NZ735399B2 - Nucleotide composition and application in food thereof - Google Patents
Nucleotide composition and application in food thereof Download PDFInfo
- Publication number
- NZ735399B2 NZ735399B2 NZ735399A NZ73539916A NZ735399B2 NZ 735399 B2 NZ735399 B2 NZ 735399B2 NZ 735399 A NZ735399 A NZ 735399A NZ 73539916 A NZ73539916 A NZ 73539916A NZ 735399 B2 NZ735399 B2 NZ 735399B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- nucleotide composition
- nucleotide
- food
- cmp
- cell
- Prior art date
Links
- 239000002773 nucleotide Substances 0.000 title claims abstract description 171
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 163
- 239000000203 mixture Substances 0.000 title claims abstract description 143
- 235000013305 food Nutrition 0.000 title claims abstract description 52
- 210000003229 CMP Anatomy 0.000 claims abstract description 48
- UDMBCSSLTHHNCD-KQYNXXCUSA-N Adenosine monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 claims abstract description 47
- 229950006790 Adenosine phosphate Drugs 0.000 claims abstract description 46
- 210000003996 CFU-GM Anatomy 0.000 claims abstract description 44
- RQFCJASXJCIDSX-UUOKFMHZSA-N Guanosine monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O RQFCJASXJCIDSX-UUOKFMHZSA-N 0.000 claims abstract description 44
- DJJCXFVJDGTHFX-XVFCMESISA-N Uridine monophosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-XVFCMESISA-N 0.000 claims abstract description 44
- 235000013928 guanylic acid Nutrition 0.000 claims abstract description 44
- 235000013365 dairy product Nutrition 0.000 claims abstract description 30
- 210000001842 enterocyte Anatomy 0.000 claims abstract description 11
- 230000001737 promoting Effects 0.000 claims abstract description 7
- 230000003647 oxidation Effects 0.000 claims abstract description 6
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims description 51
- IERHLVCPSMICTF-XVFCMESISA-N CMP group Chemical group P(=O)(O)(O)OC[C@@H]1[C@H]([C@H]([C@@H](O1)N1C(=O)N=C(N)C=C1)O)O IERHLVCPSMICTF-XVFCMESISA-N 0.000 claims description 15
- 239000011734 sodium Substances 0.000 claims description 10
- 150000001720 carbohydrates Chemical class 0.000 claims description 8
- 235000014633 carbohydrates Nutrition 0.000 claims description 8
- 239000004615 ingredient Substances 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 235000018102 proteins Nutrition 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- KEAYESYHFKHZAL-UHFFFAOYSA-N sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 claims description 8
- 235000019197 fats Nutrition 0.000 claims description 7
- 229910052708 sodium Inorganic materials 0.000 claims description 7
- 229940078795 Lactoferrin Drugs 0.000 claims description 6
- 102000010445 Lactoferrin Human genes 0.000 claims description 6
- 108010063045 Lactoferrin Proteins 0.000 claims description 6
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims description 6
- 235000013373 food additive Nutrition 0.000 claims description 6
- 239000002778 food additive Substances 0.000 claims description 6
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 6
- 235000021242 lactoferrin Nutrition 0.000 claims description 6
- DFPAKSUCGFBDDF-UHFFFAOYSA-N nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims description 5
- 229930003268 Vitamin C Natural products 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 235000019154 vitamin C Nutrition 0.000 claims description 4
- 239000011718 vitamin C Substances 0.000 claims description 4
- 150000003700 vitamin C derivatives Chemical class 0.000 claims description 4
- MBWXNTAXLNYFJB-NKFFZRIASA-N 2-methyl-3-[(2E,7R,11R)-3,7,11,15-tetramethylhexadec-2-en-1-yl]-1,4-dihydronaphthalene-1,4-dione Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CCC[C@H](C)CCC[C@H](C)CCCC(C)C)=C(C)C(=O)C2=C1 MBWXNTAXLNYFJB-NKFFZRIASA-N 0.000 claims description 3
- 229960000304 Folic Acid Drugs 0.000 claims description 3
- 229940055726 Pantothenic Acid Drugs 0.000 claims description 3
- MBWXNTAXLNYFJB-ODDKJFTJSA-N Phylloquinone Natural products C1=CC=C2C(=O)C(C\C=C(C)/CCC[C@H](C)CCC[C@H](C)CCCC(C)C)=C(C)C(=O)C2=C1 MBWXNTAXLNYFJB-ODDKJFTJSA-N 0.000 claims description 3
- 229960001898 Phytomenadione Drugs 0.000 claims description 3
- 229960002477 Riboflavin Drugs 0.000 claims description 3
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 claims description 3
- 229940045997 Vitamin A Drugs 0.000 claims description 3
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 claims description 3
- 229930003451 Vitamin B1 Natural products 0.000 claims description 3
- 229930003779 Vitamin B12 Natural products 0.000 claims description 3
- 229930003471 Vitamin B2 Natural products 0.000 claims description 3
- 229940011671 Vitamin B6 Drugs 0.000 claims description 3
- 229930003629 Vitamin B6 Natural products 0.000 claims description 3
- 229940046008 Vitamin D Drugs 0.000 claims description 3
- 229930003316 Vitamin D Natural products 0.000 claims description 3
- 229940046009 Vitamin E Drugs 0.000 claims description 3
- 229930003427 Vitamin E Natural products 0.000 claims description 3
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 claims description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 3
- 229960002685 biotin Drugs 0.000 claims description 3
- 235000020958 biotin Nutrition 0.000 claims description 3
- 239000011616 biotin Substances 0.000 claims description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 3
- 239000011575 calcium Substances 0.000 claims description 3
- 229910052791 calcium Inorganic materials 0.000 claims description 3
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 claims description 3
- 229960002079 calcium pantothenate Drugs 0.000 claims description 3
- 239000000460 chlorine Substances 0.000 claims description 3
- 229910052801 chlorine Inorganic materials 0.000 claims description 3
- ZAMOUSCENKQFHK-UHFFFAOYSA-N chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 3
- 125000003346 cobalamin group Chemical group 0.000 claims description 3
- RYGMFSIKBFXOCR-UHFFFAOYSA-N copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 3
- 229910052802 copper Inorganic materials 0.000 claims description 3
- 239000010949 copper Substances 0.000 claims description 3
- 235000019152 folic acid Nutrition 0.000 claims description 3
- 239000011724 folic acid Substances 0.000 claims description 3
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 claims description 3
- 239000011630 iodine Substances 0.000 claims description 3
- 229910052740 iodine Inorganic materials 0.000 claims description 3
- 229910052742 iron Inorganic materials 0.000 claims description 3
- FYYHWMGAXLPEAU-UHFFFAOYSA-N magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims description 3
- 239000011777 magnesium Substances 0.000 claims description 3
- 229910052749 magnesium Inorganic materials 0.000 claims description 3
- 235000005152 nicotinamide Nutrition 0.000 claims description 3
- 239000011570 nicotinamide Substances 0.000 claims description 3
- GHOKWGTUZJEAQD-ZETCQYMHSA-N pantothenic acid Natural products OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 claims description 3
- 235000019161 pantothenic acid Nutrition 0.000 claims description 3
- 239000011713 pantothenic acid Substances 0.000 claims description 3
- OAICVXFJPJFONN-UHFFFAOYSA-N phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 3
- 239000011574 phosphorus Substances 0.000 claims description 3
- 229910052698 phosphorus Inorganic materials 0.000 claims description 3
- 235000019175 phylloquinone Nutrition 0.000 claims description 3
- 239000011772 phylloquinone Substances 0.000 claims description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 3
- 239000011591 potassium Substances 0.000 claims description 3
- 229910052700 potassium Inorganic materials 0.000 claims description 3
- 229960003471 retinol Drugs 0.000 claims description 3
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 claims description 3
- 229960003495 thiamine Drugs 0.000 claims description 3
- 235000019155 vitamin A Nutrition 0.000 claims description 3
- 239000011719 vitamin A Substances 0.000 claims description 3
- 235000010374 vitamin B1 Nutrition 0.000 claims description 3
- 239000011691 vitamin B1 Substances 0.000 claims description 3
- 235000019163 vitamin B12 Nutrition 0.000 claims description 3
- 239000011715 vitamin B12 Substances 0.000 claims description 3
- 235000019164 vitamin B2 Nutrition 0.000 claims description 3
- 239000011716 vitamin B2 Substances 0.000 claims description 3
- 235000019158 vitamin B6 Nutrition 0.000 claims description 3
- 239000011726 vitamin B6 Substances 0.000 claims description 3
- 150000003697 vitamin B6 derivatives Chemical class 0.000 claims description 3
- 235000019166 vitamin D Nutrition 0.000 claims description 3
- 239000011710 vitamin D Substances 0.000 claims description 3
- 150000003710 vitamin D derivatives Chemical class 0.000 claims description 3
- 235000019165 vitamin E Nutrition 0.000 claims description 3
- 239000011709 vitamin E Substances 0.000 claims description 3
- 150000003712 vitamin E derivatives Chemical class 0.000 claims description 3
- HCHKCACWOHOZIP-UHFFFAOYSA-N zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 3
- 239000011701 zinc Substances 0.000 claims description 3
- 229910052725 zinc Inorganic materials 0.000 claims description 3
- 229960003966 nicotinamide Drugs 0.000 claims description 2
- 235000008452 baby food Nutrition 0.000 abstract description 10
- 210000004027 cells Anatomy 0.000 description 114
- 230000000694 effects Effects 0.000 description 59
- 239000002609 media Substances 0.000 description 52
- 239000000843 powder Substances 0.000 description 47
- 230000000052 comparative effect Effects 0.000 description 40
- 210000004080 Milk Anatomy 0.000 description 29
- 235000013336 milk Nutrition 0.000 description 29
- 239000008267 milk Substances 0.000 description 29
- 206010057249 Phagocytosis Diseases 0.000 description 26
- 238000004166 bioassay Methods 0.000 description 26
- 230000008782 phagocytosis Effects 0.000 description 26
- 230000000968 intestinal Effects 0.000 description 23
- 239000000243 solution Substances 0.000 description 22
- 210000002540 Macrophages Anatomy 0.000 description 21
- 230000012010 growth Effects 0.000 description 18
- 230000004792 oxidative damage Effects 0.000 description 18
- 230000001681 protective Effects 0.000 description 15
- 230000004083 survival Effects 0.000 description 15
- 241000186000 Bifidobacterium Species 0.000 description 14
- 210000004698 Lymphocytes Anatomy 0.000 description 14
- 102000019197 Superoxide Dismutase Human genes 0.000 description 14
- 108010012715 Superoxide Dismutase Proteins 0.000 description 14
- 235000013902 inosinic acid Nutrition 0.000 description 14
- 210000002966 Serum Anatomy 0.000 description 13
- 229940118019 Malondialdehyde Drugs 0.000 description 12
- 238000011161 development Methods 0.000 description 12
- 230000002708 enhancing Effects 0.000 description 12
- WSMYVTOQOOLQHP-UHFFFAOYSA-N malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 12
- 230000003308 immunostimulating Effects 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- 210000000683 Abdominal Cavity Anatomy 0.000 description 10
- 210000000952 Spleen Anatomy 0.000 description 10
- 238000007792 addition Methods 0.000 description 10
- 239000003153 chemical reaction reagent Substances 0.000 description 10
- 230000036039 immunity Effects 0.000 description 10
- 108010062580 Concanavalin A Proteins 0.000 description 9
- 210000000822 Killer Cells, Natural Anatomy 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 9
- 235000013350 formula milk Nutrition 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 210000004369 Blood Anatomy 0.000 description 8
- 210000004693 NK cell Anatomy 0.000 description 8
- 239000008280 blood Substances 0.000 description 8
- 238000004113 cell culture Methods 0.000 description 8
- 239000006285 cell suspension Substances 0.000 description 8
- 230000002949 hemolytic Effects 0.000 description 8
- 239000007759 RPMI Media 1640 Substances 0.000 description 7
- 230000004663 cell proliferation Effects 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- 230000035755 proliferation Effects 0.000 description 7
- 239000002994 raw material Substances 0.000 description 7
- 150000003839 salts Chemical group 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 230000001954 sterilising Effects 0.000 description 7
- 206010018910 Haemolysis Diseases 0.000 description 6
- 239000005862 Whey Substances 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000001580 bacterial Effects 0.000 description 6
- 235000005687 corn oil Nutrition 0.000 description 6
- 239000002285 corn oil Substances 0.000 description 6
- 238000003304 gavage Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 239000004005 microsphere Substances 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 235000012424 soybean oil Nutrition 0.000 description 6
- 239000003549 soybean oil Substances 0.000 description 6
- 238000004659 sterilization and disinfection Methods 0.000 description 6
- 235000020238 sunflower seed Nutrition 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 5
- 241000283690 Bos taurus Species 0.000 description 5
- 230000037250 Clearance Effects 0.000 description 5
- 210000003743 Erythrocytes Anatomy 0.000 description 5
- 210000001035 Gastrointestinal Tract Anatomy 0.000 description 5
- GUBGYTABKSRVRQ-UUNJERMWSA-N Lactose Natural products O([C@@H]1[C@H](O)[C@H](O)[C@H](O)O[C@@H]1CO)[C@H]1[C@@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1 GUBGYTABKSRVRQ-UUNJERMWSA-N 0.000 description 5
- 210000003324 RBC Anatomy 0.000 description 5
- 230000000240 adjuvant Effects 0.000 description 5
- 239000002671 adjuvant Substances 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- 230000003078 antioxidant Effects 0.000 description 5
- 230000037396 body weight Effects 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 239000000969 carrier Substances 0.000 description 5
- 230000001413 cellular Effects 0.000 description 5
- 230000035512 clearance Effects 0.000 description 5
- 230000004727 humoral immunity Effects 0.000 description 5
- 230000001965 increased Effects 0.000 description 5
- GUBGYTABKSRVRQ-XLOQQCSPSA-N lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 5
- 239000008101 lactose Substances 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 230000003287 optical Effects 0.000 description 5
- 210000000056 organs Anatomy 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 238000001694 spray drying Methods 0.000 description 5
- 230000000638 stimulation Effects 0.000 description 5
- 230000001131 transforming Effects 0.000 description 5
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 4
- 241000287523 Ara Species 0.000 description 4
- SBJKKFFYIZUCET-JLAZNSOCSA-N Dehydro-L-ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(=O)C1=O SBJKKFFYIZUCET-JLAZNSOCSA-N 0.000 description 4
- 241000287828 Gallus gallus Species 0.000 description 4
- 240000007842 Glycine max Species 0.000 description 4
- 235000010469 Glycine max Nutrition 0.000 description 4
- FDGQSTZJBFJUBT-UHFFFAOYSA-N Hypoxanthine Chemical class O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 4
- 229940067606 Lecithin Drugs 0.000 description 4
- 241000700159 Rattus Species 0.000 description 4
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical class O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 4
- -1 alkaline earth metal salts Chemical class 0.000 description 4
- 239000003963 antioxidant agent Substances 0.000 description 4
- 235000006708 antioxidants Nutrition 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 4
- 235000020669 docosahexaenoic acid Nutrition 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000012467 final product Substances 0.000 description 4
- 235000021255 galacto-oligosaccharides Nutrition 0.000 description 4
- 150000003271 galactooligosaccharides Chemical class 0.000 description 4
- 238000000265 homogenisation Methods 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 239000000787 lecithin Substances 0.000 description 4
- 235000010445 lecithin Nutrition 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- PVNIIMVLHYAWGP-UHFFFAOYSA-N nicotinic acid Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 239000003921 oil Substances 0.000 description 4
- 235000019198 oils Nutrition 0.000 description 4
- 150000002978 peroxides Chemical class 0.000 description 4
- 235000020183 skimmed milk Nutrition 0.000 description 4
- 210000000813 small intestine Anatomy 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000006188 syrup Substances 0.000 description 4
- 235000020357 syrup Nutrition 0.000 description 4
- OPTASPLRGRRNAP-UHFFFAOYSA-N Cytosine Chemical class NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N HCl Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 210000003494 Hepatocytes Anatomy 0.000 description 3
- 229940039696 Lactobacillus Drugs 0.000 description 3
- 241000186660 Lactobacillus Species 0.000 description 3
- 210000004185 Liver Anatomy 0.000 description 3
- VMGAPWLDMVPYIA-HIDZBRGKSA-N N'-amino-N-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 3
- 241000283898 Ovis Species 0.000 description 3
- 210000003371 Toes Anatomy 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 102000007544 Whey Proteins Human genes 0.000 description 3
- 108010046377 Whey Proteins Proteins 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000001464 adherent Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000000875 corresponding Effects 0.000 description 3
- 235000020247 cow milk Nutrition 0.000 description 3
- 210000002249 digestive system Anatomy 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 238000001543 one-way ANOVA Methods 0.000 description 3
- 238000009928 pasteurization Methods 0.000 description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 3
- 230000003389 potentiating Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 159000000000 sodium salts Chemical class 0.000 description 3
- 210000001519 tissues Anatomy 0.000 description 3
- 229960001322 trypsin Drugs 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 235000021119 whey protein Nutrition 0.000 description 3
- UCSJYZPVAKXKNQ-HZYVHMACSA-N 1-[(1S,2R,3R,4S,5R,6R)-3-carbamimidamido-6-{[(2R,3R,4R,5S)-3-{[(2S,3S,4S,5R,6S)-4,5-dihydroxy-6-(hydroxymethyl)-3-(methylamino)oxan-2-yl]oxy}-4-formyl-4-hydroxy-5-methyloxolan-2-yl]oxy}-2,4,5-trihydroxycyclohexyl]guanidine Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- IYXQQOMXWUAAOK-JJSXZHHUSA-N 2-[(2S,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]-1-[4-(bromomethyl)phenyl]-2-hydroxyethanone Chemical compound OC([C@H]1O[C@H]([C@@H]([C@@H]1O)O)N1C=2N=CN=C(C=2N=C1)N)C(=O)C1=CC=C(CBr)C=C1 IYXQQOMXWUAAOK-JJSXZHHUSA-N 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N 2-mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 101700050663 ART1 Proteins 0.000 description 2
- 101710036216 ATEG_03556 Proteins 0.000 description 2
- 210000001015 Abdomen Anatomy 0.000 description 2
- 210000003815 Abdominal Wall Anatomy 0.000 description 2
- 229960000643 Adenine Drugs 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Natural products NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 210000000628 Antibody-Producing Cells Anatomy 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 101710025159 CMPK1 Proteins 0.000 description 2
- 102100011801 CMPK1 Human genes 0.000 description 2
- 229940113118 Carrageenan Drugs 0.000 description 2
- 244000038022 Chenopodium capitatum Species 0.000 description 2
- 235000004391 Chenopodium capitatum Nutrition 0.000 description 2
- 210000003022 Colostrum Anatomy 0.000 description 2
- 229940104302 Cytosine Drugs 0.000 description 2
- DYDCUQKUCUHJBH-UWTATZPHSA-N D-cycloserine Chemical compound N[C@@H]1CONC1=O DYDCUQKUCUHJBH-UWTATZPHSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 229940088598 Enzyme Drugs 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 210000003608 Feces Anatomy 0.000 description 2
- 210000002683 Foot Anatomy 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N Guanine Chemical class O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 229920002907 Guar gum Polymers 0.000 description 2
- 206010022114 Injury Diseases 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- OYHQOLUKZRVURQ-IXWMQOLASA-N Linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 101700015696 PHL12 Proteins 0.000 description 2
- 229920001914 Ribonucleotide Polymers 0.000 description 2
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 2
- 101700003161 TXLR2 Proteins 0.000 description 2
- 210000001541 Thymus Gland Anatomy 0.000 description 2
- 206010053613 Type IV hypersensitivity reaction Diseases 0.000 description 2
- 229940035893 Uracil Drugs 0.000 description 2
- 229940029983 VITAMINS Drugs 0.000 description 2
- 229940021016 Vitamin IV solution additives Drugs 0.000 description 2
- 230000003187 abdominal Effects 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive Effects 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical class C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 101710024925 alv Proteins 0.000 description 2
- 230000002429 anti-coagulation Effects 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 101700053124 asa1 Proteins 0.000 description 2
- 238000010009 beating Methods 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 235000010418 carrageenan Nutrition 0.000 description 2
- 239000000679 carrageenan Substances 0.000 description 2
- 229920001525 carrageenan Polymers 0.000 description 2
- 235000021277 colostrum Nutrition 0.000 description 2
- 230000000295 complement Effects 0.000 description 2
- 230000003247 decreasing Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 2
- INTPYBRGLGSMRA-WFIJOQBCSA-L disodium;[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound [Na+].[Na+].O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP([O-])([O-])=O)O1 INTPYBRGLGSMRA-WFIJOQBCSA-L 0.000 description 2
- 229940079593 drugs Drugs 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N edta Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 239000006153 eosin methylene blue Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000001605 fetal Effects 0.000 description 2
- 150000004676 glycans Polymers 0.000 description 2
- 239000000665 guar gum Substances 0.000 description 2
- 235000010417 guar gum Nutrition 0.000 description 2
- 229960002154 guar gum Drugs 0.000 description 2
- 239000003228 hemolysin Substances 0.000 description 2
- 101700027420 hly Proteins 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000003859 lipid peroxidation Effects 0.000 description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 2
- 235000021243 milk fat Nutrition 0.000 description 2
- 235000013384 milk substitute Nutrition 0.000 description 2
- 229960003512 nicotinic acid Drugs 0.000 description 2
- 235000001968 nicotinic acid Nutrition 0.000 description 2
- 239000011664 nicotinic acid Substances 0.000 description 2
- MYMOFIZGZYHOMD-UHFFFAOYSA-N oxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 2
- 101700005116 plc Proteins 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 150000004804 polysaccharides Polymers 0.000 description 2
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 2
- 230000002062 proliferating Effects 0.000 description 2
- VBICKXHEKHSIBG-UHFFFAOYSA-N rac-1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- 125000002652 ribonucleotide group Chemical group 0.000 description 2
- 101710010431 shlA Proteins 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 230000000576 supplementary Effects 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 230000005951 type IV hypersensitivity Effects 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229930003231 vitamins Natural products 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 229920000160 (ribonucleotides)n+m Polymers 0.000 description 1
- KEQXNNJHMWSZHK-UHFFFAOYSA-L 1,3,2,4$l^{2}-dioxathiaplumbetane 2,2-dioxide Chemical compound [Pb+2].[O-]S([O-])(=O)=O KEQXNNJHMWSZHK-UHFFFAOYSA-L 0.000 description 1
- LOTKRQAVGJMPNV-UHFFFAOYSA-N 1-Fluoro-2,4-dinitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(F)C([N+]([O-])=O)=C1 LOTKRQAVGJMPNV-UHFFFAOYSA-N 0.000 description 1
- 125000001917 2,4-dinitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C(=C1*)[N+]([O-])=O)[N+]([O-])=O 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N 289-95-2 Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- 229960003190 Adenosine Monophosphate Drugs 0.000 description 1
- 210000003567 Ascitic Fluid Anatomy 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- CJDPJFRMHVXWPT-UHFFFAOYSA-N Barium sulfide Chemical compound [S-2].[Ba+2] CJDPJFRMHVXWPT-UHFFFAOYSA-N 0.000 description 1
- 210000000601 Blood Cells Anatomy 0.000 description 1
- 101700052094 CAMK1 Proteins 0.000 description 1
- 125000006414 CCl Chemical group ClC* 0.000 description 1
- 102000011632 Caseins Human genes 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 210000000170 Cell Membrane Anatomy 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- 229960004397 Cyclophosphamide Drugs 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N D-sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 210000003298 Dental Enamel Anatomy 0.000 description 1
- 210000002969 Egg Yolk Anatomy 0.000 description 1
- 210000001513 Elbow Anatomy 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 210000003195 Fascia Anatomy 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 102100000204 GUK1 Human genes 0.000 description 1
- 101710025899 GUK1 Proteins 0.000 description 1
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 1
- 101700050903 IPMK Proteins 0.000 description 1
- 102100009138 IPMK Human genes 0.000 description 1
- 210000000987 Immune System Anatomy 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010021972 Inflammatory bowel disease Diseases 0.000 description 1
- GRSZFWQUAKGDAV-KQYNXXCUSA-N Inosinic acid Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(NC=NC2=O)=C2N=C1 GRSZFWQUAKGDAV-KQYNXXCUSA-N 0.000 description 1
- 240000007946 Iris tectorum Species 0.000 description 1
- 229920000161 Locust bean gum Polymers 0.000 description 1
- 230000035633 Metabolized Effects 0.000 description 1
- 210000004251 Milk, Human Anatomy 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 101710035826 PRKAA2 Proteins 0.000 description 1
- 102100012041 PRKAA2 Human genes 0.000 description 1
- 101710015127 PRKAB1 Proteins 0.000 description 1
- 229940049954 Penicillin Drugs 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 206010036590 Premature baby Diseases 0.000 description 1
- 108010078762 Protein Precursors Proteins 0.000 description 1
- 102000014961 Protein Precursors Human genes 0.000 description 1
- KDCGOANMDULRCW-UHFFFAOYSA-N Purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J Pyrophosphate Chemical class [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 241000219061 Rheum Species 0.000 description 1
- 101700050102 SLAC Proteins 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 210000003491 Skin Anatomy 0.000 description 1
- 229960005322 Streptomycin Drugs 0.000 description 1
- RWQNBRDOKXIBIV-UHFFFAOYSA-N Thymine Chemical class CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 1
- 229910004338 Ti-S Inorganic materials 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 101700049232 UCK2 Proteins 0.000 description 1
- 210000003462 Veins Anatomy 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K [O-]P([O-])([O-])=O Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 230000002378 acidificating Effects 0.000 description 1
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 1
- 102000004965 antibodies Human genes 0.000 description 1
- 108090001123 antibodies Proteins 0.000 description 1
- 210000000436 anus Anatomy 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 229960000626 benzylpenicillin Drugs 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 238000003889 chemical engineering Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000012495 crackers Nutrition 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 229960003077 cycloserine Drugs 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000003111 delayed Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- QLBHNVFOQLIYTH-UHFFFAOYSA-L dipotassium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [K+].[K+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O QLBHNVFOQLIYTH-UHFFFAOYSA-L 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- KURVIXMFFSNONZ-WFIJOQBCSA-L disodium;[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound [Na+].[Na+].O[C@@H]1[C@H](O)[C@@H](COP([O-])([O-])=O)O[C@H]1N1C(=O)NC(=O)C=C1 KURVIXMFFSNONZ-WFIJOQBCSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000007580 dry-mixing Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000002919 epithelial cells Anatomy 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000005755 formation reaction Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229940075507 glyceryl monostearate Drugs 0.000 description 1
- 239000001963 growth media Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N iso-propanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 230000000670 limiting Effects 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 235000010420 locust bean gum Nutrition 0.000 description 1
- 239000000711 locust bean gum Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 239000001788 mono and diglycerides of fatty acids Substances 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000001717 pathogenic Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- NXBZXYJXNIFCRJ-UHFFFAOYSA-M potassium;2,4,5,7-tetrabromo-9-(2-methoxycarbonylphenyl)-6-oxoxanthen-3-olate Chemical compound [K+].COC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C([O-])=C(Br)C=C21 NXBZXYJXNIFCRJ-UHFFFAOYSA-M 0.000 description 1
- OZAIFHULBGXAKX-UHFFFAOYSA-N precursor Substances N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 1
- 230000002335 preservative Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000000529 probiotic Effects 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 101700009937 pyrH Proteins 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 230000002829 reduced Effects 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 230000001235 sensitizing Effects 0.000 description 1
- 239000001187 sodium carbonate Substances 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 239000012453 solvate Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 230000002522 swelling Effects 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-I triphosphate(5-) Chemical class [O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O UNXRWKVEANCORM-UHFFFAOYSA-I 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/16—Agglomerating or granulating milk powder; Making instant milk powder; Products obtained thereby
-
- 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
- A23L33/13—Nucleic acids or derivatives thereof
-
- 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
- A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
Abstract
The invention relates to a nucleotide composition consisting of 58-72% CMP, 6-14% AMP, 10-18% UMP and 8-14% GMP used for promoting repair of enterocytes after damage caused by oxidation. The composition may be in the form of a food product, such as infant food or a dairy product.
Description
NUCLEOTIDE COMPOSITION AND APPLICATION IN FOOD THEREOF
The present application claims the priority of Chinese application No. 201510099850.6 with
the title of “NUCLEOTIDE COMPOSITION AND APPLICATION IN FOOD THEREOF”
filed on March 6, 2015, of which the content is incorporated herein by reference in its entirety.
Technical field
Provided is a nucleotide composition as food additive. Provided is also a food comprising the
nucleotide composition and use of the nucleotide composition for manufacture of a food.
Background
In the recent years, many experiments have been provided showing the important function of
nucleotides in living body. For example, nucleotides may be used to enhance the functions of
immune system and gastrointestinal tract of the body. Meanwhile, cowmilk-based milk
substitute is an importance supplementary source of food for infant. Due to the low content of
nucleotide and derivative thereof in cow milk, it is important to add foreign nucleotides into
cowmilk-based milk substitute for growth and development (e.g. gastrointestinal tract
development) and immune enhancement of the body, especially for that of the infant.
US 4,994,442 discloses addition of various nucleotides into baby formula food could enhance
immune response of T cell. However, the nucleotide components (CMP, AMP, UMP, GMP and
IMP) are each added into the formula food in an equal amount. EP 1549158 discloses a baby
milk composition, comprising 3.2-15.4 mg/L of CMP, 1.8-11.0 mg/L of UMP, 1.8-8.0 mg/L of
GMP, 0.1-2.2 mg/L of IMP and 2.5-13.2 mg/L of AMP. However, this composition is only
useful for premature baby and none of possible effects was described.
On the basis of the prior art, to sufficiently exert the function of nucleotides on growth and
development and optimize the effect of nucleotides as food additive, the present inventor has
performed extensive experiments to adjust the components and proportions of nucleotides in
nucleic acid composition so as to maximize the effect of the composition as nucleotides
supplementary additive. Meanwhile, the present inventor surprisingly found that, as compared
to the products of the prior art, the nucleotide composition according to the present invention
has specific components and proportions and could provide better effects.
Summary
In the first aspect, provided is a nucleotide composition which is used as food additive.
In an embodiment, the nucleotide composition according to the invention substantially consists
of or consists of CMP, AMP, UMP, GMP and IMP, wherein on the weight basis, the proportions
of the components are: CMP: 58~70%, AMP: 7.5~12.5%, UMP: 12~16.5%, GMP: 10~13%
and IMP: 0~2.5%, provided that sum of the components is 100%.
In a preferable embodiment, the nucleotide composition according to the invention substantially
consists of or consists of CMP, AMP, UMP, GMP and IMP, wherein on the weight basis, the
proportions of the components are: CMP: 60~65%, AMP: 8~12%, UMP: 14~16%, GMP:
11~12% and IMP: 0~2%, provided that sum of the components is 100%.
In another embodiment, the nucleotide composition according to the invention substantially
consists of or consists of CMP, AMP, UMP and GMP, wherein on the weight basis, the
proportions of the components are: CMP: 58~72%, AMP: 6~14%, UMP: 10~18% and GMP:
8~14%, provided that sum of the components is 100%.
In a further embodiment, the nucleotide composition according to the invention substantially
consists of or consists of CMP, AMP, UMP and GMP, wherein on the weight basis, the
proportions of the components are: CMP: 60~70%, AMP: 8~12%, UMP: 12~16% and GMP:
~12%, provided that sum of the components is 100%.
In a further embodiment, the nucleotide composition according to the invention substantially
consists of or consists of CMP, AMP, UMP and GMP, wherein on the weight basis, the
proportions of the components are: CMP: 60~65%, AMP: 10~12%, UMP: 14~16% and GMP:
11~12%, provided that sum of the components is 100%.
In a further embodiment, the nucleotide composition according to the invention substantially
consists of or consists of CMP, AMP, UMP and GMP, wherein on the weight basis, the
proportions of the components are: CMP: 65~70%, AMP: 8~10%, UMP: 12~14% and GMP:
~11%, provided that sum of the components is 100%.
In a preferable embodiment, the nucleotide composition according to the invention substantially
consists of or consists of CMP, AMP, UMP and GMP, wherein on the weight basis, the
proportions of the components are: CMP: 60%, AMP: 12%, UMP: 16% and GMP: 12%.
In a preferable embodiment, the nucleotide composition according to the invention substantially
consists of or consists of CMP, AMP, UMP and GMP, wherein on the weight basis, the
proportions of the components are: CMP: 65%, AMP: 10%, UMP: 14% and GMP: 11%.
In a preferable embodiment, the nucleotide composition according to the invention substantially
consists of or consists of CMP, AMP, UMP and GMP, wherein on the weight basis, the
proportions of the components are: CMP: 70%, AMP: 8%, UMP: 12% and GMP: 10%.
In various embodiments of the first aspect, the food is preferably an infant food and more
preferably the food is in the form of dairy product, for example the form of milk powder or
liquid dairy product, such as the milk powder or liquid dairy product useful for infant.
In the second aspect, provided is a food comprising the nucleotide composition according to
the invention. In a preferable embodiment, the food is an infant food. In a more preferable
embodiment, the food is in the form of dairy product, for example the form of milk powder or
liquid dairy product, such as the milk powder or liquid dairy product useful for infant.
In the third aspect, provided is a process for preparing a food, comprising adding the nucleotide
composition according to the invention into the raw material of the food. In a preferable
embodiment, the food is an infant food. In a more preferable embodiment, the food is in the
form of dairy product, for example the form of milk powder or liquid dairy product, such as the
milk powder or liquid dairy product useful for infant.
In the fourth aspect, provided is also use of the nucleotide composition according to the
invention for the manufacture of food. In a preferable embodiment, the food is an infant food.
In a more preferable embodiment, the food is in the form of dairy product, for example the form
of milk powder or liquid dairy product, such as the milk powder or liquid dairy product useful
for infant. In a preferable embodiment, the food is used to provide the effect of
immunostimulation. In another preferable embodiment, the food is used to promote growth and
development (such as, gastrointestinal tract development) and promote repair of intestinal tract
after damage (such as, promote repair of cells after damage, particularly enterocytes). In a
further preferable embodiment, the food is used to promote growth of intestinal beneficial
microorganisms.
In the fifth aspect, provided is a method for enhancing immunity in a subject, promoting growth
and development (such as, gastrointestinal tract development), promoting repair of intestinal
tract after damage (such as, promote repair of cells , particularly enterocytes after damage, e.g.
damage caused by oxidation) and/or promoting growth of intestinal beneficial microorganisms
in a subject and/or any combination thereof, comprising administering the subject the
nucleotide composition or food according to the invention. In a preferable embodiment, the
subject is human, preferably human infant.
Figures
Figure 1 shows the results of cell survival rate experiment.
Figure 2 shows the results of SOD activity experiment.
Figure 3 shows the results of LDH activity experiment.
Figure 4 shows the results of MDA content experiment.
Figure 5 shows the results of cell proliferative experiment.
Detailed description
Unless specifically defined otherwise, all the technical and scientific terms used herein have
the same meanings as those commonly used for a person skilled in the relevant field. Unless
specifically defined otherwise, the ratios, proportions (including percentages) used herein are
calculated on weight basis.
Nucleotide
The term “nucleotide” used herein refers to the compound formed from purine or pyrimidine
base, ribose or ribodesose and phosphate group. For example, according to the sugar group,
nucleotides can be categorized as ribonucleotide and deoxyribonucleotide. For example,
according to the base group, nucleotides can be categorized as adenine nucleotide, guanine
nucleotide, cytosine nucleotide, uracil nucleotide, thymine nucleotide, hypoxanthine nucleotide
and the like. When there is one phosphate group in nucleotide molecule, it is called
monophosphate nucleotide (NMP). The phosphate group of 5'-nucleotide may be further
phosphorylated to be diphosphate nucleotide (NDP) and triphosphate nucleotide (NTP).
The term “nucleotide” used herein also encompasses cytosine (C), uracil (U), adenine (A),
guanine (G) and/or hypoxanthine (I) present in the nucleotide composition according to the
invention in various forms, for example ribonucleoside, ribonucleotide, RNA phosphate and
derivatives or precursors in any other forms, provided that they can be transferred or
metabolized into their corresponding nucleotide forms in vivo or in vitro.
For example, in the field of food addition, the nucleotides used are mainly CMP (cytosine
nucleotide), UMP (uracil nucleotide), AMP (adenine nucleotide), GMP (guanine nucleotide),
IMP (hypoxanthine nucleotide) or the like. For example, the commercially available 5'-mixed
nucleotides comprise 5'-adenosine monophosphate (AMP), 5'-cytidine monophosphate (CMP),
5'-guanosine monophosphate (GMP), 5'-uridine monophosphate (UMP) and 5'-inosine
monophosphate (IMP). Generally, when they are used in combination, the 5'-mixed nucleotides
may for example be present in two types: one type is that 5'- adenosine monophosphate and 5'-
cytidine monophosphate are in the forms of free acid while other three nucleotides are in the
forms of sodium salt, i.e. two acid and three sodium type; the other is that all the nucleotides
are in the form of sodium salt, i.e. five sodium type. Accordingly, the term “nucleotide” used
herein encompasses its salt form, for example alkaline metal salts or alkaline earth metal salts,
such as sodium salt, potassium salt, calcium salt or the like, e.g. monosodium salt, disodium
salt or the like, such as CMPNa , AMPNa , UMPNa , GMPNa , IMPNa and CMPK , AMPK ,
2 2 2 2 2 2 2
UMPK , GMPK, IMPK or the like. It will be understood by a person skilled in the art, the
components in the nucleotide composition according to the invention can be present each
optionally and independently in the forms of various salts, comprising but not limited to the
above-mentioned “two acid and three sodium type”; or the components and the salts thereof
can be present in any combination, for example only GMP is in the form of salt or only CMP is
in the form of salt. Optionally, all the components in the nucleotide composition according to
the invention can be present in the form of salt, comprising but not limited to the above-
mentioned “five sodium type”.
Likewise, the term “nucleotide” used herein comprises the solvate thereof in various forms
(such as hydrate). Accordingly, when the nucleotide composition according to the invention
comprises other forms such as those listed above of the nucleotide components, the proportions
of such forms in the composition shall be calculated according to their corresponding nucleotide
molecules. For example, when CMP is present in the form of disodium salt (CMPNa ), the
weight ratio shall be calculated after being transferred into CMP.
Nucleotide composition
In an embodiment, the nucleotide composition according to the invention substantially consists
of or consists of the following components: CMP, AMP, UMP, GMP and IMP. In another
embodiment, the nucleotide composition according to the invention substantially consists of or
consists of the following components: CMP, AMP, UMP and GMP.
In an embodiment, in the nucleotide composition according to the invention, CMP is present in
an amount of 58~72% by weight, preferably 60~70% by weight, for example 60~65% by
weight and 65~70% by weight. It will be understood that the ranges comprise all the point
values therein, for example but not limited to 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,
67%, 68%, 69%, 70%, 71% or the like; as well as various subranges formed by such point
values, for example but not limited to 60~63%, 60~68%, 63~70% or the like. In a specific
embodiment, in the nucleotide composition according to the invention, CMP is present in an
amount of 60%, 65% or 70% by weight.
In an embodiment, in the nucleotide composition according to the invention, AMP is present in
an amount of 6~14% by weight, preferably 8~12% by weight, for example 8~10% by weight
and 10~12% by weight. It will be understood that the ranges comprise all the point values
therein, for example but not limited to 7%, 8%, 9%, 10%, 11%, 12%, 13% or the like; as well
as various subranges formed by such point values, for example but not limited to 8~9%, 8~11%,
9~12% or the like. In a specific embodiment, in the nucleotide composition according to the
invention, AMP is present in an amount of 8%, 10% or 12% by weight.
In an embodiment, in the nucleotide composition according to the invention, UMP is present in
an amount of 10~18% by weight, preferably 12~16% by weight, for example 12~14% by
weight and 14~16% by weight. It will be understood that the ranges comprise all the point
values therein, for example but not limited to 11%, 12%, 13%, 14%, 15%, 16%, 17% or the
like; as well as various subranges formed by such point values, for example but not limited to
12~15%, 14~15% or the like. In a specific embodiment, in the nucleotide composition
according to the invention, UMP is present in an amount of 12%, 14% or 16% by weight.
In an embodiment, in the nucleotide composition according to the invention, GMP is present in
an amount of 8~14% by weight, preferably 10~12% by weight, for example 10~11% by weight
and 11~12% by weight. It will be understood that the ranges comprise all the point values
therein, for example but not limited to 9%, 10%, 11%, 12%, 13% or the like; as well as various
subranges formed by such point values, for example but not limited to 8~11%, 10~14% or the
like. In a specific embodiment, in the nucleotide composition according to the invention, GMP
is present in an amount of 10%, 11% or 12% by weight.
In an embodiment, in the nucleotide composition according to the invention, IMP is present in
an amount of 0~2.5% by weight, preferably 0~2% by weight, more preferably 0% (i.e. the
nucleotide composition according to the invention does not comprise IMP). It will be
understood that the ranges comprise all the point values therein, for example but not limited to
1%, 2% or the like as well as various subranges formed by such point values, for example but
not limited to 0~1%, 1~2% or the like.
It will be understood by a person skilled in the art that, the ranges and specific values listed
above for various components can be optionally combined and selected, provided that sum of
the selected components is 100%. For example, in the nucleotide composition according to the
invention, when the content of CMP is 60~70%, such as 60~65% or 65~70%, the content of
AMP can be 8~12%, such as 8~10% or 10~12%. Likewise, when the contents of CMP and
AMP are within any above ranges, the content of UMP can be 12~16%, such as 12~14% or
14~16%. Similarly, when the contents of CMP, AMP and UMP are within any above ranges,
the content of GMP can be 10~12%, such as 10~11% or 11~12%. According to the same
principle, when the contents of CMP, AMP, UMP and GMP are within any above ranges, the
content of IMP can be 0~2.5%, such as 0~2% or 0% (i.e. the nucleotide composition according
to the invention does not comprise IMP). A person skilled in the art will understand that the
above selections shall comply with the requirement that sum of the selected components is
100%.
The expression “substantially consist of” means in addition to the defined nucleotide
components, if necessary, the nucleotide composition according to the invention can optionally
comprise food or physiologically acceptable carrier, excipient or adjuvant, for example
preservative, antioxidant, binder, thickener, diluent or the like and various impurities which
possibly exist. The carrier, excipient or adjuvant and impurities are inert for the active
ingredients in the composition (nucleotide components) and their presence or amount would
not disturb or significantly disturb the functions of the active ingredients. Moreover, when
calculating the proportions of the nucleotide components in the nucleotide composition
according to the invention, the carrier, excipient or adjuvant and impurities which are optionally
present will not be considered. In addition, a person skilled in the art will understand that the
expression “substantially consist of …” encompasses the meaning of “consist of …”.
The term “optional” or “optionally” used herein refers a subject matter which may or may not
exist. For example, “optionally comprising food or physiologically acceptable carrier, excipient
or adjuvant” means the nucleotide composition according to the invention may or may not
comprise such carrier, excipient or adjuvant.
Food
The term “food” used herein has the common meaning to a person skilled in the art and for
example refers to edible or drinkable material for human, including the food which is processed,
semi-processed, unprocessed or the like.
The term “infant food” used herein refers to the food, except breast milk, suitable for infant,
which is added with various ingredients due to the requirement of infant’s growth and
development, for example nucleotides, fatty acids, vitamins, carbohydrates, vegetable oils,
microelements or the like, such as the nucleotide composition according to the invention.
Examples of the food are dairy product (e.g. milk powder and liquid dairy product), puree, rice
flour or the like. According a specific embodiment, the infant food is in the form of dairy
product, for example milk powder or liquid dairy product, such as infant milk powder or liquid
dairy product added with the nucleotide composition according to the invention.
The term “infant” used herein generally refers to human subject of 0~3 years. Application of
the nucleotide composition according to the invention, however, is not limited to this year range.
If necessary, the nucleotide composition according to the invention or the food containing the
same may be administered to older human subject, for example 4, 5, 6, 7, 8, 9, 10 or older.
In another aspect, provided is a process for preparing a food, comprising adding the nucleotide
composition according to the invention into the food. In an embodiment, the food is an infant
food. In a preferable embodiment, the food is in the form of dairy product, for example the
forms of milk powder or liquid dairy product, such as the milk powder or liquid dairy product
for infant.
Throughout the description, the terms “liquid dairy product” and “liquid milk” have the same
meaning and are interchangeably used, referring to dairy product in liquid form and comprising
various nutrient ingredient and energy useful for human, such as those listed below.
According to an embodiment, when preparing the milk powder according to the invention, the
nucleotide composition according to the invention is used in an amount of 0.2~0.58 weight
portion (based on 1000 weight portion of milk powder). For example, the milk powder, based
on 1000 weight portion of the milk powder, comprise: skimmed milk powder 120~160 weight
portion, lactose 240~280 weight portion, desalted whey powder 180~210 weight portion, whey
protein powder (WPC34%) 90~120 weight portion, sunflower seed oil 155~180 weight portion,
corn oil 35~55 weight portion, soybean oil 40~60 weight portion, nucleotide composition
according to the invention 0.35~0.5 weight portion, soyabean lecithin 1~2.5 weight portion,
bifidobacteria 0.1~0.15 weight portion, oligofructose powder 4~5 weight portion,
galactooligosaccharide syrup 10~12 weight portion, nutriologically acceptable amount of
vitamins and nutriologically acceptable amount of microelement.
In a specific embodiment, the main process for preparing the milk powder according to the
invention mainly comprise: dosing, preheating, homogenizing, concentrating and sterilizing,
spray-drying, dry-mixing and obtaining the final product, wherein the nucleotide composition
according to the invention is added together with DHA, ARA and bifidobacteria into milk
powder after spray-drying, which are then mixed.
According to another embodiment, the liquid dairy product according to the invention comprise
the following ingredients (based on 100g liquid dairy product): protein beyond lactoferrin
1.75g ~4.26g, fat 1.75g ~4.97g, energy 250kJ ~355kJ, vitamin A 42.5μg ~191.7μg RE,
vitamin D 0.625μg ~ 2.6625μg, vitamin E≥0.375mg α-TE, vitamin K1≥2.5μg, vitamin
B1≥27.5μg, vitamin B2≥27.5μg, vitamin B6≥27.5μg, vitamin B12≥0.1μg, nicotinic acid (or
nicotinamide)≥275μg, folic acid≥2.5μg, pantothenic acid≥175μg, vitamin C≥4.5mg,
biotin≥1μg, sodium≤71mg, potassium 45mg ~ 244.95mg, copper 17.5μg ~ 124.25μg,
magnesium ≥3.5mg, iron 0.625mg ~1.775mg, zinc 0.25mg ~1.065mg, calcium ≥42.5mg,
phosphorus ≥20.75mg, iodine ≥3.5μg, chlorine ≤184.6mg, lactoferrin 5 ~ 13mg and
carbohydrate, wherein the amount of the nucleotides according to the invention is 2.64 ~
7.66mg and the amount of carbohydrate is such that the energy provided by carbohydrate,
protein and fat is 250kJ ~355kJ.
The fat used is provided by one or more of anhydrous milk fat, soybean oil, corn oil, sunflower
seed in any ratio and combination. In addition, to improve the properties of liquid milk system
like stability, some food acceptable additive for example emulsion stabilizer can be added.
Preferably, the food additive and the amount thereof herein comprise but not limited to one or
more of carrageenan 0.005wt% ~0.05wt%, glycerin monostearate 0.01wt% ~1wt%, guar gum
0.01wt% ~0.1wt%, locust bean gum 0.01wt% ~0.1wt% or any combination thereof.
Finally, provided is use of the nucleotide composition according to the invention for the
manufacture of food. In an embodiment, the food is an infant food. In a preferable embodiment,
the food is in the form of dairy product, for example the form of milk powder or liquid dairy
product, such as the milk powder or liquid dairy product useful for infant.
Immunostimulation
In a preferable embodiment, the nucleotide composition according to the invention and the food
containing the same can provide the effect of immunostimulation after consumption. The effect
of “immunostimulation” herein refers to the function of enhance the immunity in a subject, for
example in the aspects of improving transformation function of lymphocytes, enhancing
phagocytosis function of macrophagocytes, enhancing NK cells activity, improving immune
response and antibody production or the like, such those provided in the Examples of the
description. In a preferable embodiment, the subject is human and preferably human infant.
Promotion of growth and development and promotion of repair of intestinal tract after damage
The nucleotide composition according to the invention and the food containing the same can
promote growth and development (for example, promote development of gastrointestinal tract)
and promote repair of intestinal tract after damage in a subject after consumption. For example,
promotion of growth and development and mature of intestinal tract (e.g. small intestine), repair
of intestinal tract (e.g. small intestine) after damage, protection of intestinal tract (e.g. small
intestine) cells from the attack of free radical, reduction of intestinal tract (e.g. small intestine)
inflammation occurrence. The inventor, from the experiment in vitro found that the nucleotide
composition according to the invention can promote growth of enterocyte, promote
proliferation of enterocyte, and protect enterocyte and hepatocyte from damage. Such damage
is for example oxidative damage, such as that cause by reactive oxygen species. In a preferable
embodiment, the subject is human and preferably human infant.
Promotion of growth of intestinal beneficial microorganisms
The nucleotide composition according to the invention and the food containing the same can
promote growth of intestinal beneficial microorganisms (e.g. bifidobacteria and lactobacillus)
in a subject after consumption, for example in the aspects of being beneficial for growth of
bifidobacteria, increase of contents of bifidobacteria and lactobacillus in intestinal tract and
faeces (such bacterial flora can inhibit reproduction of acid-abominating pathogenic bacteria
and E. coli.), reduction of proportion of intestinal tract harmful bacteria, thereby benefiting the
health of the subject. The present inventor found that, addition of the composition into intestinal
tract condition or a similar condition in vitro, by observing the multiplication amount of
probiotics and lactobacillus in the environment, especially that of bifidobacteria, nucleotide
composition according to the invention show significant growth promotion to intestinal
beneficial microorganisms. In a preferable embodiment, the subject is human and preferably
human infant.
Example
In the Examples, the test samples have the components and proportions as follows (on the
weight basis):
Nucleotide composition according to the invention (also see claim 5):
Sample 1: CMP: 60%, AMP: 12%, UMP: 16% and GMP: 12%
Sample 2: CMP: 65%, AMP: 10%, UMP: 14% and GMP: 11%
Sample 3: CMP: 70%, AMP: 8%, UMP: 12% and GMP: 10%
Nucleotide composition of the prior art:
Comparative Sample 1: CMP: 33.7%, AMP: 20.3%, UMP: 23.1%, GMP: 7.6% and IMP: 15.3%
Comparative Sample 2: CMP: 60%, AMP: 14.5%, UMP: 18.2% and GMP: 7.3%
Comparative Sample 3: CMP: 42.7%, AMP: 13.4%, UMP: 24.2% and GMP: 19.7%
Example 1: immunostimulation effect (I)
The purpose of this example is to test the effect of immunostimulation of the nucleotide
composition according to the invention and the principle and steps are based on the
requirements and specification of “technical standards of examination and evaluation for
healthcare food (2003)”, Second part “functional test method” Chapter I.
1. Materials and methods
1.1. Main reagents
Main reagents used in this example: RPMI-1640 medium (Gibco); fetal calf serum (Gibco);
Concanavalin A (ConA) (Sigma); Lymphocyte Separation liquid, whole blood and tissue
diluent, cell wash solution (Tian Jin Hao Yang Biological Manufacture CO., LTD); 5% chicken
red blood cell suspension (prepared by the lab); MTT kit, Hank's solution (Beyotime Institute
of Biotechnology); Giemsa dye liquor (Zhuhai BASO Biotech CO., LTD); EDTA anticoagulant
tube (BD); 96-well cell culture plate (Corning); Baicheng colostrum capsule (Shanghai
Fuzheng Biotech CO., LTD); nucleotide samples (Nanjing Tongkaizhaoye Biotech CO., LTD).
1.2. Main instruments
CO incubator (Sanyo MCO-18AIC(UV)); clean bench (AIRTECH); inverted microscope
(OLYMPUS); Hitachi horizontal centrifuge (himac-CT6EL); microplate reader (BIO-RAD
Model 680); OLYMPUS microscope (BX51); DNP thermostatic incubator (Shanghai Jinghong
Lab instrument CO., LTD).
1.3.Animals
160 KM male mice of 5~6 weeks, provided by Shanghai SLAC Laboratory Animal Co., Ltd.
1.4. Animals grouping
The KM mice were adapted for 7 days and then randomly divided into 8 groups, 20 animals
each group: blank control group, positive control group, high, medium and low dosage groups
of Sample 1 and high, medium and low dosage groups of Comparative Sample 1. The animals
in each groups received gavage of sterile water dissolved with each sample (below) and the
blank control group received gavage of sterile water at the same volume, one administration
per day, for 30 days.
-1 -1
The high dosage group received a dose of 67.37 mg·kg ·d , the medium dosage group received
-1 -1 -1 -1
a dose of 41.82 mg·kg ·d , the low dosage group received a dose of 23.23 mg·kg ·d and the
-1 -1
positive control group received a dose of 150.17 mg·kg ·d .
The positive control group: colostrum capsule.
Test group: Sample 1 is nucleotide composition according the invention. Comparative Sample
1 is nucleotide composition which was prepared according to nucleotide components and
proportions of a commercially available infant food added with nucleotides.
1.5. Assay
Lymphocyte transformation assay:
At 38 day, 10 mice were randomly taken from each group. Blood was taken by removal of
eyeball with 2mL EDTA anticoagulant tube and lymphocytes were separated under sterilization
with Ficoll density gradient centrifugation. 1 mL anti-coagulate fresh blood was mixed
uniformly with whole blood and tissue diluent at 1:1, which was carefully added to the liquid
surface of lymphocyte separation liquid at the same volume. After centrifugation at 1500 rpm
for 15 min with horizontal centrifuge, cyclic milk-white lymphocyte layer was collected, which
was then washed twice with cell wash solution. RPMI-1640 full liquid medium was used to
prepare a concentration of 1×10 /mL, which was added into 96-well cell culture plate, 100 μL
each well and 6 wells per mice, wherein 3 wells were added with ConA (final concentration
5μg/mL) and other 3 wells without addition as control. After 68 h incubation in CO incubator
at 5%CO and 37°C, MTT (5mg/mL) solution (10 μL/well) was added, after a further
incubation for 4 h, 100 μL Formazan solution was added to each well, followed by further
incubation in CO incubator at 37°C until full dissolution of Formazan. OD value at 570 nm
was determined in microplate reader and Stimulation Index (SI) was calculated: SI = ConA
stimulation tube OD average/control tube OD average.
Enterocoelia macrophage phagocytosis assay:
At 38 day, 10 mice were randomly taken from each group. Each mouse was injected 1 mL of
% chicken red blood cell suspension at abdominal cavity. After 30 min, the animal was killed
by cervical dislocation. To abdominal cavity was injected 1 mL normal saline and the abdomen
was rubbed gently for 1 min. The skin of abdominal wall was cut with the open slot at muscular
layer. A tubularis was used to suck 1 mL of peritoneal fluid from abdominal cavity, which was
placed dropwise on a clean slide. The slide was placed in an enamel box with wet gauze pad
and then incubated in 37°C incubator for 30 min. The slide was taken out and washed to remove
supernatant and cells which were not attached to the slide and dried in the air under room
temperature. After fixation with 1:1 acetone/methanol solution, the slide was placed in Giemsa
dye solution for 15 ~30 min and then washed and dried in the air. Macrophages were counted
under high magnification, and 100 cells were counted per slide to calculate phagocytosis
percentage and phagocytosis index.
Phagocytosis percentage (%) = (number of macrophages taking chicken red blood cells /
number of counted macrophages) ×100
Phagocytosis index = (total number of chicken red blood cells which have been taken / number
of counted macrophages)
1.6. Statistical analysis
Data was shown as ±s, variance analysis was performed with SPSS 13.0 statistical software,
pairwise comparison was performed with SNK method, and test level is α=0.05.
2. Results
2.1. Influence of samples on lymphocyte proliferation
Table 1: Influence of samples on mice lymphocyte proliferation ( ±s, n=10)
Group Stimulation index (SI)
Blank control group 1.24±0.19
Positive control group 2.13±0.47
Sample 1 high dosage group 2.09±0.23
Sample 1 medium dosage group 1.87±0.26
Sample 1 low dosage group 1.46±0.34
Comparative Sample 1 high dosage group 1.51±0.18
Comparative Sample 1 medium dosage group 1.30±0.22
Comparative Sample 1 low dosage group 1.27±0.15
Note: significant as compared to blank control group (p<0.05)
According to table 1, SI values of high, medium and low dosage groups of Sample 1 were
significantly higher than that of blank control group (p<0.05) and showed dose dependence. SI
value of high dosage group of Comparative Sample 1 is significantly higher than that of blank
control group (p<0.05), SI values of medium and low dosage groups of Comparative Sample 1
were not significantly distinguished from those of blank control group (p>0.05). SI value of
low dosage group of Sample 1 is significantly higher than that of medium dosage group of
Comparative Sample 1 (p<0.05).
The results showed that the nucleotide composition according to the invention can significantly
enhance mice lymphocyte proliferation and showed a better effect over comparative sample.
2.2. Influence of samples on macrophages phagocytosis
Table 2: Influence of samples on mice macrophage phagocytosis ( ±s, n=10)
Grouping Phagocytosis percentage (%) Phagocytosis index
Blank control group 35.2±4.2 0.43±0.06
Positive control group 52.7±8.6 0.62±0.10
Sample 1 high dosage group 50.1±2.5 0.59±0.01
Sample 1 medium dosage group 48.7±3.2 0.57±0.13
Sample 1 low dosage group 46.7±3.1 0.53±0.07
Comparative Sample 1 high dosage group 49.8±2.0 0.50±0.22
Comparative Sample 1 medium dosage
34.9±1.7 0.44±0.12
group
Comparative Sample 1 low dosage group 35.1±3.9 0.42±0.19
Note: significant as compared to blank control group (p<0.05)
According to table 2, phagocytosis percentage and phagocytosis index of high, medium and
low groups of Sample 1 are significantly higher than those of blank control group (p<0.05).
Phagocytosis percentage and phagocytosis index of high dosage group of Comparative Sample
1 are significantly higher than those of blank control group (p<0.05). Phagocytosis percentage
and phagocytosis index of medium and low dosage groups of Comparative Sample 1 are not
significantly distinguished from those of blank control group (p>0.05). Phagocytosis
percentage and phagocytosis index of low dosage group of Sample 1 are significantly higher
than those of medium dosage group of Comparative Sample 1 (p<0.05).
The results showed that the nucleotide composition according to the invention can significantly
enhance mice enterocoelia macrophage phagocytosis function and showed a better effect over
comparative sample.
3. Conclusion
According to the experiment results, nucleotide composition according to the invention can
significantly improve lymphocytes transformation function, enhance enterocoelia macrophage
phagocytosis function, whereby showing the effect of enhance immunity function and such
immunoregulation effect is significantly better than comparative sample.
In another aspect, more potent immunoregulation effect means the same or similar effect can
be achieved with less dosage, which shows a significant advantage with respect to cost in large
scale industrial production.
On this basis, the above experiments are repeated, wherein Sample 1 were replaced with Sample
2 and Sample 3. As for Sample 2 and Sample 3, the results similar as Sample 1 were obtained.
Example 2: Immunostimulation effect (II)
The purpose of this example is to test the effect of immunostimulation of the nucleotide
composition according to the invention and the principle and steps are based on the
requirements and specification of “technical standards of examination and evaluation for
healthcare food (2003)”, Second part “functional test method” Chapter I.
1. Materials and methods
1.1. Main instruments
high speed refrigerated centrifuge (SIGMA 3-30K), shaker (Vortex4 digital), pipettor
(eppendorf, Germany), Whole blood cell analyzer (Urit, U-2900PLUS), CO incubator
(Thermo 311), microplate reader (Biotek H4), spectrophotometer (Shanghai JingKe 722s),
clean bench (Shangyu Xingxing Instrument CO., LTD SW-CJ-2D), autoclave sterilizer (Sanyo
Japan MLSPC 75L), cryogenic refrigerator (Hangzhou Aipu Instrument CO., LTD DW-
40L058), inverted microscope (Nikon Japan Eclipse Ti-S), vernier caliper (Standard Gage, US),
electronic scales (Sartorius BSA124S-CW), Toe Volume Meter (Jinan Yiyan Science
Development CO., LTD YLS-7C), Hemolytic plaque automatic image analyzer (Beijing Antai
Yongxin Medical Technology CO., LTD AT-Spot 5100), flow cytometry (BD, US
FACSCalibur).
1.2 Main reagents
cyclophosphamide (Jiangsu Hengrui Medicine CO., LTD), Dipotassium ethylene diamine
tetraacetate (Beijing Reagent factory), RpMI1640 liquid cell medium (Shanghai Yuanlong
Biology Technology Company), bovine calf serum, 2-mercaptoethanol, penicillin,
streptomycin, ConA solution (Beijing Mengyimei Biology Technology CO., LTD), sterile
Hank’s solution, BrdUp marker solution (Beijing Mengyimei Biology Technology CO., LTD),
dinitrofluorobenzene (Beijing Qingshengda Chemical Engineering Technology CO., LTD),
barium sulfide, sheep red blood cell (SRBC), guinea pig serum, Indian ink (Shanghai Yuanmu
Biotechnology CO., LTD), YAC-1 cell (Shanghai Enzyme Research Biotechnology CO., LTD),
Tris-HCL buffer (Beijing Mengyimei Biology Technology CO., LTD).
1.3 Animals
770 healthy male BALB/c mice of 3-4 years and weight of 11-13g were provided by Beijing
Huafukang Bioscience CO., LTD and were accommodated in level 2 Animal house of Chinese
Academy of Medical Science, Union Institute of Materia Medica: room temperature (25±2°C),
relative humidity (55±2)%, 12h/12h illumination, free access to food and water, 4-5 animals
per cage. The test was initiated after adaption for 3 days.
770 animals were fed in 5 big immune groups, 154 in each big group, which was randomly
divided into 11 groups (high and low dosage for 5 nucleotide samples, 10 groups in total and
one group as control group), 14 in each group. Immune group 1: delayed type hypersensitivity;
Immune group 2: mice lymphocyte transformation assay, NK cell activity assay, ratio of the
organs to body; Immune group 3: half value of hemolysis, antibody producing cell number;
Immune group 4: carbon clearance test; Immune group 5: mice abdominal cavity macrophage
taking fluorescent microsphere assay. Each sample was administered by gavage for 28 days,
once per day, with the gavage volume as 0.2 mL/10g. Control group received distilled water by
gavage. After administration, the mice were killed and determined for various immune
indicators.
1.4 Test samples
Samples 1-3 were nucleotide composition according to the invention. Comparative Samples 2
and 3 were other two nucleotide compositions disclosed in the prior art. Each sample was set
with low and high dosage groups as 120.7 mg/kg and 1207.0 mg/kg, respectively.
1.5 Test items and indicator
1.5.1 Organs/body weight ratio: The mice were weighed initially and at 28 day after
administration as initial weight and final weight. The mice were killed by dislocation and spleen
and thymus were taken and removed off fascia. The bloodiness on the surface was sucked by
filter paper and the organs were weighed to calculate spleen/body weight ratio and thymus/body
weight ratio.
1.5.2 Delayed type hypersensitivity(toe thickening, DTH): After successive administration for
28 days, each mouse was injected in abdominal cavity 2% packed-cell volume of SRBC (v/v,
formulated in normal saline) SRBC 0.2 mL. Four days after sensitization, the thickness of left
rear foot plantar was measured and average value was calculated according to 3 measurements
at the same site. At the measuring site, 20 μL of 20% SRBC was injected subcutaneously. 24 h
after injection, the thickness of left rear foot plantar was measured and average value was
calculated according to 3 measurements. The difference in thickness before and after the
challenge (swelling degree of toe) was used to present DTH degree.
1.5.3 Mice lymphocytes transformation induced by ConA (MTT method): After successive
administration for 28 days, the mice were killed. After sterilization in 75% alcohol in beaker,
the spleen was taken under sterile condition and placed in a dish with four layers of 3 cm×3 cm
gauze (autoclaved), to which was added appropriate amount of Hank’s solution. The spleen was
packaged with gauze and ground with elbow forceps to prepare single cell suspension, which
was washed twice with Hank’s solution and centrifuged at 1000 rpm for 10 min. The cells were
suspended in 2 mL full liquid medium and viable cells were counted to adjust the cell
concentration to be 5×10 /mL. The cell suspensions were added into 24 well culture plate in
duplicated wells, 1 mL in each well. 75 μL of ConA solution (corresponding to 7.5 μg/mL) was
added into one well and the other well was used as control. The cells were incubated at 5% CO ,
37°C for 72 h. 4 h prior to end of incubation, 0.7 mL supernatant was sucked gently from each
well and then 0.7 mL of RPMI 1640 liquid culture medium free of bovine calf serum was added.
At the same time, MTT (5 mg/mL) 50 μL/well was added and incubation was continued for 4
h. After incubation, 1 mL acidic isopropanol was added to each well with beating for
homogeneity such that purple crystal was dissolved completely. The system was split into 96
well culture plate, each well having 3 parallel wells. Optical density value at 570 nm was
determined with enzyme linked immunosorbent detector. Proliferation capacity of lymphocyte
was expressed as optical density value of well with ConA subtract that of the well without ConA.
1.5.4 Antibody producing cell assay: After successive administration for 28 days, sheep blood
was washed three times with normal saline and each mouse was injected in abdominal cavity
2% packed-cell volume (v/v, formulated in normal saline) of SRBC 0.2 mL for immunization.
Four days after immunization with SRBC, the mice were killed and spleen was taken to prepare
a cell suspension of 5×10 cells/mL. Agarose was heated for dissolution and mixed with double
amounts of Hank’s solution, which was then split into tube, 0.5 mL in each tube. Into the tube
was added 20% packed-cell volume (v/v, formulated in normal saline) of SRBC 50 μL, spleen
cell suspension 200 μL. After quick mixing, the mixture was poured on 6 well plate coated with
agarose thin layer. After solidification of the agar, the plate was placed in CO incubator for 1h,
then complement diluted with SA buffer (1:10) was added and incubation was performed for 2
h. Number of hemolytic plaque was counted.
1.5.5 Serum hemolysin half value of hemolysis (HC ): After successive administration for 28
days, sheep blood was washed three times with normal saline and each mouse was injected in
abdominal cavity 2% packed-cell volume (v/v, formulated in normal saline) of SRBC 0.2 mL
for immunization. After four days, blood was taken by removal of eyeball into 1.5 mL
centrifuge tube, which was placed at 4°C for 1 h to allow sufficient separation of serum. The
tube was centrifuged at 2000rpm for 10 min and serum was collected. The serum was diluted
100 folds with SA buffer. The diluted serum was added into 96 well plate, 100 μL in each well,
to which was successively added 10%(v/v) SRBC 50 μL, complement 100 μL diluted with SA
buffer (1:8). The place was incubated in 37°C thermostatic water bath for 30min and then
centrifuged at 1500 rpm for 10 min. 50 μL of supernatants from sample wells and blank control
well were added into another 96 well plate, to which was added 150 μL of VanKampen-Zijlstra’s
reagent. At the same time, half hemolysis well was set, to which was added 10%(v/v)SRBC
12.5 μL and then VanKampen-Zijlstra’s reagent to 200 μL. The mixture was mixed
homogenously with a shaker and allowed to stand for 10 min. Optical density values of each
wells at 540 nm were determined with Automatic microplate reader.
The amount of hemolysin is shown as half value of hemolysis (HC ) and calculated as:
Sample HC = (sample optical density value/ optical density value upon half hemolysis of
SRBC) × dilution ratio
1.5.6 Mice abdominal cavity macrophage taking fluorescent microsphere assay: After
successive administration for 28 days, four days before end of gavage, each mice was injected
0.2 mL of 2% SRBC to activate macrophage. On the day of test, mice were killed with
dislocation of the cervical spine, of which the abdominal cavity was injected Hank's solution
added with bovine calf serum, 3mL/animal. The abdomen was kneaded softly for 20 times to
wash macrophages from abdominal cavity sufficiently. The abdominal wall was cut with a small
open slot, and 2 mL of abdominal washing liquid was sucked and filtered into tube with 75 m
filter. Macrophages number was adjusted to 4~6×10 /mL. A pipettor was used to transfer 1 mL
abdominal washing liquid into 6 well culture well, to which was added preconditioned
fluorescent microspheres (1×10 /plate). The plate was incubated in CO incubator at 37°C in
dark for 120 min. After incubation, the supernatant (containing cell which were not adherent
and excess fluorescent microspheres) was discarded. 1.0 mL of PBS buffer was used to wash
gently for two times. After removal of supernatant, 0.3 mL PBS buffer at 4°C was added. The
adherent cells were scraped with cell scraper and filtered with 75 μm filter after gentle beating.
The cells were ready for analysis.
The results were calculated as
phagocytosis percentage (%) = (number of macrophages taking fluorescent microspheres /
counted number of macrophages) ×100
phagocytosis index = (total number of fluorescent microsphere which have been taken / counted
number of macrophages)
1.5.7 NK cell activity assay (lactic dehydrogenase (LDH) method):
After successive administration for 28 days, 24 h before test, the target cells YAC-1 were
subjected to passage culture. Before use, the cells were washed twice with Hank’s solution and
adjusted to the concentration of 1×10 /mL(target cell) with RPMI 1640 full liquid medium
containing 10% bovine calf serum. The mice were killed by cervical dislocation and spleen was
taken under sterile condition to prepare spleen cell suspension, which was washed twice with
Hank’s solution, centrifuged at 1000 rpm for 10 min and resuspended with 2 mL of RPMI 1640
full liquid medium containing 10% bovine calf serum. Trypan blue was used to perform viable
cells dyeing counting (viable cells should be over 95%) and the cell concentration was adjusted
to 1×10 /mL(effector cell ) such that the ratio of effector cells to target cells was 100:1. 100 μL
of target cells and effector cells were respectively added into U shape 96 well culture plate. The
target natural releasing well was added with target cells and liquid medium 100μL, respectively
and the target maximum releasing well was added with target cells and 1% NP40 100μL,
respectively. These wells were set with three parallel wells and incubated in 5% CO incubator
at 37°C for 4 h. 96 well culture plate was centrifuged at 1500 rpm for 5 min, 100 μL of
supernatant was sucked from each well and placed in flat 96 well culture plate, to which was
added 100 μL of LDH base solution. The reaction was performed for 3 min and to each well
was added 30 μL of 1 mol/L HCl solution to quench the reaction. OD value at 490 nm was
determined with microplate reader and NK activity was calculated according to the following
formula.
NK cell activity % = (reaction well OD – natural releasing well OD) / (maximum releasing well
OD - natural releasing well OD) × 100%
1.5.8 Carbon clearance assay: The animals were successively administered for 28 days and
weighed. Indian ink was injected through tail vein. 2, 10 min after ink injection, 20 μl of blood
was taken and added into 2 ml of 0.1% sodium carbonate solution and OD value at 600 nm was
determined. The mice were killed and liver and spleen were taken, and the bloodiness on the
surface was sucked by filter paper and the organs were weighed.
Phagocytosis index was used to present the mice carbon clearance capacity and calculated
according to the following formula:
LgOD1 −LgOD2
t2 −t1
bodyweight
phagocytosis index = κ
liverweight +spleenweight
1.6 Criteria: As for the four major aspects of cellular immunity, humoral immunity, monocyte-
macrophage function and NK cell activity, if any two aspects had positive results, the sample
should be considered as having the function of enhance immunity. In cellular immunity function
assay items, if two test results were both positive, or two dosage group of any one test had
positive result, the cellular immunity function assay had positive result. In humoral immunity
function assay items, if two test results were both positive, or two dosage group of any one test
had positive result, the humoral immunity function assay had positive result. In monocyte-
macrophage function assay items, if two test results were both positive, or two dosage group of
any one test had positive result, the monocyte-macrophage function assay had positive result.
As for NK cell activity assay, if one or more dosage group had positive result, the NK cell
activity assay had positive result.
2. Results:
After oral administration of test samples for 28 day, the immune function items measurements
were as follows:
Body weight and organs ratios were not significantly distinguished from normal groups.
The positive and negative results were shown in Table 3.
Table 3
Monocyte-macrophage
cellular immunity humoral immunity
function
Spleen Carbon NK cell
Grouping Delayed type Antibody Half value of macrophage
lymphocyte clearance activity
hypersensiti producing hemolysis phagocytosis
transforma assay
vity cells HC50 assay
tion assay
Sample 1
positive negative positive positive positive negative positive
low dosage
Sample 2
negative negative negative positive positive positive positive
low dosage
Sample 3
negative negative negative negative negative positive negative
low dosage
comparative
Sample 2 negative negative negative negative negative negative negative
low dosage
comparative
Sample 3 negative negative negative negative negative positive positive
low dosage
Sample 1
positive negative positive negative positive positive positive
high dosage
Sample 2
negative negative negative negative positive positive positive
high dosage
Sample 3
negative negative negative negative positive positive positive
high dosage
comparative
Sample 2 positive negative negative negative positive negative positive
high dosage
comparative
Sample 3 positive negative negative negative positive positive positive
high dosage
According to the results in Table 3, from the criteria for the four major aspects (i.e. the four
major aspects in section 1.6), Samples 1, 2, 3 and Comparative Sample 3 were considered as
positive and thus had the immunostimulation effect. On the contrary, Comparative Sample 2
was not considered as positive.
On this basis, as for Samples 1-3 and Comparative Sample 3, from point of the above dosage
groups (section 1.6), the nucleotide composition according to the invention had more
immunostimulation positive indicators, showing more potent and more comprehensive
immunostimulation effect. For example, 1) Sample 1 had the cellular immunity effect, while
other samples did not; 2) Sample 1 had humoral immunity effect, while other samples did not;
3) Sample 1 and 2 were positive for carbon clearance assay, while other samples were not; 4)
Samples 2, 3 and Comparative Sample 3 had macrophage phagocytosis function, while other
samples did not; 5) Sample 1, 2, 3 and Comparative Sample 3 were positive for NK cell activity.
From the above results, the nucleotide composition according to the invention was effective in
stimulation of immunity function and showed better effect over the compositions of the prior
art. Likewise, as stated above in Example 1, more potent immunoregulation effect means the
same or similar effect can be achieved with less dosage, which shows a significant advantage
with respect to cost in large scale industrial production.
Example 3: Protection from damage and repair of the nucleotide composition
The purpose of this example is to test the effect of the nucleotide composition according to the
invention in providing protection from damage and repair.
1. Materials and methods
1.1 Instruments and reagents
RPMI-1640 medium and DMEM high sugar medium were purchased from Gibco. 5’-AMP
(A1752), 5’-CMP-Na (C1006), 5’-GMP-Na (G8377), 5’-UMP-Na (U6375), Thiazolyl Blue
2 2 2
(MTT), insulin were purchased from Sigma. Fetal calf serum was purchased from Sijiqin
Hanzhou. The kits used in the example such as LDH, SOD, MDA kits were all purchased from
Nanjing Jiancheng Bioengineering Institute. Trypsin, double-antibody, BCA protein assay kit
were purchased from Beyotime. Other reagents such as dimethylsulfoxide (DMSO), hydrogen
peroxide were domestic products with analytical purity and were purchased from Sinopharm
Chemical Reagent CO., LTD, Shanghai. 25 cm culture bottle, 96 well cell culture plate, 6 well
cell culture plate, 60 mm cell culture dish, 50 mL centrifuge tube, 15 mL centrifuge tube were
purchased from Corning, US. Disposable Syringe Filter was purchased from Millipore. The
main instruments were Heal Force biosafety cabinet (Heal Force Bio-Meditech Holdings
Limited, Hongkong, China), Countstar cell counter (Inno-Alliance Biotech, US), EVOS FL
fluorescence microscope (Thermo Fisher), microplate reader (Finnpipette), 721 visible
spectrophotometer (Shanghai INESA Analytical Instrument CO., LTD), thermostatic water bath
(Shanghai Boxun), ultrasonic cracker (Ningbo Xinzhi Bioscience CO., LTD), inverted
microscope (Nikon, Japan), refrigerated centrifuge (Thermo Fisher).
1.2 Materials
Rat small intestinal crypt epithelial cell (IEC-6 cell) was obtained from Chinese Academy of
Medical Science, Cell Research Institute. Rat normal hepatocyte (BRL 3A Cell) was obtained
from Chinese Academy of Sciences, Cell bank at Shanghai.
1.3 Test sample
Samples 1-3 were nucleotide composition according to the invention. Comparative Samples 2
and 3 were nucleotide compositions disclosed in the prior art (above). Each sample was set with
three concentrations of low, medium and high (62.5, 250, 1000 μmol/L, respectively).
1.4 Model and indicators
Oxygen damage is one of the most common and typical damage to living body (e.g. intestinal
tract damage). It has been known that many diseases like those of digestive system of human
and animals are closely associated with radical and reactive oxygen species, for example,
oxygen damage has been shown to be one of the causes of inflammatory bowel disease. The
redox reaction in cell is kept balance under normal physiological status and oxygen radicals and
antioxidant system in body are important for the balance. If oxygen radicals are produced
greatly or the function of antioxidant system is reduced, damage will be caused to tissue cells.
SOD (superoxide dismutase) is an important antioxidase, which can clear O produced by
peroxide and protect cells from damage and thus is essential to oxidation/antioxidant balance
in cell. As for the damage caused by oxide/peroxide, lactic dehydrogenase (LDH) is the
indicator for cell membrane integrity or cell necrosis. In addition, oxide/peroxide in cells can
attack polyunsaturated fatty acids in biofilms, which leads to lipid peroxidation and formation
of lipid peroxide, for example malondialdehyde (MDA). Oxygen radicals not only cause cell
damage by peroxidation of polyunsaturated fatty acids in biofilms, but lead to cell damage by
decomposition product of lipid hydroperoxide, and thus the amount of MDA can generally
reflect the degree of lipid peroxidation, thereby showing the degree of cell damage. Therefore,
the concentrations, activities or changes thereof of the substances and cell survival rate can be
used to evaluate the protective effects of the nucleotide composition according to the invention.
See, “Study on Healing effect of Rheum Tanguicum Polysaccharides (RTP) on intestinal
epithelial cell injury and its mechanism”, Thesis of Ph. D. Liu Lin-na, Fourth Military Medical
University, May 1, 2005; “Protection of Longyanshe Polysaccharide on CCl – induced Damage
of Primary Cultured Hepatocytes in Rats”, Duan Xiaoqun et. al, China Pharmacy, 2006 vol 17,
No 15, pp 1132-1143; “In vitro Protective Effects of Baoganning On Injury of Liver Cells
Induced by Hydrogen Peroxide in Rats” Zhao Jinjun et. al, Journal of Guangzhou University of
Traditional Chinese Medicine, 2002, vol 19, No 3, pp 211-213.
In addition, according to the inventor’s experiments, H O at 600 μmol/L will significantly
lower survival rate of BRL 3A cells (P <0.01). Therefore, in oxidative damage test of BRL 3A,
H O at 600 μmol/L was used to induce cell oxidative damage. Similarly, H O at 100 μmol/L
2 2 2 2
was used to induce cell oxidative damage for IEC-6 cell.
According to the above information, in this example, the protective effect of the composition
according to the invention on cell oxidative damage was investigated by treating cells with
nucleotide composition and inducing cell oxidative damage via H O . For BRL 3A cell, cell
survival rate and SOD activity were tested. For IEC-6 cell, LDH activity and MDA content
were tested.
1.5 Cell culture and sample preparation
IEC-6 cell was cultured in 37°C, 5%CO incubator with RPMI-1640 medium containing
5%FBS, 2 mg/L insulin and passage was performed every four days. BRL 3A was cultured in
37°C, 5%CO incubator with DMEM high sugar medium containing 10%FBS and passage was
performed every five days. The nucleotide composition was formulated as 50 mmol/L of stock
solution with PBS, which was filtered for sterilization and stored at -20°C for use.
1.6 Procedures
As for cell survival rate, the cells at logarithmic phase were inoculated in 96 well plate at 5000
/well. After adherence, the cells were incubated with medium containing various concentrations
of nucleotide compositions for 24 h (each concentration set with 6 repeats). In the test, there
were normal control without H O and positive control with H O only. After 24 h, H O was
2 2 2 2 2 2
added to induce cell oxidative damage, respectively. After 2h, MTT method was used to
determine cell survival rate. Cell survival rate = OD value of test group / OD value of normal
control ×100%.
For other indicators, the cells at logarithmic phase were inoculated in 60 mm cell culture dish
at 7×10 /dish. After adherence, the cells were incubated with medium containing various
concentrations of nucleotide compositions for 24 h (each concentration set with 3 repeats). In
the test, there were normal control without H O and positive control with H O only. After 24
2 2 2 2
h, H O of a certain concentration was added to induce cell oxidative damage, respectively and
culture was continued. After 2 h, liquid medium was sucked and centrifuged and the supernatant
was taken for relevant analysis. The cells were digested with trypsin and washed with PBS once
after centrifugation and then resuspended in 700 μl PBS, which were then cracked with
supersonic and tested for all the indicators within 2 days.
1.7 Data analysis
All the data were shown as average ±standard deviation ( x ±s) and statistics analysis was
performed with SPSS17.0 software, inter-group difference comparisons utilized one-way
analysis of variance. P<0.05 means statistical significance and P<0.01 means high statistical
significance.
2. Results
2.1 Cell survival rate assay
Cell survival rate results of BRL 3A cell were shown in Figure 1. According to this Figure,
H O treatment significantly lowered the cell survival rate of BRL 3A (P <0.01). Treating cells
with nucleotide composition samples, cell survival rates were increased in various degrees,
especially Samples 1 and 2. Statistical results showed, for all the concentration groups of
Sample 1, low and medium concentration groups of Sample 2 and low concentration group of
Sample 3, the cell survival rates were significantly higher than those of H O control (P <0.01
or P <0.05). In addition, for all the concentration groups of Sample 1 and low concentration
group of Sample 2, the cell survival rates were significantly higher than those of all the
concentration groups of Comparative Samples 2 and 3 (P <0.01 or P <0.05). Accordingly,
treatment of cells with the nucleotide composition according to the invention can effectively
increase of survival rate the cell with oxidative damage, indicating protective effect against
oxidative damage and further showing promotion repair of cells after damage. Moreover, the
protective effect is better than the nucleotide composition of the prior art.
2.2 SOD activity assay
SOD activity results of BRL 3A cell were shown in Figure 2. According to this Figure, H O
treatment significantly lowered the SOD activities in cells (P <0.01). Treating cells with
nucleotide composition samples, SOD activities in cells were increased in various degrees. For
all the concentration groups of Sample 1 (P <0.05), medium concentration group of Sample 2
(P <0.05), medium and high concentration groups of Sample 3 (P <0.01), the SOD activities
in cells were significantly higher than those of H O control. Moreover, for medium
concentration group of Sample 3, the SOD activities in cells were significantly higher than
those of medium and high concentration groups of Comparative Sample 2 (P <0.05) and all
concentration groups of Comparative Sample 3 (P <0.05). As for high concentration group of
Sample 3, SOD activities in cells were significantly higher than those of high concentration
group of Comparative Sample 2 (P < 0.05) and low and high concentration groups of
Comparative Sample 3 (P <0.01). Accordingly, the nucleotide composition according to the
invention can significantly increase concentration of antioxidative active substance (e.g. SOD)
in cells with oxidative damage, indicating protective effect against oxidative damage. Moreover,
the protective effect is better than the nucleotide composition of the prior art.
2.3. LDH activity assay
LDH activity results of IEC-6 cell were shown in Figure 3. According to this Figure, H O
treatment significantly made LDH content in liquid medium supernatant higher than that of the
normal control (P < 0.01). Treating cells with nucleotide composition, LDH contents in
supernatant were lowered in various degrees. As for low concentration group of Sample 2 and
medium concentration group of Sample 3, the LDH content decreased most significantly,
showing no statistical difference as compared to normal control. Additionally, as compared to
H O control, in medium concentration group of Sample 1 (P < 0.05), low and high
concentration groups of Sample 2 (P <0.01 or P <0.05), low and medium concentration groups
of Sample 3 (P <0.01), the LDH contents were lowered significantly. Moreover, as compared
to high concentration of Comparative Sample 2, the LDH contents of low concentration group
of Sample 2 (P <0.05) and low and medium concentration group of Sample 3 (P <0.01) were
lowered significantly. Accordingly, the nucleotide composition according to the invention can
effectively lower the concentration of the substance indicative of undesired indicator (e.g. LDH)
in supernatant of cells with oxidative damage, indicating protective effect against oxidative
damage. Moreover, the protective effect is better than the nucleotide composition of the prior
art.
2.4. MDA concentration assay
MDA concentration results of IEC-6 cell were shown in Figure 4. According to this Figure,
H O treatment of cells significantly increased MDA contents in cells (P <0.01). Treating cells
with nucleotide composition samples, as for all concentration groups of Samples 1-3, MDA
contents are decreased in some degree and were significantly lower than H O control. On the
contrary, medium and high concentration groups of Comparative Samples 2 and 3 were not
significantly distinguished from H O control. Accordingly, the nucleotide composition
according to the invention can effectively lower the concentration of the substance indicative
of undesired indicator (e.g. lipid peroxide MDA) in cells with oxidative damage, indicating
protective effect against oxidative damage. Particularly, the nucleotide composition according
to the invention can show significant and consistent protective effects under all the
concentration ranges. On the contrary, the nucleotide composition in the prior art cannot show
protective effect under relatively high concentrations (e.g. medium and high concentrations).
This fact further shows advantage of the nucleotide composition according to the invention over
the prior art.
3. Discussion
According to the above results, treatment with the nucleotide composition according to the
invention can increase survival rate of the cells with oxidative damage, increase
activity/concentration of the factor with protective effect from oxidation (e.g. SOD) in cells,
lower activity/concentration of the factor indicative of undesired effect (e.g. LDH and MDA).
Such facts means the nucleotide composition according to the invention can provide protective
effect against cell oxidative damage and the effect is better than the nucleotide composition in
the prior art. Meanwhile, by increasing of the activity of the factor with protective effect and
lower the content of undesired factor, the nucleotide composition can promote recovery of the
balance condition of cell before damage, whereby promoting repair after damage. Moreover,
effects of the nucleotide composition according to the invention can not only be shown on
enterocytes but also on other part of the digestive system (e.g. liver), thereby showing positive
effect on the whole digestive system.
Example 4: Cell proliferative effect
The instruments and reagents used in this example refer to example 3, wherein IEC-6 cell was
used to verify promotion of cell proliferation of the nucleotide composition according to the
invention.
In this example, MTT method was used to detect the influence of nucleotide composition on
IEC-6 cell. IEC-6 cells at logarithmic phase were digested with trypsin, which after
centrifugation were prepared into cell suspension. Using cell counter, the cells were inoculated
in 96 well plate at 5000/well, which was incubated overnight at 37°C, 5%CO such that the
cells were adherent. The liquid medium was exchanged the next day and each well was added
with 200 μL of cell liquid medium containing various concentrations of nucleotide
compositions (each sample was set with three concentrations of 62.5, 250, 1000 μmol/L). Each
concentration of each sample was set with 6 repeats. Control group without addition of
nucleotide composition and zero well without cells (only addition of normal medium) were also
set. The liquid medium was exchanged every 24 h. At 24 h of nucleotide composition treatment,
to each well of 96 well plate was added 10 μL of MTT with gentle shake and the plate was
incubated in incubator for 4 h. The supernatant was discarded after 4 h and 150 μL/well of
DMSO was added. The plate was shaked in dark. After dissolution of bluish violet formazan
crystal, the plate was placed in microplate reader and OD value was determined at 490 nm
wavelength. Zero was set with the blank group without cell and curve was drawn with OD value.
All the data were shown as average ±standard deviation ( x ±s) and statistics analysis was
performed with SPSS17.0 software, inter-group difference comparisons utilized one-way
analysis of variance. P<0.05 means statistical significance and P<0.01 means high statistical
significance.
The results were shown in Figure 5. According to this Figure, after addition of nucleotide
sample, proliferation of IEC-6 cell was significantly changed as compared to control. As for
high concentration group of Sample 1, all concentration groups of Sample 3, the cell
proliferation speeds were significantly higher than those of normal control (P <0.01); the cell
proliferation speed of medium concentration group of Sample 2 was faster than normal control
(P <0.05). The cell proliferation speed of low concentration group of Comparative Sample 2
was significantly lower than those of high concentration group of Sample 1, low and medium
concentration groups of Sample 2, low, medium and high concentration groups of Sample 3 (P
<0.01). The cell proliferation speed of high concentration group of Sample 2 was significantly
higher than that of low concentration group of Comparative Sample 2 (P <0.05). Accordingly,
the nucleotide composition according to the invention can promote cell proliferation, especially
promote intestinal tract cell proliferation and growth, thereby showing the effect of promotion
of enterocytes growth and enhance enterocytes proliferation. The effect was better than the
nucleotide composition in the prior art.
Example 5: Effect on intestinal tract beneficial bacterial flora
This example shows the nucleotide composition according to the invention has stimulation on
intestinal tract beneficial bacterial flora. Each sample was set with 2 concentrations (low dosage:
1g/100ml; high dosage: 2g/100ml) and there was no nucleotide in control group.
1. Materials and methods
1.1. Main reagents
eosin methylene blue agar (EMB); Tryptose Sulfite Cycloserine Agar (TSC); Bifidobacterium
Agar medium (BBL); sodium hydroxide; hydrochloric acid; Egg Yolk Emulsion 50%; D-
cycloserine; nucleotide samples (Nanjing Tongkaizhaoye).
1.2. Main instruments
Automatic vertical steam sterilizer (LDZX-30FBS, Shanghai Shenan medical apparatus
factory); CO incubator (HH.CP-TW9, Shanghai Shenxian Thermostatic Equipment);
electronic scales (XS1003S, Swiss); clean bench (VS-130L-U, Suzhou Antai Airtech CO.,
LTD.); Colony Counter (DIGITAL S 4905000, Selecta Spain); Sealed culture box (C-32,
Mitsubishi Japan).
2. Procedures
2.1. Obtaining intestinal tract flora
In sterile bench, 0.5 g of faeces sample were taken from the anus of five SPF mice and placed
in sterile tube, to which was added three sterile glass beads. The mixture was subjected to vortex
and then to gradient dilution to 10 and used as seed solution for further test.
2.2. Preparation of liquid medium
Beef extract peptone liquid medium was used as base medium, in which nucleotide samples at
1 g/100 ml, 2 g/100 ml were added and the seed solution prepared in section 2.1 was added at
1% (volume).
2.3. Bacterial flora counting
With specific selective medium (BBL agar), the Bifidobacteria in liquid medium was cultured
under anaerobic condition for 48 h at 36±1°C and then bacterial flora counting was performed
and expressed as Lg CFU/ml.
2.4. Data statistic
Statistics analysis was performed with SPSS17.0 software and one-way analysis of variance
was used to compare the data of various groups, wherein α=0.05 was used as criterion for
difference determination. The results were shown in Table 4.
Table 4
Grouping Dosage Bifidobacteria
Control group 7.00±0.00
low dosage 7.15±0.17
Sample 1
high dosage 8.30±0.35*
low dosage 7.89±0.47*
Sample 2
high dosage 8.24±0.28*
low dosage 7.63±0.17*
Sample 3
high dosage 7.80±0.23*
* Significant increase
Statistical results showed, taking Bifidobacteria as an example, after treatment with the
nucleotide composition according to the invention, growth of Bifidobacteria was significantly
increased as compared to control (Lg CFU), indicating that the nucleotide composition
according to the invention can stimulate growth of intestinal tract beneficial bacterial flora.
Example 6: Infant milk powder preparation (1000 kg, dry addition)
skimmed milk powder
Raw materials of the milk powder according to the invention were:
, lactose , desalted whey powder , whey protein powder (
140 kg 260 kg 200 kg WPC34%)
100 kg corn oil 52 kg, soybean oil 42 kg, sunflower seed oil 170 kg, oligofructose powder 4.8
mixed nutrients 6.4 kg and
kg, galactooligosaccharide syrup 12 kg, soyabean lecithin 2 kg.
After homogenous mixing, the above raw materials were subjected to pasteurization,
homogenization, evaporation and concentration and spray drying to form semiproduct as
0.38 kg 0.1
powder, to which was added nucleotide composition according to the invention,
kg Bifidobacteria, 4.32 kg DHA, 6.0 kg ARA. After mixing with dry mixer, the
uniformly mixed milk powder was packed with nitrogen to obtain the final product.
Example 7: Infant milk powder preparation (1000 kg, dry addition)
Skimmed milk powder , lactose , desalted whey powder , whey
150 kg 250 kg 190 kg
protein powder ( ,
WPC34%) 110 kg corn oil 44 kg, soybean oil 44 kg, sunflower seed oil 175
, mixed nutrients 7 kg,
kg, oligofructose powder 4.5 kg, galactooligosaccharide syrup 11 kg
2 kg soyabean lecithin were mixed uniformly and then subjected to pasteurization,
homogenization, evaporation and concentration and spray drying to form semiproduct as
0.5 kg 0.1 kg
powder. Then nucleotide composition according to the invention,
Bifidobacteria, 5.2 kg DHA and 6.7 kg ARA After mixing with dry mixer,
were added.
the uniformly mixed milk powder was packed with nitrogen to obtain the final product.
Example 8: Infant milk powder food preparation (1000 kg, wet addition)
Skimmed milk powder , lactose , desalted whey powder , whey
150 kg 250 kg 190 kg
protein powder ( ,
WPC34%) 110 kg corn oil 43 kg, soybean oil 44.7 kg, sunflower seed oil
, mixed nutrients 7
178 kg, oligofructose powder 4.5 kg, galactooligosaccharide syrup 11 kg
kg, 0.4 kg
nucleotide composition according to the invention and 2 kg soyabean lecithin were
mixed uniformly and subjected to pasteurization, homogenization, evaporation and
0.1 kg
concentration and spray drying to form semiproduct as powder, to which were added
Bifidobacteria, 3.9 kg DHA and 5.4 kg ARA. After mixing with dry mixer, the
uniformly mixed milk powder was packed with nitrogen to obtain the final product.
Example 9: Infant liquid milk preparation (based on 100g liquid milk)
The liquid milk according to the invention contained the follows nutritional ingredients:
Protein 2.2 g, fat 3.0g, lactose 9.0 g, vitamin A 80 μg RE, vitamin D 1.5 μg, vitamin E 1mg α-
TE, vitamin K1 6 μg, vitamin B1 70 μg, vitamin B2 70 μg, vitamin B6 200 μg, vitamin B12 0.5
μg, nicotinic acid 350 μg, folic acid 3.5 μg, pantothenic acid 200 μg, vitamin C 7 mg, biotin 2
μg, sodium 50 mg, potassium 70 mg, copper 80 μg, magnesium 30 mg, iron 1 mg, zinc 0.7 mg,
calcium 80 mg, phosphorus 45 mg, iodine 20 μg, chlorine 100 mg, lactoferrin 10 mg and 2.7
mg nucleotide composition according to the invention, wherein the energy is about 300kJ.
Fat is provided by anhydrous milk fat, soybean oil, corn oil, sunflower seed in any ratio and
combination, wherein based on 100 g of total ingredients, 0.5 g of linoleic acid was included.
The preparing process was as follows (raw materials and process comply with relevant national
standards):
1. Receiving, checking and optimizing the raw materials.
2. (1) Dissolving protein (including casein, whey protein, lactoferrin), B vitamins and vitamin
C, and nucleotide composition according to the invention with warm water;
(2) Dissolving carbohydrates raw materials and food additives like carrageenan, glyceryl
monostearate, guar gum and the like with warm water; dissolving minerals with warm
water; dissolving other raw materials with fatty feed liquid at 40°C ~50°C; and mixing
the above three dissolved feed liquids uniformly;
(3) Subjecting the feed liquid obtained in step (1) to membrane filtration for sterilization;
(4) Subjecting the feed liquid obtained in step (2) which has been mixed uniformly to UHT
(ultra - high temperature instantaneous sterilization) for sterilization;
(5) Mixing the feed liquids obtained in step (3) and step (4) under sterile condition and
metered to total amount of the ingredients with water;
(6) Subjecting the above mixed feed liquids to sterile homogenization; and
(7) Filling under sterile condition and packing.
Unless otherwise indicated, all numbers expressing quantities of ingredients, cell culture,
treatment conditions, and so forth used in the specification, including claims, are to be
understood as being modified in all instances by the term “about”. Accordingly, unless
otherwise indicated to the contrary, the numerical parameters are approximations and may vary
depending upon the desired properties sought to be obtained by the present invention. Unless
otherwise indicated, the term “at least” preceding a series of elements is to be understood to
refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the specific embodiments of
the invention described herein. Such equivalents are intended to be encompassed by the
appended claims.
Many modifications and variations of this invention can be made without departing from its
spirit and scope, as will be apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only and are not meant to be limiting in any
way. It is intended that the specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the appended claims.
Claims (7)
1. Use of a nucleotide composition, for the manufacture of a medicament for promoting repair of enterocytes after damage caused by oxidation, wherein the nucleotide composition consists of CMP, AMP, UMP and GMP, and the proportions of each of the nucleotides, on a weight basis relative to the total weight of the nucleotide composition, are CMP: 58-72%, AMP: 6-14%, UMP: 10-18% and GMP: 8-14%, provided that sum of the proportions is 100%.
2. The use according to claim 1, wherein the proportions of the components of the nucleotide composition are CMP: 60-70%, AMP: 8-12%, UMP: 12-16% and GMP: 10-12%.
3. The use according to claim 1, wherein the proportions of the components of the nucleotide composition are CMP: 60-65%, AMP: 10-12%, UMP: 14-16% and GMP: 11-12%.
4. The use according to claim 1, wherein the proportions of the components of the nucleotide composition are CMP: 65-70%, AMP: 8-10%, UMP: 12-14% and GMP: 10-11%.
5. The use according to claim 1, wherein the proportions of the components of the nucleotide composition are CMP: 60%, AMP: 12%, UMP: 16% and GMP: 12%; CMP: 65%, AMP: 10%, UMP: 14% and GMP: 11%; or CMP: 70%, AMP: 8%, UMP: 12% and GMP: 10%.
6. Use of a food comprising the nucleotide composition according to any one of claims 1-5, for the manufacture of a medicament for promoting repair of enterocytes after damage caused by oxidation, wherein the food is in the form of a liquid dairy product and comprises based on 100g liquid dairy product, energy 250kJ-355kJ and the following ingredients: protein beyond lactoferrin 1.75g-4.26g, fat 1.75g-4.97g, vitamin A 42.5μg-191.7μg RE, vitamin D 0.625μg- 2.6625μg, vitamin E≥0.375mg α-TE, vitamin K1≥2.5μg, vitamin B1≥27.5μg, vitamin B2≥27.5μg, vitamin B6≥27.5μg, vitamin B12≥0.1μg, nicotinic acid or nicotinamide≥275μg, folic acid≥2.5μg, pantothenic acid≥175μg, vitamin C≥4.5mg, biotin≥1μg, sodium≤71mg, potassium 45mg-244.95mg, copper 17.5μg-124.25μg, magnesium ≥3.5mg, iron 0.625mg- 1.775mg, zinc 0.25mg-1.065mg, calcium ≥42.5mg, phosphorus ≥20.75mg, iodine ≥3.5μg, chlorine ≤184.6mg, lactoferrin 5-13mg and carbohydrates; and wherein the nucleotide composition according to any one of claims 1-5 is present in an amount of 2.64-7.66mg and the amount of carbohydrates is such that the energy provided by carbohydrates, protein and fat is 250kJ-355kJ.
7. Use of the nucleotide composition according to any one of claims 1-5, wherein the medicament is in the form of a food or a food additive.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510099850.6A CN105982074A (en) | 2015-03-06 | 2015-03-06 | Nucleotide composition and application thereof in foods |
CN201510099850.6 | 2015-03-06 | ||
PCT/CN2016/074813 WO2016141812A1 (en) | 2015-03-06 | 2016-02-29 | Nucleotide composition and application in food thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ735399A NZ735399A (en) | 2020-11-27 |
NZ735399B2 true NZ735399B2 (en) | 2021-03-02 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP3209308B1 (en) | Activated bifidobacteria and methods of use thereof | |
CN107019701B (en) | Methods of using human milk oligosaccharides to reduce the incidence of necrotizing enterocolitis in infants, toddlers, or children | |
EP2934189B1 (en) | Human milk oligosaccharides to ameliorate symptoms of stress | |
EP3335576A1 (en) | Human milk oligosaccharides to promote growth of beneficial bacteria | |
TW201304692A (en) | Synbiotic combination of probiotic and human milk oligosaccharides to promote growth of beneficial microbiota | |
AU2016228670B2 (en) | Nucleotide composition and application in food thereof | |
CN113662199A (en) | Human milk oligosaccharides for preventing gastrointestinal damage and/or promoting gastrointestinal healing | |
CN102458152A (en) | Synergistic mixture of beta-galacto-oligosaccharides with beta-1,3 and beta-1,4/1,6 linkages | |
CN106259952B (en) | 1-3 years old baby formulas milk powder and preparation method thereof containing nucleotide and dietary fiber | |
TWI639387B (en) | Galactooligosaccharides for preventing injury and/or promoting healing of the gastrointestinal tract | |
AU2017307952A1 (en) | Nutritional compositions and infant formulas comprising a mix of oligosaccharides and optionally Bifidobacterium lactis for preventing, treating or reducing the severity of non-rotavirus-associated diarrhoea | |
CN105828641A (en) | Nutritional compositions for reducing intestinal pathogens | |
BR112013011642B1 (en) | method for inhibiting pathogens using a nutritional composition | |
KR20180122380A (en) | Pycalibacter sp. | |
NZ735399B2 (en) | Nucleotide composition and application in food thereof | |
Kazemi et al. | Evaluation the effect of royal jelly on the growth of two members of gut microbiota; Bacteroides fragillis and Bacteroides thetaiotaomicron. | |
Penders | Early diet and the infant gut microbiome: how breastfeeding and solid foods shape the microbiome | |
CN109329942A (en) | A kind of composition and preparation method thereof with adjusting intestinal flora function | |
CN118044614A (en) | Use of nutritional composition for promoting intestinal and brain bi-directional development through intestinal brain axis | |
CN116287008A (en) | Peptide, peptide capsule and application thereof in aspects of medicines for treating colonitis | |
TW201244721A (en) | Methods for decreasing the incidence of necrotizing enterocolitis in infants, toddlers, or children using human milk oligosaccharides |