperplasia or hypertrophy of mice. The kidney index of the high-fat group and the model group is lower than that of the normal group (p < 0.05), wherein the kidney index of the model group is basically consistent with that of the low-dose group and the high-dose experimental group (p > 0.05), which indicates that the control of the 1,3C-2D-TAG on the weight of the mice is not completed through kidney metabolism, but the kidney index of the 1,2,3C-TAG group of the mice is obviously higher than that of the normal group, the model group and each experimental group (p < 0.05), and the influence of the model group and the model group on the weight control of the mice can be speculated, and the 1,2,3C-TAG mainly realizes the influence on the weight of the mice through the metabolism of the kidneys. It appears that structural lipids Sn-1,3 fatty acids have a large influence on the metabolism of animal fat, and that the unsaturated type of Sn-2 fatty acids also has an influence on the change of body weight.
TABLE 4 influence of different feed groups on weight gain in C57BL/6 mice
| Group of | Wet liver weight/body weight (%) | Kidney wet weight/body weight (%) |
| Control group | 3.92±0.13 c | 1.04±0.05 b |
| High fat group | 4.86±0.18 a | 0.95±0.03 c |
| 1,2,3C-TAG group | 4.57±0.26 ab | 1.40±0.05 a |
| Low dose group | 4.24±0.09 bc | 0.90±0.06 c |
| Medium dose group | 4.26±0.12 bc | 1.02±0.04 b |
| High dose group | 4.32±0.11 b | 1.00±0.04 bc |
(4) Influence of structural lipids on lipid metabolism in mice
(1) The collected blood was centrifuged at 4000rmp for 15min at 4℃and the supernatant was collected and assayed for glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST) and Blood Urea Nitrogen (BUN) in each blood sample by an automatic blood analyzer.
Blood Urea Nitrogen (BUN), the main end product of protein metabolism, is discharged outside the body mainly by glomerular filtration, and is one of the reference indexes for judging the impairment of renal function. As shown in FIG. 11, the BUN concentration (16.4 mmol/L) was higher in the high-fat group mice than in the normal group (13.8 mmol/L), indicating that the high-fat diet had an effect on the normal functioning of the kidneys of the mice. Compared with the high-fat group, the BUN of the low-dose 1,3C-2D-TAG experimental group has no significant change. The medium dose 1,3C-2D-TAG experimental group had a significant decrease in BUN (15.1 mmol/L) in the higher lipid group (p < 0.05), indicating that the medium dose 1,3C-2D-TAG did not cause a nephrotoxic response in mice.
Liver is the main organ of lipid, sugar and protein metabolism, glutamic-oxaloacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT) are one of the representative indexes of liver function, can intuitively reflect the damage degree of liver cells, and indicate that the higher the damage degree of liver is. Figures 12-13 show that both high-fat groups AST and ALT were significantly higher than normal (p < 0.05), indicating that hyperlipidemia induced by high-fat feeds would impair mouse liver function and affect lipid metabolism. There was a significant increase in AST for the low-dose 1,3C-2D-TAG experimental group compared to the normal group, but no significant change (p > 0.05) for the medium-dose and high-dose experimental groups. Similar to AST, there was no significant change (p > 0.05) in ALT normal groups in both medium and high dose experimental groups. These results indicate that the medium and high doses of 1,3C-2D-TAG do not cause liver toxicity in mice.
(2) Determination of Triglyceride (TG) and Total Cholesterol (TC) content: and respectively taking triglyceride and total cholesterol kits, marking blank holes, standard holes and sample holes in a clean 96-well plate, sucking distilled water by using a 10uL pipette to be injected into the first three holes of the 96-well plate to serve as blank holes, injecting 2.5uL of each hole, injecting 2.5uL of calibrator into each hole of the three holes after the blank holes to serve as standard holes, and the rest being sample holes, wherein the sample holes are 5 groups of samples of an experimental group, dropwise adding 5 samples of each group in sequence, namely a blank group, a low concentration group (15%), a medium concentration group (30%) and a high concentration group (45%), dropwise adding 3 holes into each sample to serve as parallel tests, injecting 2.5uL of each hole into each sample, sucking 250uL of working solution by using a 500uL pipette to be injected into each hole of the blank holes, the standard holes and the sample holes, fully and uniformly mixing, placing the mixture in a 37 ℃ for 10 minutes, setting a wavelength to be 510nm, and measuring absorbance values by using an enzyme-labeling instrument.
As shown in fig. 14 to 15, the TG and TC of the mice in the high-fat group were significantly higher than those in the normal group compared to the blank group, reflecting that the high-fat feed can effectively increase the TG and TC content in the blood of the mice. The low dose 1,3C-2D-TAG group had a decreasing effect on mouse TG and TC compared to the high-fat group, but the effect was not significant (p > 0.05). The medium and high dose 1,3C-2D-TAG groups were able to significantly reduce the TG and TC levels in mice, indicating that 1,3C-2D-TAG has an effect of inhibiting the rise in total cholesterol and triglycerides in mice.
(3) Determination of high density lipoprotein cholesterol (HDL-C) and Low density lipoprotein cholesterol (LDL-C) content: the method comprises the steps of taking a high-density lipoprotein cholesterol kit, marking a clean 96-well plate as a blank hole, a standard hole and a sample hole respectively, sucking distilled water by a 10uL pipette to be injected into the first three holes of the 96-well plate as the blank hole, injecting 2.5uL of calibrator into each hole of the 96-well plate as the standard hole after the blank hole, injecting 2.5uL of calibrator into each hole of the three holes, taking the rest as the sample hole, taking the sample holes as 5 groups of samples of an experimental group, sequentially dripping 5 samples of the blank group, a low concentration group (15%), a medium concentration group (30%) and a high concentration group (45%), dripping 3 holes of each sample into each group, performing parallel test, injecting 2.5uL of sample into each hole, sucking 180uL of working solution R1 into each hole of the blank hole, the standard hole and the sample hole by a 250uL pipette, fully and uniformly mixing, placing the sample holes in a 37 ℃ oven for 5 minutes, setting the wavelength to be 546nm, measuring the absorbance value A1 of each hole by an enzyme marker, taking out 96, sucking the 5 samples of the sample into the blank hole, and measuring the absorbance value of each hole by a standard meter, and uniformly mixing the absorbance value of the enzyme marker, and setting the absorbance value of each sample in the air meter at 37 nm for 2 minutes.
High density lipoprotein is a complex lipoprotein composed of lipid and protein and its carried regulating factor, and mainly reflects the ability of resisting atherosclerosis, and is a protecting factor for coronary heart disease. From FIGS. 16 to 17, the HDL-C of the normal group was lower than that of the high-fat group, the model group and the experimental group after 6 weeks of feeding, wherein the HDL-C of the high-dose group was significantly higher than that of the model group and the medium-low-dose group (p < 0.05), which indicates that the high dose 1,3C-2D-TAG had an elevating effect on the HDL-C of the C57BL/6 mice and exhibited a good dose-effect relationship. 1,2,3C-TAG also significantly increased HDL-C content (p < 0.05) compared to the blank.
Compared with HDL-C, the LDL-C content of mice in the model group and the low-dose 1,3C-2D-TAG group is obviously increased (p < 0.05) compared with that of mice in other groups, the LDL-C content of mice in the high-dose 1,3C-2D-TAG group and 1,2,3C-TAG group is not obviously different from that of mice in the blank group, and the LDL-C content of mice in the medium-dose 1,3C-2D-TAG group is the lowest, which shows that the medium-high dose 1,3C-2D-TAG has a certain control effect on the LDL-C content of the mice. Taken together, 1,3C-2D-TAG has a certain improvement effect on high-fat mice atherosclerosis induced by high-fat feed, and particularly the regulation effect of medium-dose groups on rat blood fat is better.
(5) Influence of structural fat on liver and kidney tissues of mice
The liver, kidney and adipose tissues of the mice were washed and fixed in a 4% paraformaldehyde solution. The liver organ is cut to a right leaf longitudinal section of about 4-5 mm 3 The method comprises the steps of carrying out a first treatment on the surface of the Half of the kidney was cut longitudinally along the largest section, multiplied in a 2mL centrifuge tube containing formalin, immersed for 10min, taken out and sectioned, followed by HE staining (hematoxylin-staining).
Dehydrating: sequentially dehydrating ethanol solutions (70%, 80%, 90%) with different concentrations for 30min, and dehydrating twice with ethanol solution (95%, 100%) for 20min each time to gradually remove water in tissue blocks; wax penetration: the mixture was transparent for 15min with xylene alcohol solution (mixing ratio 1:1) and xylene paraffin wax mixture solution (mixing ratio 1:1), and then was put into paraffin wax solution at 55℃for wax permeation for 60min. Embedding: the paraffin mould is placed on a table top after being heated slightly on an alcohol lamp, a little paraffin is poured in, the section of the organ material is placed in the paraffin mould downwards, then an embedding box is placed in, and molten paraffin is poured in lightly. Slicing: the fixed paraffin block is placed on a slicing machine, a tissue slice with the thickness of 5-10 mu m is cut, and the slice is unfolded and stuck on a glass slide. Dewaxing: placing the slices in a water bath at 60 ℃ to melt paraffin, respectively soaking with xylene to remove paraffin in the slices for 30min, respectively soaking with 100% absolute ethyl alcohol and 80% absolute ethyl alcohol for 5min, eluting with distilled water for 5min, and dyeing; dyeing: placing the slice into hematoxylin solution for dyeing for 5min, repeatedly washing with distilled water for 5min, dehydrating with ethanol of different concentrations for 10min, and placing into 0.5% eosin solution for dyeing for 3min; and (3) water penetration: dehydrating with 80% ethanol and 95% ethanol for 3min, respectively, and transparentizing with xylene for 5min; sealing piece: the transparent sections were mounted by dropping neutral gum and covering with a cover slip. The sections after HE staining were observed under a 100-fold inverted fluorescence microscope and photographed.
Fig. 18 shows that in the liver pathology section of the blank group, the individual liver cells are complete, the boundary is clear, the cells are full and full, the cell nuclei are not damaged basically, the sizes are basically consistent, the shape is round, and the cells are uniformly and orderly arranged around the central vein in radioactivity. The mice in the control group have serious injury to the liver cell nucleus, disordered arrangement, loose tissue structure, large-area cavitation, 1,3C-2D-TAG structural fat and 1,2,3C-TAG group mice have slightly enlarged liver cells, relatively uniform arrangement, compact tissue structure, few or no cavitation, and similar cell morphology and structure as those of the blank group mice. Medium chain fatty acids are more soluble than long chain fatty acids, providing energy directly in the liver, whereas long chain fatty acids are mainly metabolized by absorption through the lymphatic system after intestinal wall micellar transport. Based on this, the metabolism and absorption of Sn-1, 3-position medium chain fatty acids are mainly concentrated in liver organs, and medium chain fatty acids can make energy metabolism faster. The kidney section of the mice shows that the liver cell boundary of the mice in the model group is clear, the kidney cells are slightly enlarged, the cell nucleus is slightly reduced, the nuclear membrane is thickened, the chromatin is deepened, the cytoplasm is filled with a large amount of fat drops with different sizes, the cell nucleus is pushed to one side, and the number of the fat drops in the visual field is large. Some of the kidney cells in the 1,3C-2D-TAG experimental group were damaged, but to a relatively lesser extent, and individual cells were suspected of having slight edema.
Comparing the blank liver and kidney sections (fig. 19) it is clear that the low dose group has a more disturbed hepatocyte arrangement, a different cell nucleus size, a more distinct cell boundary, and a small proportion of cavitation. The liver cells of the medium-dose group are orderly arranged, the arrangement is compact, the cell gaps are obvious, the cells around the central vein are arranged in a regular sequence, the cells are distributed in a regular sporadic shape, and the overall morphology of the liver cells is relieved. The liver cell morphology of the high-dose group is closer to that of the mice in the blank group, the arrangement is relatively regular, the fat drops are relatively small in size and quantity, the liver cell boundary is clear, and the liver sinus is visible. The size and number of fat droplets in hepatocytes of the high dose 1,3C-2D-TAG group were not significantly different from those of the blank group, indicating that the accumulation in cells was reduced after the synthesis of triglyceride in hepatocytes was controlled. Compared with the low dose group, the kidney cell nuclear membrane of the high dose 1,3C-2D-TAG group is clear, smooth and complete, the nucleolus is clear and visible, the cytoplasmic staining is fresh and uniform, the central vein and the venous sinus are not found to be expanded and engorged, the fibrous tissue proliferation and inflammatory cell infiltration are not found, the kidney cell morphology is more similar to that of the blank group, and the result proves that each dose 1,3C-2D-TAG has a certain quantitative effect relationship on the liver and kidney tissue.
(6) Influence of structured fat on mouse leptin
Leptin is a hormone secreted by adipose tissue that binds to leptin receptors on hypothalamic nerve cells, producing a range of physiological effects such as satiety, limiting energy intake and expenditure in the body to regulate body weight and body fat mass. Fig. 20 shows that the high fat model group leptin content is 1.47ng/mL, significantly higher than normal and experimental groups (p < 0.05), indicating that obesity results in hormonal imbalance to some extent, and thus does not lead to a limiting choice of energy intake in mice. Although the low dose 1,3C-2D-TAG structured lipid group leptin content was lower than that of the high lipid model group, it was significantly higher than that of the medium and high dose groups (p < 0.05), indicating that 1,3C-2D-TAG had the effect of modulating hormonal imbalance at certain doses. Leptin regulates body mass and self-stabilization of body fat in vivo by regulating appetite of the body. Studies have shown that the presence of substances antagonizing leptin function (triglycerides) or the concomitant presence of abnormal leptin-mediated nerve signaling pathways (reduced leptin receptor transport efficiency) in the blood circulation of obese subjects suffered from various degrees of disruption of the body's homeostatic regulatory system, manifested as high levels of leptin in obese subjects and thus leptin resistance. The leptin content of 1,2,3C-TAG group is not significantly different from that of normal group, 1,3C-2D-TAG medium-dose group and 1,3C-2D-TAG high-dose group, which shows that medium-chain fatty acid can reduce leptin resistance, thereby having a certain positive effect on body regulation and body fat self-stabilization system control. It was thus readily found that medium and high doses of 1,3C-2D-TAG structural fat significantly reduced the lean mass concentration in diet-induced rats, effectively alleviating leptin resistance caused by obesity.
Fatty acid differences at different positions of triglycerides are an important reason for the efficient utilization of fatty acids. In this process, the Sn-1, 3-fatty acid of the MLM structural ester of Sn-2 long chain polyunsaturated fatty acid is preferentially hydrolyzed by pancreatic lipase in the digestive tract to produce free fatty acid and efficiently absorbed 2-MAG. For 1,3C-2D-TAG structural fat, sn-1,3 medium chain fatty acid oil fatty acid has small volume and high solubility, is not easy to be converted into TAG again due to higher hydrophilicity, is transported to the liver mainly through portal veins (non-lymphatic system), is easier to digest and absorb for rapidly supplying energy in the body (does not form saponification complex with calcium or magnesium ions), and medium carbon chain fatty acid cannot be stored in adipose tissues, so that the probability and risk of hyperlipidemia of mice are reduced. The hydrolyzed 2D-MAG long chain fatty acid is prevented from oxidation under the protection of glycerol, is absorbed by mucous membrane cells in small intestine and is well absorbed through intestinal wall. Therefore, the long-chain unsaturated fatty acid is grafted on the Sn-2 position of the TAG, so that the advantage of timely energy supply of the medium-chain fatty acid can be effectively utilized, and the condition that the long-chain polyunsaturated fatty acid is difficult to absorb can be overcome.
While specific embodiments of the invention have been described in detail in connection with the examples, it should not be construed as limiting the scope of protection of the patent. Various modifications and variations which may be made by those skilled in the art without the creative effort are within the scope of the patent described in the claims.
Claims (10)
- Use of sn-2 long chain polyunsaturated fatty acid structured fat for the preparation of a food product for reducing body weight and body fat percentage; the Sn-2 long chain polyunsaturated fatty acid structural grease is grease formed by connecting a long carbon chain fatty acid with a Sn-2 position of a glycerin carbon chain and connecting a medium carbon chain fatty acid with a Sn-1,3 position, wherein the carbon number of the long carbon chain fatty acid is 15-25, and the carbon number of the medium carbon chain fatty acid is 6-10.
- 2. The use according to claim 1, characterized in that: the carbon number of the long carbon chain fatty acid is 22, and the carbon number of the medium carbon chain fatty acid is 8.
- 3. The use according to claim 2, characterized in that: the structural formula of the Sn-2 long-chain polyunsaturated fatty acid structural fat is shown as formula I:wherein R is
- The preparation method of the Sn-2 long-chain polyunsaturated fatty acid structural ester is characterized by comprising the following steps of:(1) Preparation of Sn-2 long chain monoglyceride 2D-MAGThe algae oil and Sn-1,3 specific hydrolase are dissolved in absolute ethyl alcohol according to the mass ratio of 8-10:2-5, and stirred and reacted for 4-16 h at 30-55 ℃; then adding a reactant after sn-1,3 specific hydrolase removal into a mixed solution formed by mixing n-hexane and KOH alcohol solution according to the volume ratio of 3:1, standing after violent oscillation, collecting ethanol phase and evaporating to remove organic solvent, thus obtaining 2D-MAG;(2) Preparation of Sn-2 long chain polyunsaturated fatty acid 1,3C-2D-TAG2D-MAG, octanoic acid, lipase and 4A molecular sieve are dissolved in an organic solvent together according to the mass ratio of 80-120:140-160:20-30:1-3, and stirred and reacted for 4-6 h at 35-45 ℃ to obtain 1,3C-2D-TAG.
- 5. The method of preparing as claimed in claim 4, wherein the 2D-MAG is purified prior to use, the purification comprising the steps of:dissolving 2D-MAG in n-hexane according to a feed liquid ratio of 1g to 15ml, then adding 85% ethanol water solution, standing and layering; taking the lower extraction layer, evaporating to remove the organic phase, dissolving the obtained material in a mixed solution formed by mixing ethanol and n-hexane according to the volume ratio of 9:1, centrifuging for 4-6 min at the speed of 3000-4000 rpm, taking the hexane phase of the upper 2D-MAG, and evaporating to remove the organic phase.
- 6. The method of manufacturing according to claim 4, wherein: the organic solvent is n-hexane.
- 7. The method of manufacturing according to claim 4, wherein: the Sn-1,3 specific hydrolase is Lipozyme RM IM or Novozym 435; the lipase is Lipozyme RM IM, lipozyme TL IM or Novozym 435.
- 8. The method of manufacturing according to claim 4, wherein: the stirring rate of the stirring reaction in the step (1) was 200rpm, the shaking time was 2min, and the standing time was 5 mm.
- 9. The method of claim 4, further comprising a step of purifying 1,3C-2D-TAG, the method comprising the steps of:s1: mixing silica gel and alumina with equal mass, dispersing the mixture in n-hexane according to the feed liquid ratio of 2g to 5mL, and filling the obtained slurry into a chromatographic column with the thickness of 300 multiplied by 30mm to obtain a separation and purification column;s2: loading 1,3C-2D-TAG obtained by the reaction into a separation and purification column, eluting with eluent, monitoring the eluent flowing out of the separation and purification column by TLC and HPLC, collecting the eluent containing 1,3C-2D-TAG structural lipid, and removing the organic phase by evaporation.
- 10. The Sn-2 long chain polyunsaturated fatty acid structural ester produced by the production method according to any one of claims 4 to 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310433524.9A CN116458645A (en) | 2023-04-20 | 2023-04-20 | Sn-2 long chain polyunsaturated fatty acid structural fat and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310433524.9A CN116458645A (en) | 2023-04-20 | 2023-04-20 | Sn-2 long chain polyunsaturated fatty acid structural fat and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116458645A true CN116458645A (en) | 2023-07-21 |
Family
ID=87176697
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310433524.9A Pending CN116458645A (en) | 2023-04-20 | 2023-04-20 | Sn-2 long chain polyunsaturated fatty acid structural fat and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116458645A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117233293A (en) * | 2023-11-14 | 2023-12-15 | 湖南加农正和生物技术有限公司 | Method for detecting DHA in milk |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1816614A (en) * | 2003-07-09 | 2006-08-09 | 日清奥利友集团株式会社 | Process for producing symmetrical triglyceride |
| CN104186705A (en) * | 2014-08-18 | 2014-12-10 | 江苏科技大学 | Enzymatic acidolysis-based method for synthesizing structured lipids from palmitic acid triglycerides |
| US20160177220A1 (en) * | 2014-06-27 | 2016-06-23 | Commonwealth Scientific And Industrial Research Organisation | Lipid compositions comprising triacylglycerol with long-chain polyunsaturated fatty acids at the sn-2 position |
| CN111893143A (en) * | 2020-07-23 | 2020-11-06 | 广州福汇食品科技有限公司 | Preparation method and application of grease capable of customizing sn-2-site fatty acid |
-
2023
- 2023-04-20 CN CN202310433524.9A patent/CN116458645A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1816614A (en) * | 2003-07-09 | 2006-08-09 | 日清奥利友集团株式会社 | Process for producing symmetrical triglyceride |
| US20160177220A1 (en) * | 2014-06-27 | 2016-06-23 | Commonwealth Scientific And Industrial Research Organisation | Lipid compositions comprising triacylglycerol with long-chain polyunsaturated fatty acids at the sn-2 position |
| CN104186705A (en) * | 2014-08-18 | 2014-12-10 | 江苏科技大学 | Enzymatic acidolysis-based method for synthesizing structured lipids from palmitic acid triglycerides |
| CN111893143A (en) * | 2020-07-23 | 2020-11-06 | 广州福汇食品科技有限公司 | Preparation method and application of grease capable of customizing sn-2-site fatty acid |
Non-Patent Citations (3)
| Title |
|---|
| 王强: "Sn-2位长链多不饱和脂肪酸结构脂修饰及其氧化稳定性差异研究", 中国博士学位论文全文数据库 工程科技Ⅰ辑, 15 January 2021 (2021-01-15), pages 17 - 18 * |
| 王强等: "长链多不饱和脂肪酸结构脂合成方法及影响因素研究进展", 食品与发酵工业, vol. 46, no. 08, 24 February 2020 (2020-02-24), pages 285 - 292 * |
| 王瑛瑶;魏翠平;栾霞;段章群;张帆;: "MLM型结构脂质特性及氧化稳定性研究", 中国粮油学报, vol. 28, no. 03, 25 March 2013 (2013-03-25), pages 49 - 52 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117233293A (en) * | 2023-11-14 | 2023-12-15 | 湖南加农正和生物技术有限公司 | Method for detecting DHA in milk |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109907126B (en) | Grease composition of medium-long carbon chain triglyceride and preparation method thereof | |
| Deng et al. | Enzymatic production of alkyl esters through alcoholysis: A critical evaluation of lipases and alcohols | |
| Wang et al. | From microalgae oil to produce novel structured triacylglycerols enriched with unsaturated fatty acids | |
| EP0191217B1 (en) | Process for producing glycerides in the presence of lipases | |
| CN111019979B (en) | Grease rich in low saturated fatty acid diglyceride and preparation method thereof | |
| CN116458645A (en) | Sn-2 long chain polyunsaturated fatty acid structural fat and preparation method and application thereof | |
| CN103952448B (en) | A kind of method utilizing enzyme-chemically method directional preparation OPO | |
| CN104186705A (en) | Enzymatic acidolysis-based method for synthesizing structured lipids from palmitic acid triglycerides | |
| CN111560403A (en) | Triglyceride type polyunsaturated fatty acid and preparation method and application thereof | |
| Meng et al. | Positional distribution of Δ5-olefinic acids in triacylglycerols from Torreya grandis seed oil: Isolation and purification of sciadonic acid | |
| KR102172422B1 (en) | A method of increasing the content of triglycerides having saturated fatty acids at the sn-2 position in vegetable oil and a method of converting triglyceride having an unsaturated fatty acid at the sn-2 position to triglyceride having a saturated fatty acid at the sn-2 position | |
| CN109666709B (en) | Method for preparing diglyceride by using high-acid-value grease as raw material | |
| Kuan et al. | A novel clean process for the combined production of fatty acid ethyl esters (FAEEs) and the ethyl ester of polyunsaturated fatty acids (PUFAs) from microalgae oils | |
| Diao et al. | Preparation and characterization of diacylglycerol via ultrasound-assisted enzyme-catalyzed transesterification of lard with glycerol monolaurate | |
| US20230189834A1 (en) | Method for manufacturing sn-2 palmitic triacylglycerols | |
| EP2403955B1 (en) | Method for producing ricinoleic acid ester by selective enzymatic transesterification | |
| WO2013086243A2 (en) | Phospholipid compositions enriched for palmitoleic, myristoleic or lauroleic acid, their preparation and their use in treating metabolic and cardiovascular disease | |
| Noble | Lipid metabolism in the chick embryo | |
| CN111838676A (en) | Medium-long chain fatty acid grease rich in 1, 3-dilaurate-2-palmitic acid triglyceride and preparation and application thereof | |
| Ma et al. | Integrated production of biodiesel and concentration of polyunsaturated fatty acid in glycerides through effective enzymatic catalysis | |
| Turan et al. | Effects of reaction parameters on the incorporation of caprylic acid into soybean oil for production of structured lipids | |
| CN109082447B (en) | Preparation method of mixed ester rich in OPO structure ester | |
| US20050171368A1 (en) | Processes for the production of triglycerides of conjugated linoleic acid | |
| KR101055646B1 (en) | Method for reducing saturated fat acid and oil compound reduced saturated fat acid | |
| Otero et al. | Enzymatic interesterification between pine seed oil and a hydrogenated fat to prepare semi-solid fats rich in pinolenic acid and other polyunsaturated fatty acids |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |