JP6865456B2 - Method of utilizing marine heterotrophic algae having an amino acid composition suitable for feed as a protein component of feed - Google Patents
Method of utilizing marine heterotrophic algae having an amino acid composition suitable for feed as a protein component of feed Download PDFInfo
- Publication number
- JP6865456B2 JP6865456B2 JP2016099054A JP2016099054A JP6865456B2 JP 6865456 B2 JP6865456 B2 JP 6865456B2 JP 2016099054 A JP2016099054 A JP 2016099054A JP 2016099054 A JP2016099054 A JP 2016099054A JP 6865456 B2 JP6865456 B2 JP 6865456B2
- Authority
- JP
- Japan
- Prior art keywords
- algae
- mass
- feed
- amino acid
- composition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000195493 Cryptophyta Species 0.000 title claims description 66
- 239000000203 mixture Substances 0.000 title claims description 43
- 150000001413 amino acids Chemical class 0.000 title claims description 31
- 238000000034 method Methods 0.000 title claims description 12
- 235000004252 protein component Nutrition 0.000 title description 2
- 239000004467 fishmeal Substances 0.000 claims description 47
- 235000019733 Fish meal Nutrition 0.000 claims description 46
- 229940024606 amino acid Drugs 0.000 claims description 30
- 241000251468 Actinopterygii Species 0.000 claims description 25
- 235000019688 fish Nutrition 0.000 claims description 25
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 claims description 20
- 238000009360 aquaculture Methods 0.000 claims description 19
- 244000144974 aquaculture Species 0.000 claims description 19
- 241001306132 Aurantiochytrium Species 0.000 claims description 18
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- 102000004169 proteins and genes Human genes 0.000 claims description 16
- 235000015170 shellfish Nutrition 0.000 claims description 15
- 235000014102 seafood Nutrition 0.000 claims description 13
- 229960003080 taurine Drugs 0.000 claims description 10
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 5
- 239000004472 Lysine Substances 0.000 claims description 5
- 229930182817 methionine Natural products 0.000 claims description 5
- 239000004475 Arginine Substances 0.000 claims description 4
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 4
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 4
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 4
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 4
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 4
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 4
- 239000004473 Threonine Substances 0.000 claims description 4
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 4
- 229960000310 isoleucine Drugs 0.000 claims description 4
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 4
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 4
- 239000004474 valine Substances 0.000 claims description 4
- ZSLUVFAKFWKJRC-IGMARMGPSA-N 232Th Chemical compound [232Th] ZSLUVFAKFWKJRC-IGMARMGPSA-N 0.000 claims 1
- 229910052776 Thorium Inorganic materials 0.000 claims 1
- 239000003797 essential amino acid Substances 0.000 description 29
- 235000020776 essential amino acid Nutrition 0.000 description 29
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 21
- 229910052799 carbon Inorganic materials 0.000 description 21
- 239000002609 medium Substances 0.000 description 16
- 238000012360 testing method Methods 0.000 description 16
- 235000018102 proteins Nutrition 0.000 description 13
- 238000012258 culturing Methods 0.000 description 12
- 241000238557 Decapoda Species 0.000 description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 10
- 229960001031 glucose Drugs 0.000 description 10
- 239000008103 glucose Substances 0.000 description 10
- 238000009395 breeding Methods 0.000 description 8
- 230000001488 breeding effect Effects 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 238000009313 farming Methods 0.000 description 6
- 239000013535 sea water Substances 0.000 description 6
- 108010082495 Dietary Plant Proteins Proteins 0.000 description 5
- 235000012054 meals Nutrition 0.000 description 5
- 239000008188 pellet Substances 0.000 description 5
- 240000008042 Zea mays Species 0.000 description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 4
- 235000005822 corn Nutrition 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 108010068370 Glutens Proteins 0.000 description 3
- 238000012136 culture method Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 235000021312 gluten Nutrition 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- 241000238552 Penaeus monodon Species 0.000 description 2
- 235000019764 Soybean Meal Nutrition 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N 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 2
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- JAZBEHYOTPTENJ-JLNKQSITSA-N all-cis-5,8,11,14,17-icosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O JAZBEHYOTPTENJ-JLNKQSITSA-N 0.000 description 2
- MBMBGCFOFBJSGT-KUBAVDMBSA-N all-cis-docosa-4,7,10,13,16,19-hexaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(O)=O MBMBGCFOFBJSGT-KUBAVDMBSA-N 0.000 description 2
- 235000021120 animal protein Nutrition 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 235000020669 docosahexaenoic acid Nutrition 0.000 description 2
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 2
- 235000020673 eicosapentaenoic acid Nutrition 0.000 description 2
- 229960005135 eicosapentaenoic acid Drugs 0.000 description 2
- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 description 2
- 239000003925 fat Substances 0.000 description 2
- 235000019197 fats Nutrition 0.000 description 2
- 239000013505 freshwater Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 239000013028 medium composition Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 239000004455 soybean meal Substances 0.000 description 2
- 229940031439 squalene Drugs 0.000 description 2
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 230000001228 trophic effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- DVSZKTAMJJTWFG-SKCDLICFSA-N (2e,4e,6e,8e,10e,12e)-docosa-2,4,6,8,10,12-hexaenoic acid Chemical compound CCCCCCCCC\C=C\C=C\C=C\C=C\C=C\C=C\C(O)=O DVSZKTAMJJTWFG-SKCDLICFSA-N 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- GZJLLYHBALOKEX-UHFFFAOYSA-N 6-Ketone, O18-Me-Ussuriedine Natural products CC=CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O GZJLLYHBALOKEX-UHFFFAOYSA-N 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 241000003595 Aurantiochytrium limacinum Species 0.000 description 1
- 241000341438 Aurantiochytrium mangrovei Species 0.000 description 1
- 241001454694 Clupeiformes Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- IMQLKJBTEOYOSI-GPIVLXJGSA-N Inositol-hexakisphosphate Chemical compound OP(O)(=O)O[C@H]1[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O IMQLKJBTEOYOSI-GPIVLXJGSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 241001491670 Labyrinthula Species 0.000 description 1
- 241000530454 Litopenaeus schmitti Species 0.000 description 1
- 241001157775 Litopenaeus stylirostris Species 0.000 description 1
- 241001124325 Marsupenaeus japonicus Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000131739 Oncorhynchus masou rhodurus Species 0.000 description 1
- 241001282110 Pagrus major Species 0.000 description 1
- 241000269979 Paralichthys olivaceus Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- IMQLKJBTEOYOSI-UHFFFAOYSA-N Phytic acid Natural products OP(O)(=O)OC1C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C1OP(O)(O)=O IMQLKJBTEOYOSI-UHFFFAOYSA-N 0.000 description 1
- 241001600434 Plectroglyphidodon lacrymatus Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 241000844671 Taraba Species 0.000 description 1
- 241001441724 Tetraodontidae Species 0.000 description 1
- 241001504592 Trachurus trachurus Species 0.000 description 1
- 101710162629 Trypsin inhibitor Proteins 0.000 description 1
- 229940122618 Trypsin inhibitor Drugs 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 238000010162 Tukey test Methods 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 235000019513 anchovy Nutrition 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 235000021053 average weight gain Nutrition 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000004067 bulking agent Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229940077731 carbohydrate nutrients Drugs 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 230000001112 coagulating effect Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
- 229940090949 docosahexaenoic acid Drugs 0.000 description 1
- KAUVQQXNCKESLC-UHFFFAOYSA-N docosahexaenoic acid (DHA) Natural products COC(=O)C(C)NOCC1=CC=CC=C1 KAUVQQXNCKESLC-UHFFFAOYSA-N 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000003746 feather Anatomy 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 229940093915 gynecological organic acid Drugs 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 235000002949 phytic acid Nutrition 0.000 description 1
- 229940068041 phytic acid Drugs 0.000 description 1
- 239000000467 phytic acid Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229940073490 sodium glutamate Drugs 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
Images
Landscapes
- Feed For Specific Animals (AREA)
- Fodder In General (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
本発明は、飼料に好適なアミノ酸組成である微細藻類を飼料のタンパク質成分として利用する方法に関する。 The present invention relates to a method of utilizing microalgae having an amino acid composition suitable for feed as a protein component of feed.
魚介類養殖用飼料で使用されている主なタンパク質源は魚粉であり、我が国はチリおよびペルーから主にカタクチイワシ、マアジ魚粉を輸入している。しかし、水産資源の減少や、世界的に養殖・畜産業が盛んになったことによる魚粉の需要の増大により、魚粉価格が高騰している。その輸入価格は、平成１６年には７６円／kgであったのに対し、平成２５年には１５４円／ｋｇと２倍にまで高騰している。 The main protein source used in the feed for fish and shellfish farming is fish meal, and Japan mainly imports anchovy and horse mackerel fish meal from Chile and Peru. However, the price of fishmeal is rising due to the decrease in fishery resources and the increase in demand for fishmeal due to the global aquaculture and livestock industry. The import price was 76 yen / kg in 2004, but has doubled to 154 yen / kg in 2013.
魚粉は、魚介類養殖用飼料のみならず、ニワトリやブタなどの飼料に配合され、さまざまな家畜の動物性タンパク質源として利用されている。 Fish meal is blended not only in feed for aquaculture of seafood but also in feed for chickens and pigs, and is used as an animal protein source for various livestock.
近年では魚粉に頼らない低魚粉飼料の開発が盛んに行われている。低魚粉飼料は、魚粉の代わりに大豆粕やコーングルテンミール等の植物性タンパク質やミートミール、フェザーミール等の動物性タンパク質が用いられているが、それらはアミノ酸組成が魚粉と大きく異なり、大豆粕ではメチオニンが、コーングルテンミールではリジンが第１制限アミノ酸となっている。それゆえ、低魚粉飼料には結晶のアミノ酸を飼料に添加する必要があり、製造コストを押し上げることが障害となっている。また、植物性タンパク質源には、トリプシンインヒビター、フィチン酸、難消化性糖質などの生理阻害物質が含まれているため、配合量を高めると魚類の成長を阻害し、腸管の炎症等の抗病性の低下を招くことも報告されている。 In recent years, the development of low fish meal feed that does not rely on fish meal has been actively carried out. In low fish meal feed, vegetable proteins such as soybean meal and corn gluten meal and animal proteins such as meat meal and feather meal are used instead of fish meal, but their amino acid composition is significantly different from that of fish meal, and soybean meal. Methionine is the first limiting amino acid in corn gluten meal, and lysine is the first limiting amino acid in corn gluten meal. Therefore, it is necessary to add crystalline amino acids to the feed for low fish meal feed, and it is an obstacle to increase the production cost. In addition, since the vegetable protein source contains physiological inhibitors such as trypsin inhibitor, phytic acid, and indigestible sugar, increasing the amount of the vegetable protein source inhibits the growth of fish and prevents inflammation of the intestinal tract. It has also been reported to lead to reduced morbidity.
ブリやマダイ等の海水魚は、必須アミノ酸に加え、十分量のタウリンを体内で合成することができないため、餌飼料からタウリンを摂取しなければならない。植物性タンパク質はタウリンが少なく、低魚粉飼料ではタウリン不足による成長の低下や緑肝症、脱鱗がしばしば観察され、市場価値を下げることが問題であった。平成２１年にはタウリンの重要性が認められて飼料添加物に認定され、低魚粉飼料の開発に拍車がかかった。 Saltwater fish such as yellowtail and red sea bream cannot synthesize a sufficient amount of taurine in the body in addition to essential amino acids, so taurine must be ingested from the diet. Vegetable protein is low in taurine, and in low fish meal feed, decreased growth due to taurine deficiency, green liver disease, and scales are often observed, and it has been a problem to reduce the market value. In 2009, the importance of taurine was recognized and it was certified as a feed additive, spurring the development of low fish meal feed.
発明者らは、上記植物性タンパク質供給源のように生理阻害物質を含有せず、タウリンを含有する培養微細藻類の、低魚粉飼料への配合を検討した。斯かる低魚粉飼料に配合され得る微細藻類の例として、ラビリンチュラ類（Ｌａｂｙｒｉｎｔｈｕｌｏｍｙｃｅｔｅｓ）に属する藻類が挙げられる。ラビリンチュラ類は様々な炭化水素や油脂を生産するものが報告されており、ドコサヘキサエン酸（ＤＨＡ）やエイコサペンタエン酸（ＥＰＡ）等の高度不飽和脂肪酸を多量に蓄積する性質を有するもの（ＳＲ２１株、特許文献１）や、スクアレンを生産するものが知られている（非特許文献１、２、３）。それらの増殖速度は光合成藻類に比べて極めて速く、７日間の培養で乾燥重量１００ｇ／Ｌの藻体を生産することができ（非特許文献４）、高タンパク、高脂質要求の魚類にとって理想的な飼料原料である。 The inventors examined the formulation of cultured microalgae containing taurine, which does not contain a physiological inhibitor like the above-mentioned vegetable protein source, into a low fish meal feed. Examples of microalgae that can be blended in such low fish meal feed include algae belonging to Labyrinthulomyces. Labyrinthulas have been reported to produce various hydrocarbons and fats and oils, and have the property of accumulating a large amount of highly unsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) (SR21 strain). , Patent Document 1) and those that produce squalene are known (Non-Patent Documents 1, 2, and 3). Their growth rate is extremely faster than that of photosynthetic algae, and algae with a dry weight of 100 g / L can be produced by culturing for 7 days (Non-Patent Document 4), which is ideal for high-protein, high-lipid-requiring fish. It is a raw material for feed.
本発明は、アミノ酸組成を最適化した微細藻類を魚介類養殖用飼料のタンパク質源として利用する手法および養殖方法を提供することを目的とする。 An object of the present invention is to provide a method and a method for utilizing microalgae having an optimized amino acid composition as a protein source for a feed for aquaculture of seafood.
発明者らは、微細藻類のアミノ酸組成を、培養方法や培地組成を調整することで、魚粉に近いアミノ酸組成に最適化させる技術を開発した。斯かる技術は、生産コスト増大を伴わずに低魚粉飼料の品質を向上させ、魚粉の価格、供給量に左右されることなく、魚介類養殖用飼料の安定生産および水産資源の保全に寄与することに繋がる。 The inventors have developed a technique for optimizing the amino acid composition of microalgae to an amino acid composition close to that of fish meal by adjusting the culture method and medium composition. Such technology improves the quality of low-fish meal feed without increasing production costs, and contributes to the stable production of feed for fish and shellfish farming and the conservation of marine resources regardless of the price and supply of fish meal. It leads to.
ＤＨＡを蓄積するオーランチオキトリウム・リマシナム（Ａｕｒａｎｔｉｏｃｈｙｔｒｉｕｍ ｌｉｍａｃｉｎｕｍ）４Ｗ−１ｂ株を通常のＧＴＹ培地で培養したところ、収量は乾燥藻体１３ｇ／Ｌ、藻体の魚粉を基準とした必須アミノ酸指数（後述）は８３．８であった。しかし、同株を、ＧＴＹ培地に炭素源としてグルコースを流加して培養を行ったところ、収量が乾燥藻体５０ｇ／Ｌに顕著に増大し、更に、藻体の魚粉を基準とした必須アミノ酸指数は９６．４まで上昇した。これは、オーランチオキトリウム藻類を、炭素源を流加しながら培養することで、収量増大に加えて、そのアミノ酸組成を魚粉のアミノ酸組成に近付けることが出来ることを示す。 When Aurantiochytrium limacinum 4W-1b strain, which accumulates DHA, was cultured in a normal GTY medium, the yield was 13 g / L of dried algae, and the essential amino acid index based on the fish meal of the algae (described later). ) Was 83.8. However, when this strain was cultured by fed-batch glucose as a carbon source into GTY medium, the yield increased remarkably to 50 g / L of dried algae, and further, essential amino acids based on algae fish meal. The index rose to 96.4. This indicates that by culturing Aurantiochytrium algae while feeding a carbon source, in addition to increasing the yield, the amino acid composition can be brought closer to that of fish meal.
また、炭素源流加を要さずに良好なアミノ酸組成をとる藻類も存在する。スクアレンを細胞内に蓄積するオーランチオキトリウムｔｓｕｋｕｂａ−３株（受託番号：ＦＥＲＭ ＡＰ−２２０１４７）は、ＧＴＹ培地で培養すると６ｇ／Ｌの藻体が生産可能であり、必須アミノ酸指数は９１．２と極めて魚粉に近いアミノ酸組成となり、魚粉の代替タンパク質源として有望である。 There are also algae that have a good amino acid composition without the need for carbon source infusion. Aurantiochytrium tsukuba-3 strain (accession number: FERM AP-220147), which accumulates squalene in cells, can produce 6 g / L algae when cultured in GTY medium, and has an essential amino acid index of 91.2. It has an amino acid composition extremely close to that of fishmeal, and is promising as an alternative protein source for fishmeal.
発明者らは、このようなアミノ酸組成に優れるオーランチオキトリウム属藻類を配合した飼料を与えて魚介類を養殖することにより、魚粉主体の飼料と同等の飼育成績が得られることも明らかにした。 The inventors have also clarified that by feeding fish and shellfish with a feed containing Aurantiochytrium algae having an excellent amino acid composition, breeding results equivalent to those of a fish meal-based feed can be obtained. ..
従って、本願は、以下の発明を提供する。
１．タンパク質を構成する全アミノ酸中、メチオニンの占める割合が１．５−４．６質量％、イソロイシンの占める割合が２．２−６．５質量％、フェニルアラニンの占める割合が２．３−６．９質量％、リジンの占める割合が３．９−１１．６質量％、である、海産従属栄養性藻類。
２．タンパク質を構成する全アミノ酸中、トレオニンの占める割合が２．５−７．６質量％、バリンの占める割合が２．９−８．８質量％、ロイシンの占める割合が３．４−１０．２質量％、ヒスチジンの占める割合が１．７−５．０質量％、アルギニンの占める割合が２．８−８．５質量％である、項目１に記載の海産従属栄養性藻類。
３．タウリンを０．２−０．７質量％含む、項目１又は２に記載の海産従属栄養性藻類。
４．魚粉の必須アミノ酸組成に基づく必須アミノ酸指数が９０以上である、項目１〜３のいずれか１項に記載の海産従属栄養性藻類。
５．オーランチオキトリウム属の藻類である、項目１〜４のいずれか１項に記載の海産従属栄養性藻類。
６．オーランチオキトリウムＦＥＲＭ ＢＰ−１１４４２株である、項目５に記載の海産従属栄養性藻類。
７．培養槽に炭素源を流加する培養工程を含む、項目１〜６のいずれか１項に記載の海産従属栄養性藻類の生産方法。
８．項目１〜６のいずれか１項に記載の海産従属栄養性藻類を、全質量に対して、乾燥藻体換算で１．０〜７５．０質量％含有する、魚介類養殖用飼料組成物。
９．項目８に記載の飼料組成物を給餌する工程を含む、魚介類の養殖方法。
Therefore, the present application provides the following inventions.
1. 1. Of all the amino acids that make up the protein, methionine accounts for 1.5-4.6% by mass, isoleucine accounts for 2.2-6.5% by mass, and phenylalanine accounts for 2.3-6.9. Marine dependent trophic algae, which are 3.9-11.6 mass% by mass and lysine accounts for 3.9-11.6 mass%.
2. Of all the amino acids that make up a protein, threonine accounts for 2.5-7.6% by mass, valine accounts for 2.9-8.8% by mass, and leucine accounts for 3.4-10.2. The marine dependent trophic algae according to item 1, wherein the proportion of mass%, histidine is 1.7-5.0 mass%, and the proportion of arginine is 2.8-8.5 mass%.
3. 3. The marine heterotrophic alga according to item 1 or 2, which contains 0.2-0.7% by mass of taurine.
4. The marine heterotrophic alga according to any one of items 1 to 3, wherein the essential amino acid index based on the essential amino acid composition of fish meal is 90 or more.
5. The marine heterotrophic alga according to any one of items 1 to 4, which is an alga of the genus Aurantiochytrium.
6. The marine heterotrophic alga according to item 5, which is Aurantiochytrium FERM BP-11442 strain.
7. The method for producing a marine heterotrophic alga according to any one of items 1 to 6, which comprises a culturing step of feeding a carbon source into a culturing tank.
8. A feed composition for aquaculture of seafood containing 1.0 to 75.0% by mass of the marine heterotrophic algae according to any one of items 1 to 6 in terms of dry algae with respect to the total mass.
9. A method for culturing seafood, which comprises a step of feeding the feed composition according to item 8.
本発明により、魚粉の代替として利用可能な優れたアミノ酸組成を有する微細藻類を養殖飼料のタンパク質源として利用することができれば、水産資源量や魚粉価格に左右されずに、安定的に高品質の魚介類養殖用飼料を供給することが可能になる。下記実施例にて示すように、本発明に係るオーランチオキトリウムの乾燥藻体を配合した養殖飼料を用いてクルマエビ飼育試験を行った結果、タウリンおよび結晶のアミノ酸の補足なしで、魚粉主体の飼料と同等の成績が得られることが見出された。従って、本発明に係る藻類は、魚介類養殖用飼料のタンパク質源として非常に優れていると言え、水産養殖において画期的な進歩をもたらすものである。 According to the present invention, if microalgae having an excellent amino acid composition that can be used as a substitute for fishmeal can be used as a protein source for aquaculture feed, stable and high quality can be achieved regardless of the amount of fishery resources and the price of fishmeal. It will be possible to supply feed for fish and shellfish farming. As shown in the following examples, as a result of conducting a prawn breeding test using a farmed feed containing the dried algae of Aurantiochytrium according to the present invention, fish meal was mainly used without supplementation of taurine and crystalline amino acids. It was found that results equivalent to those of feed were obtained. Therefore, it can be said that the algae according to the present invention are extremely excellent as a protein source for feed for fish and shellfish farming, and bring about epoch-making progress in aquaculture.
本発明において、魚粉の代替として利用可能な微細藻類は、海産従属栄養性藻類である。当該藻類の例として、限定されないが、オーランチオキトリウム属、シゾキトリウム属、パリエティキトリウム属、ボトリオキトリウム属、スラウストキトリウム属、アプラノキトリウム属、シキオイドキトリウム属などのヤブレツボカビ科の藻類、またはこれらを起源とする藻類変異体、組換え藻類が挙げられる。好ましくは、本発明の微細藻類は、オーランチオキトリウム属藻類である。 In the present invention, the microalgae that can be used as a substitute for fish meal are marine heterotrophic algae. Examples of such algae include, but are not limited to, Aurantiochytrium, Sizochitrium, Parieticitorium, Botriochitrium, Slaustochytrium, Applanochytrium, Siquioidocytrium, and other species. Examples thereof include algae, algae variants originating from these, and recombinant algae. Preferably, the microalgae of the present invention are Aurantiochytrium algae.
本発明の微細藻類は、増殖能力の優れた株を用いるのが好ましい。そのような藻類株は、天然に採取及び分離されたものであっても、突然変異誘導及びスクリーニングを経てクローニングされたものであっても、あるいは遺伝子組み換え技術を利用して樹立されたものであってもよい。当該藻類株において改善され得る特性は、増殖効率、最適ではない培養条件（日照、栄養、温度、ｐＨ、成分組成等）に対する耐性、又は藻体が飼料として配合された場合の魚介類の成長速度等、本発明において飼料に配合されるタンパク質源として調製されるのに有利な任意の特性である。 For the microalgae of the present invention, it is preferable to use a strain having excellent growth ability. Such algae strains, whether naturally harvested and isolated, cloned through mutagenesis and screening, or established using genetic engineering techniques. You may. The properties that can be improved in the algae strain are growth efficiency, resistance to non-optimal culture conditions (sunshine, nutrition, temperature, pH, composition, etc.), or growth rate of fish and shellfish when the algae are added as feed. Etc., any property that is advantageous to be prepared as a protein source to be incorporated into feed in the present invention.
上記微細藻類の培養は、当該技術分野において確立された方法を基礎とする。即ち、通常の維持培養は、適切に成分調製した培地に藻類を播種し、定法に従い行われる。 The culture of microalgae is based on methods established in the art. That is, the usual maintenance culture is carried out according to a standard method by sowing algae in a medium in which the components are appropriately prepared.
微細藻類を培養するための培地は、本質的に、塩分、炭素供給源及び窒素供給源を含有する。一般的に、微細藻類の培養には、いわゆるＧＴＹ培地（人工海水塩１０−４０ｇ／Ｌ、Ｄ（＋）グルコース２０−２００ｇ／Ｌ、トリプトン１０−６０ｇ／Ｌ、酵母抽出物５−４０ｇ／Ｌ）が用いられる。本発明に関する培地も、基本的にはこれらの３つの要素を組み合わせて構成される。 The medium for culturing microalgae essentially contains a salt, carbon source and nitrogen source. Generally, for culturing microalgae, so-called GTY medium (artificial seawater salt 10-40 g / L, D (+) glucose 20-200 g / L, tryptone 10-60 g / L, yeast extract 5-40 g / L). ) Is used. The medium according to the present invention is also basically composed of a combination of these three elements.
炭素源としてはグルコース、フルクトース、スクロース等の糖類がある。これらの炭素源を、例えば、培地１リットル当たり２０〜２００ｇの濃度で添加する。 Carbon sources include sugars such as glucose, fructose, and sucrose. These carbon sources are added, for example, at a concentration of 20-200 g per liter of medium.
前記微細藻類の培養培地には、グルタミン酸ナトリウム、尿素等の有機窒素、又は酢酸アンモニウム、硫酸アンモニウム、塩化アンモニウム、硝酸ナトリウム、硝酸アンモニウム等の無機窒素、又は酵母抽出物、コーンスチープリカー、ポリペプトン、ペプトン、トリプトン等の生物由来消化物等の、様々な窒素源が添加され得る。 The culture medium for microalgae includes organic nitrogen such as sodium glutamate and urea, inorganic nitrogen such as ammonium acetate, ammonium sulfate, ammonium chloride, sodium nitrate and ammonium nitrate, yeast extract, corn steep liquor, polypeptone, peptone and tripton. Various nitrogen sources can be added, such as biological digests such as.
海洋性藻類を培養する場合、培地には適切な量の人工海水又は天然海水が添加される。好ましくは、人工海水は、最終的な培地の塩分濃度が海水（塩分濃度３．４％（ｗ／ｖ））の約１０％（ｖ／ｖ）〜約１００％（ｖ／ｖ）、例えば塩分濃度が約１．０〜３．０％（ｗ／ｖ）となるように添加される。 When culturing marine algae, an appropriate amount of artificial seawater or natural seawater is added to the medium. Preferably, the artificial seawater has a salinity of about 10% (v / v) to about 100% (v / v) of the seawater (salinity 3.4% (w / v)) in the final medium, for example, salt content. It is added so that the concentration is about 1.0 to 3.0% (w / v).
微細藻類の培養は、培養温度５〜４０℃、好ましくは１０〜３５℃、より好ましくは１５〜３０℃にて行われる。継代は、藻類株の増殖速度に応じて、通常１〜１０日間、好ましくは３〜７日間置きに行われる。培養は通気攪拌培養、振とう培養又は静置培養で行うことができるが、好ましくは通気攪拌培養又は振とう培養で培養する。藻類株の長期の保存には、液体培地に１．０〜３．０％濃度の寒天を加えて凝固させた寒天培地を用いてもよく、より長期の保存に際しては、藻類株は定法に従い凍結保存されてもよい。 Culturing of microalgae is carried out at a culture temperature of 5 to 40 ° C., preferably 10 to 35 ° C., more preferably 15 to 30 ° C. Subculture is usually carried out every 1 to 10 days, preferably every 3 to 7 days, depending on the growth rate of the algae strain. The culture can be carried out by aeration stirring culture, shaking culture or static culture, but is preferably cultured by aeration stirring culture or shaking culture. For long-term storage of the algae strain, an agar medium obtained by adding 1.0 to 3.0% concentration of agar to a liquid medium and coagulating it may be used, and for longer-term storage, the algae strain is frozen according to a conventional method. It may be saved.
好ましい態様において、本発明の微細藻類は、炭素源が流加される条件下で培養される。流加プロセスにおいては、微細藻類の培養系に、所望の炭素源が逐次添加される。斯かる流加により、培養系中に存在する炭素源の濃度が微細藻類による消費に拘らず一定のレベルに維持される。炭素源の流加の条件（流加系の設計、炭素源の種類、濃度、流加速度等）は、具体的な培養条件、培養する藻類種、目標とするアミノ酸組成に応じて当業者が適宜調整することが出来る。 In a preferred embodiment, the microalgae of the present invention are cultured under conditions in which a carbon source is fed. In the fed-batch process, the desired carbon source is sequentially added to the microalgae culture system. By such feeding, the concentration of the carbon source present in the culture system is maintained at a constant level regardless of the consumption by microalgae. Conditions for fed-batch of carbon source (design of fed-batch system, type of carbon source, concentration, flow acceleration, etc.) are appropriately determined by those skilled in the art according to specific culture conditions, algae species to be cultured, and target amino acid composition. It can be adjusted.
流加される炭素源としてはグルコース、フルクトース、スクロース等の糖類がある。当該炭素源は、当初の培養培地に添加されている炭素源と同一であっても異なるものであってもよい。好ましくは、流加される炭素源はグルコースである。好ましい態様において、炭素源は、１日当たり５〜１００ｇ／Ｌ、１０〜８０ｇ／Ｌ、１５〜６０ｇ／Ｌ、又は２０〜４０ｇ／Ｌの濃度で添加される。好ましくは、培養過程で添加される炭素源の量は、合計で６０〜２４０ｇ／Ｌ、８０〜２２０ｇ／Ｌ、１００〜２００ｇ／Ｌ、１２０〜１７０ｇ／Ｌ、又は１３０〜１５０ｇ／Ｌとなる。好ましい態様において、グルコースの流加量は、培養系内のグルコースが枯渇せず、かつ過剰なグルコースが藻類の増殖を阻害しないように、適宜調整される。 The carbon source to be fed-batch includes sugars such as glucose, fructose, and sucrose. The carbon source may be the same as or different from the carbon source added to the original culture medium. Preferably, the carbon source to be fed is glucose. In a preferred embodiment, the carbon source is added at a concentration of 5-100 g / L, 10-80 g / L, 15-60 g / L, or 20-40 g / L per day. Preferably, the total amount of carbon sources added during the culturing process is 60-240 g / L, 80-220 g / L, 100-200 g / L, 120-170 g / L, or 130-150 g / L. In a preferred embodiment, the glucose feed rate is appropriately adjusted so that the glucose in the culture system is not depleted and the excess glucose does not inhibit the growth of algae.
培養によって取得された微細藻類は、必須アミノ酸の組成が評価される。好ましくは、本発明の培養によって取得された微細藻類は、タンパク質を構成する全アミノ酸中、メチオニンを１．５−４．６質量％、イソロイシンを２．２−６．５質量％、フェニルアラニンを２．３−６．９質量％、リジンを３．９−１１．６質量％含有する。より好ましくは、当該藻類は、タンパク質を構成する全アミノ酸中、トレオニンを２．５−７．６質量％、バリンを２．９−８．８質量％、ロイシンを３．４−１０．２質量％、ヒスチジンを１．７−５．０質量％、アルギニンを２．８−８．５質量％含有する。尚もより好ましくは、当該藻類は、タウリンを０．２−０．７質量％含む。 The microalgae obtained by culturing are evaluated for the composition of essential amino acids. Preferably, the microalgae obtained by the culture of the present invention contains 1.5-4.6% by mass of methionine, 2.2-6.5% by mass of isoleucine, and 2% by mass of phenylalanine among all the amino acids constituting the protein. It contains .3-6.9% by mass and 3.9-11.6% by mass of lysine. More preferably, the algae contains 2.5-7.6% by mass of threonine, 2.9-8.8% by mass of valine, and 3.4-10.2% by mass of leucine in all the amino acids constituting the protein. %, 1.7-5.0% by mass of histidine, and 2.8-8.5% by mass of arginine. Even more preferably, the algae contain 0.2-0.7% by weight of taurine.
前記微細藻類の必須アミノ酸組成は、魚粉を基準にした必須アミノ酸指数の値によって評価されてもよい。好ましくは、本発明の微細藻類の魚粉を基準にした必須アミノ酸指数は、８５以上、８６以上、８７以上、８８以上、８９以上、９０以上、９１以上、９２以上、９３以上、９４以上、９５以上、９６以上、９７以上、９８以上又は９９以上である。 The essential amino acid composition of the microalgae may be evaluated by the value of the essential amino acid index based on fish meal. Preferably, the essential amino acid index based on the fish meal of the microalga of the present invention is 85 or more, 86 or more, 87 or more, 88 or more, 89 or more, 90 or more, 91 or more, 92 or more, 93 or more, 94 or more, 95. Above, 96 or above, 97 or above, 98 or above, or 99 or above.
培養によって取得された微細藻類は、飼料に配合するための適切な形態に加工される。本発明の飼料に配合する微細藻類の形態は、培養物、濃縮藻体、乾燥藻体、ホモジネート、脂質抽出後残渣等、性状は問わないが、いずれも簡素な手順で調製が可能なものである。例えば、前記培養物から遠心分離にて固形分を回収することでウェットな藻体を得て、これをスプレードライ、ドラム乾燥機等で乾燥することで乾燥藻体を取得する。特定の態様において、飼料への培養微細藻類の配合率は、乾燥藻体換算で０．５〜８０質量％、好ましくは１．０〜３０．０質量％、より好ましくは２．５〜２０．０質量％であるが、実際の配合率は、養殖される魚介類の種類や配合される藻類の種類、タンパク質含量に応じて、当業者が容易に決定できる。 The microalgae obtained by culturing are processed into a suitable form for inclusion in feed. The form of the microalgae to be blended in the feed of the present invention may be any property such as culture, concentrated algae, dried algae, homogenate, residue after lipid extraction, etc., but all of them can be prepared by a simple procedure. is there. For example, a wet algae is obtained by centrifuging the solid content from the culture, and the dried algae is obtained by drying this with a spray dry, a drum dryer, or the like. In a specific embodiment, the blending ratio of the cultured microalgae in the feed is 0.5 to 80% by mass, preferably 1.0 to 30.0% by mass, more preferably 2.5 to 20% by mass in terms of dry algae. Although it is 0% by mass, the actual blending ratio can be easily determined by a person skilled in the art according to the type of fish and shellfish to be cultivated, the type of algae to be blended, and the protein content.
本発明において、前記魚介類養殖用飼料は、魚粉と代替可能な養殖用飼料に好適なアミノ酸組成を有する微細藻類を含有する。 In the present invention, the seafood aquaculture feed contains microalgae having an amino acid composition suitable for aquaculture feed that can replace fish meal.
本発明において、魚介類養殖用飼料は、魚介類の養殖に適切な任意の飼料成分を含有する。当業者は、養殖する魚介類の種類や具体的な養殖の条件を考慮して、適切な飼料を選択し、又は飼料成分を配合することを承知している。 In the present invention, the feed for aquaculture of seafood contains any feed component suitable for aquaculture of seafood. Those skilled in the art are aware that an appropriate feed is selected or a feed component is blended in consideration of the type of fish and shellfish to be cultivated and specific aquaculture conditions.
前記飼料成分として、好ましくは、具体的な魚介類の養殖の条件に適合するように、動物又は植物由来の加工物又は抽出物、油脂、炭水化物、有機酸、ビタミン、ミネラル、抗生物質、香料、着色料、保存料、賦形剤、増量剤、増粘剤、接着剤、水和剤、崩壊剤、乳化剤、ｐＨ調整剤等の、飼料調製に通常利用される材料を含有する。 As the feed component, preferably, processed or extracts derived from animals or plants, fats and oils, carbohydrates, organic acids, vitamins, minerals, antibiotics, fragrances, etc., so as to meet specific seafood cultivation conditions. Contains materials commonly used in feed preparation such as colorants, preservatives, excipients, bulking agents, thickeners, adhesives, wettable powders, disintegrants, emulsifiers, pH regulators and the like.
前記魚介類養殖用飼料は、任意の適切な形態、例えばペースト、粉末、モイストペレット、ドライペレット、エクストルーダーペレット、フレーク、ケーキ若しくは錠剤の形態であってもよい。 The seafood farming feed may be in any suitable form, such as pastes, powders, moist pellets, dry pellets, extruder pellets, flakes, cakes or tablets.
本発明において、当該培養微細藻類を配合した飼料を与えて、魚介類が養殖される。養殖の諸条件は、養殖される魚介類の種類に応じて当業者が適宜選択することが出来る。 In the present invention, seafood is cultivated by feeding a feed containing the cultured microalgae. The conditions for aquaculture can be appropriately selected by those skilled in the art according to the type of seafood to be cultivated.
本発明の魚介類養殖用飼料を与えて養殖される魚介類としては、当該飼料によって養殖が可能なあらゆる養殖魚介類が想定され、限定されないが、ブリ、マダイ、カワハギ、カンパチ、マグロ、フグ、シマアジ、スズキ、ヒラメ、アジ、サバ、ハタ類、サーモンなどの海産養殖魚類、また、ニジマス、コイ、ウナギ、アユ、アマゴ、イワナなどの淡水養殖魚類、クルマエビ、ウシエビ、ホワイトシュリンプ、タイショウエビ、テンジクエビ、ブルーシュリンプ、オニテナガエビ等の海産および淡水エビ類、ガザミ、タラバガニ、ズワイガニ、ケガニ、シャコ等のカニ類を含む。 The fish and shellfish cultivated by feeding the feed for cultivating the fish and shellfish of the present invention are assumed to be all cultivated fish and shellfish that can be cultivated by the feed, and are not limited to shrimp, madai, kawahagi, kanpachi, tuna, pufferfish, and the like. Marine farmed fish such as Shimaji, Suzuki, Hirame, Aji, Saba, Hata, Salmon, Freshwater farmed fish such as Nijimasu, Koi, Eel, Ayu, Amago, Iwana, Kuruma shrimp, Cow shrimp, White shrimp, Taisho shrimp, Tenjiku shrimp , Blue shrimp, marine and freshwater shrimp such as onitenaga shrimp, crabs such as gazami, taraba crab, zuwai crab, kegani, shako.
以下、実施例により本発明をさらに詳細に説明するが、本発明はこれに限定するものではない。また、実施例中「％」で表示されているものは、特記されていなければ「質量％」の意味である。 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto. In addition, what is indicated by "%" in the examples means "mass%" unless otherwise specified.
実施例１．オーランチオキトリウムの培養
オーランチオキトリウム・リマシナム（Ａｕｒａｎｔｉｏｃｈｙｔｒｉｕｍ ｌｉｍａｃｉｎｕｍ）４Ｗ−１ｂ株の凍結保存品１．２ｍｌを、坂口フラスコ中の２００ｍｌのＧＴＹ培地に植菌し、１００ｒｐｍ、２５℃で３日間振盪培養した。この培養物１ｍｌを、５００ｍｌフラスコ中のＧＴＹ培地に植菌し、１００ｒｐｍ、２５℃で７日間培養した。得られた培養物から単離した藻類のアミノ酸組成を、図１に示す。
Example 1. Culture of Aurantiochytrium 1.2 ml of cryopreserved Aurantiochytrium 4W-1b strain was inoculated into 200 ml of GTY medium in a Sakaguchi flask and shaken at 100 rpm and 25 ° C for 3 days. It was cultured. 1 ml of this culture was inoculated into GTY medium in a 500 ml flask and cultured at 100 rpm at 25 ° C. for 7 days. The amino acid composition of the algae isolated from the obtained culture is shown in FIG.
炭素源を流加する培養は、上記の７日間の振盪培養において、１日当たりグルコースを２０〜４０ｇ／Ｌの濃度で添加し、培養過程で添加したグルコース量は、合計で１３０〜１５０ｇ／Ｌであった。得られた培養物から単離した藻類のアミノ酸組成を、図２に示す。 In the culture in which a carbon source is fed, glucose is added at a concentration of 20 to 40 g / L per day in the above-mentioned 7-day shaking culture, and the total amount of glucose added during the culture process is 130 to 150 g / L. there were. The amino acid composition of the algae isolated from the obtained culture is shown in FIG.
上記両単離藻類のアミノ酸組成を、魚粉の必須アミノ酸組成を基準とした、必須アミノ酸指数によって評価した。必須アミノ酸指数は、
（１００ａ／ａｅ×１００ｂ／ｂｅ×１００ｃ／ｃｅ×・・・・×１００ｊ／ｊｅ）１／ｎ
で求められ、ここで、
ａ，ｂ，ｃ・・・ｊ＝試験タンパク質の各必須アミノ酸含量（％）
ａｅ，ｂｅ，ｃｅ・・・ｊｅ＝魚粉の各必須アミノ酸含量（％）
ｎ＝必須アミノ酸の数
である。魚類における必須アミノ酸は、メチオニン、イソロイシン、フェニルアラニン、リジン、トレオニン、バリン、ロイシン、ヒスチジン、アルギニン及びトリプトファンの１０種類であるが、本試験において、魚粉の必須アミノ酸は、トリプトファンの分析を行わなかったため、残りの９種類の必須アミノ酸に関して評価を行った。いずれかの必須アミノ酸において藻類における含量が魚粉における含量を上回るとその項の値が１００を超えるが、その場合はその項の値を１００として必須アミノ酸指数を求めた。
The amino acid composition of both isolated algae was evaluated by the essential amino acid index based on the essential amino acid composition of fish meal. The essential amino acid index is
(100a / ae × 100b / be × 100c / ce × ・ ・ ・ ・ × 100j / je) 1 / n
Asked for, here,
a, b, c ... j = Essential amino acid content (%) of the test protein
ae, be, ce ... je = Each essential amino acid content of fish meal (%)
n = number of essential amino acids. The essential amino acids in fish are methionine, isoleucine, phenylalanine, lysine, threonine, valine, leucine, histidine, arginine and tryptophan. The remaining 9 essential amino acids were evaluated. When the content of any of the essential amino acids in algae exceeds the content in fish meal, the value of that term exceeds 100. In that case, the value of that term is set to 100 to obtain the essential amino acid index.
魚粉を基準とした必須アミノ酸指数は、最大で１００となり（必須アミノ酸組成が魚粉と同一）、必須アミノ酸組成が魚粉に近い程大きくなる。図１にアミノ酸組成を示す通常の培養で得られた藻類の必須アミノ酸指数は、８３．８であった。一方、図２にアミノ酸組成を示す、炭素源の流加を行った培養で得られた藻類の必須アミノ酸指数は、９６．４であった。この結果は、炭素源の流加により、培養藻類の必須アミノ酸組成が、魚粉に近いものに改質したことを示す。 The essential amino acid index based on fish meal is 100 at the maximum (the essential amino acid composition is the same as that of fish meal), and the closer the essential amino acid composition is to fish meal, the larger the index. The essential amino acid index of algae obtained by normal culture showing the amino acid composition in FIG. 1 was 83.8. On the other hand, the essential amino acid index of algae obtained by fed-batch culture with a carbon source, which shows the amino acid composition in FIG. 2, was 96.4. This result indicates that the essential amino acid composition of cultured algae was modified to be close to that of fish meal by fed-batch of carbon source.
魚介類の理想的なアミノ酸組成は、同魚介類の全魚体または筋肉中のアミノ酸組成であると言われており、藻類株、培養方法、培地成分を任意に変えることによって、同魚介類に最適なアミノ酸組成のタンパク質源を作り出すことができる。 The ideal amino acid composition of fish and shellfish is said to be the amino acid composition in the whole fish body or muscle of the fish and shellfish, and it is optimal for the fish and shellfish by arbitrarily changing the algae strain, culture method, and medium composition. It is possible to create a protein source having a different amino acid composition.
実施例２．クルマエビにおける飼育試験
オーランチオキトリウム・マングロベイ（Ａｕｒａｎｔｉｏｃｈｙｔｒｉｕｍ ｍａｎｇｒｏｖｅｉ）を定法に従い培養し、藻体を回収し、乾燥藻体を調製した。斯かる乾燥藻体の魚粉を基準とした必須アミノ酸指数を上記のようにして求めたところ、９１．２であった。クルマエビ養殖用飼料の魚粉の代わりに当該乾燥藻体を０、２．５、５．０、１０．０、２０．０％配合した飼料を作製し、それらを与えてクルマエビにおける代替タンパク源としての評価を行った。
Example 2. Breeding test in prawns Aurantiochytrium mangrovei was cultured according to a conventional method, and algae were collected to prepare dried algae. The essential amino acid index based on the fish meal of such dried algae was calculated as described above and found to be 91.2. Instead of fish meal for tiger prawn farming feed, a feed containing 0, 2.5, 5.0, 10.0, 20.0% of the dried algae was prepared, and they were given as an alternative protein source for tiger prawns. Evaluation was performed.
供試魚および飼育方法
株式会社ヒガシマルの臨海研究所で人工種苗生産した平均体重１．０９ｇのクルマエビを、１００Ｌ容角型水槽に１５尾ずつ収容して５試験区を設けた。各水槽には加温濾過海水を注水し、期間中の平均水温は１９．０℃であった。
Test fish and breeding method Five test plots were set up by accommodating 15 prawns with an average weight of 1.09 g produced by artificial seedlings at the Seaside Research Institute of Higashimaru Co., Ltd. in a 100 L square aquarium. Warm filtered seawater was injected into each tank, and the average water temperature during the period was 19.0 ° C.
表１に示した試験飼料組成に従い、試験飼料を作製した。各原料を秤量・混合した後、微粉砕し、外割で３０％の水を加えて混練した後、ペレットマシンを用いて直径約２ｍｍのペレットを成型した。これを熱風乾燥機により乾燥し試験飼料を作製した。飼育期間は５６日間とし、１日１回日没後に各試験飼料を給与した。翌朝に残餌、脱皮殻、糞を回収し、残餌量に応じて給餌量を決定した。試験区は各試験飼料につき３反復区を設けたが、飼育試験終了後は同一試験区である４５尾の供試魚をプールし、ミンチにして分析に用いた。統計処理に関して、得られた結果は一元分散分析で有意差を確認した後、Ｔｕｋｅｙの多重比較検定法で各試験区における平均値の有意差判定を行った（ｐ＜０．０５）。
飼育成績
飼育成績を表２に示した。平均体重、平均増重率、生残率、飼料効率に有意差はないが、日間摂餌率は試験区４が有意に高くなったことから、オーランチオキトリウム藻体は摂餌を促進させる効果があることが示唆された。これらの結果から、オーランチオキトリウムは成長に関して魚粉主体の飼料と差はないことが確認された。
表３に開始時および終了時における全魚体の一般成分を示した。魚粉主体の対照飼料と全ての区で差は認められなかった。
ＦＥＲＭ ＡＰ−２２０１４７
ＦＥＲＭ ＢＰ−１１４４２
FERM AP-220147
FERM BP-11442
Claims (6)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2016099054A JP6865456B2 (en) | 2016-05-17 | 2016-05-17 | Method of utilizing marine heterotrophic algae having an amino acid composition suitable for feed as a protein component of feed |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2016099054A JP6865456B2 (en) | 2016-05-17 | 2016-05-17 | Method of utilizing marine heterotrophic algae having an amino acid composition suitable for feed as a protein component of feed |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2017205045A JP2017205045A (en) | 2017-11-24 |
| JP6865456B2 true JP6865456B2 (en) | 2021-04-28 |
Family
ID=60414576
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2016099054A Active JP6865456B2 (en) | 2016-05-17 | 2016-05-17 | Method of utilizing marine heterotrophic algae having an amino acid composition suitable for feed as a protein component of feed |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6865456B2 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP7054088B2 (en) * | 2017-11-29 | 2022-04-13 | 国立研究開発法人水産研究・教育機構 | A method for producing seedlings of marine fish, which comprises feeding particles containing marine heterotrophic algae. |
| WO2020026794A1 (en) * | 2018-08-01 | 2020-02-06 | ＭｏＢｉｏｌ株式会社 | Method for culturing heterotrophic microalgae using palm oil mill effluent (pome) and method for producing dha |
| JPWO2023007656A1 (en) * | 2021-07-29 | 2023-02-02 | ||
| JP7248772B1 (en) | 2021-12-21 | 2023-03-29 | マルハニチロ株式会社 | Seriola farmed fish and method for raising Seriola farmed fish |
| CN114287521B (en) * | 2021-12-27 | 2023-06-20 | 中国海洋大学 | Compound enzymolysis preparation method of laminarin for replacing fish meal for cottonseed protein compound feed and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012077799A1 (en) * | 2010-12-09 | 2012-06-14 | 国立大学法人筑波大学 | Novel microorganism having high squalene-producing ability, and method for producing squalene by means of same |
| FR3019559B1 (en) * | 2014-04-03 | 2018-01-05 | Fermentalg | PROCESS FOR CULTIVATION OF MICROALGUES OF THE GENUS AURANTIOCHYTRIUM IN A SODIUM-CHLORIDE REDUCED CULTURE MEDIUM FOR THE PRODUCTION OF DHA |
| JP5747360B1 (en) * | 2014-07-11 | 2015-07-15 | 国立大学法人東北大学 | Nutrient recovery method of algal lipid extraction residue, algae culture method, and algae culture medium |
| WO2016010149A1 (en) * | 2014-07-18 | 2016-01-21 | 国立大学法人筑波大学 | Method of acclimating algae belonging to aurantiochytrium sp. to low-salt conditions |
-
2016
- 2016-05-17 JP JP2016099054A patent/JP6865456B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP2017205045A (en) | 2017-11-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Guedes et al. | Application of microalgae protein to aquafeed | |
| Harel et al. | Advanced DHA, EPA and ArA enrichment materials for marine aquaculture using single cell heterotrophs | |
| JP6865456B2 (en) | Method of utilizing marine heterotrophic algae having an amino acid composition suitable for feed as a protein component of feed | |
| JP6779450B2 (en) | Feed for aquaculture of seafood containing cultured microalgae that have accumulated squalene | |
| US11930832B2 (en) | Feed supplement material for use in aquaculture feed | |
| Arai | Eel, Anguilla spp. | |
| de Cruz et al. | Evaluation of microalgae concentrates as partial fishmeal replacements for hybrid striped bass Morone sp. | |
| CN107529411B (en) | Compound feed for soft-shell crabs of scylla paramamosain and preparation method thereof | |
| CN105166455A (en) | New-type environmentally protective, nutritional, and high-efficient pelteobagrus fulvidraco compound feed | |
| CN108271951B (en) | Algae-derived composite protein mixture for replacing fish meal in aquatic feed and application | |
| CN106306551A (en) | Efficient growth-promoting functional compound feed for micropterus salmoides | |
| Lim et al. | Practical feeding—penaeid shrimps | |
| CN104920833A (en) | Penaeus vannamei compound feed and processing method thereof | |
| CN102379376B (en) | Forage capable of improving broodstock reproductive performance of Cherax quadricarionatus | |
| Radhakrishnan et al. | Different animal feeds and their role in aquaculture | |
| Fall et al. | Effects of partial substitution of fishmeal by crustacean (Callianassa) meal on the growth performance, feed efficiency and survival rate of Nile tilapia (Oreochromis niloticus) | |
| Bilen et al. | Effects of different protein sources on growth performance and food consumption of goldfish, Carassius auratus | |
| Verma et al. | Effect of special fish feed prepared using food industrial waste on Labeo rohita | |
| Jaseera et al. | Thraustochytrids in aquaculture: Can it replace fish meal in Aquafeed? | |
| Aktaş et al. | The changes in the molecular weight profiles and biochemical compositions of potential feed ingredients for sustainable aquaculture | |
| Khan et al. | Alternative sources of proteins in farm animal feeding | |
| JPH0723718A (en) | Feed for culture fish | |
| Arai | Practical Feeding—Eels | |
| KR101879776B1 (en) | Feed composition in the formulated feed for abalone | |
| WO2021130078A1 (en) | Aquaculture feed |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A80 | Written request to apply exceptions to lack of novelty of invention |
Free format text: JAPANESE INTERMEDIATE CODE: A80 Effective date: 20160615 |
|
| A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20190514 |
|
| A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20190515 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20190516 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20190514 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20190515 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20200220 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20200324 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20200525 |
|
| A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20200821 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20200821 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20200929 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20201130 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20210302 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20210330 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6865456 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313113 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |