CN111019996A - Method for preparing active polypeptide by liquid fermentation of camellia seed meal - Google Patents
Method for preparing active polypeptide by liquid fermentation of camellia seed meal Download PDFInfo
- Publication number
- CN111019996A CN111019996A CN202010056581.6A CN202010056581A CN111019996A CN 111019996 A CN111019996 A CN 111019996A CN 202010056581 A CN202010056581 A CN 202010056581A CN 111019996 A CN111019996 A CN 111019996A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- seed meal
- camellia seed
- polypeptide
- liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000855 fermentation Methods 0.000 title claims abstract description 180
- 230000004151 fermentation Effects 0.000 title claims abstract description 180
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 95
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 95
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 95
- 235000012054 meals Nutrition 0.000 title claims abstract description 74
- 235000018597 common camellia Nutrition 0.000 title claims abstract description 71
- 238000000034 method Methods 0.000 title claims abstract description 60
- 239000007788 liquid Substances 0.000 title claims abstract description 55
- 240000001548 Camellia japonica Species 0.000 title claims description 64
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 34
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 34
- 239000001963 growth medium Substances 0.000 claims abstract description 34
- 244000199866 Lactobacillus casei Species 0.000 claims abstract description 33
- 235000013958 Lactobacillus casei Nutrition 0.000 claims abstract description 33
- 229940017800 lactobacillus casei Drugs 0.000 claims abstract description 33
- 239000000287 crude extract Substances 0.000 claims abstract description 12
- 230000008569 process Effects 0.000 claims abstract description 12
- 239000007787 solid Substances 0.000 claims abstract description 12
- 238000009630 liquid culture Methods 0.000 claims abstract description 8
- 230000004913 activation Effects 0.000 claims abstract description 7
- 230000003321 amplification Effects 0.000 claims abstract description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 3
- 238000011081 inoculation Methods 0.000 claims description 26
- 241000526900 Camellia oleifera Species 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 13
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 10
- 230000001954 sterilising effect Effects 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 8
- 229930091371 Fructose Natural products 0.000 claims description 6
- 239000005715 Fructose Substances 0.000 claims description 6
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 6
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 6
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 229940099596 manganese sulfate Drugs 0.000 claims description 6
- 239000011702 manganese sulphate Substances 0.000 claims description 6
- 235000007079 manganese sulphate Nutrition 0.000 claims description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- 239000012880 LB liquid culture medium Substances 0.000 claims description 5
- 239000000284 extract Substances 0.000 claims description 3
- 239000004698 Polyethylene Substances 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- -1 polyethylene Polymers 0.000 claims description 2
- 229920000573 polyethylene Polymers 0.000 claims description 2
- 238000007873 sieving Methods 0.000 claims description 2
- 241000209507 Camellia Species 0.000 abstract description 7
- 239000002994 raw material Substances 0.000 abstract description 6
- 230000003078 antioxidant effect Effects 0.000 abstract description 4
- 241001122767 Theaceae Species 0.000 description 16
- 230000000694 effects Effects 0.000 description 15
- 102000004190 Enzymes Human genes 0.000 description 14
- 108090000790 Enzymes Proteins 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 244000005700 microbiome Species 0.000 description 8
- 229920000604 Polyethylene Glycol 200 Polymers 0.000 description 7
- 238000002835 absorbance Methods 0.000 description 7
- 229940024606 amino acid Drugs 0.000 description 7
- 150000001413 amino acids Chemical class 0.000 description 7
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 7
- 230000008901 benefit Effects 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 239000012488 sample solution Substances 0.000 description 6
- 238000006460 hydrolysis reaction Methods 0.000 description 5
- 230000000813 microbial effect Effects 0.000 description 5
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 239000006227 byproduct Substances 0.000 description 3
- 239000010495 camellia oil Substances 0.000 description 3
- 230000007073 chemical hydrolysis Effects 0.000 description 3
- 239000002131 composite material Substances 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 235000019750 Crude protein Nutrition 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 241001148470 aerobic bacillus Species 0.000 description 2
- 230000000433 anti-nutritional effect Effects 0.000 description 2
- OHJMTUPIZMNBFR-UHFFFAOYSA-N biuret Chemical compound NC(=O)NC(N)=O OHJMTUPIZMNBFR-UHFFFAOYSA-N 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 229940113115 polyethylene glycol 200 Drugs 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229930182490 saponin Natural products 0.000 description 2
- 150000007949 saponins Chemical class 0.000 description 2
- 230000002000 scavenging effect Effects 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- FJPHHBGPPJXISY-KBPBESRZSA-N (2s)-2-[[(2s)-2-[[2-[(2-aminoacetyl)amino]acetyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-5-(diaminomethylideneamino)pentanoic acid Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CNC(=O)CN)CC1=CC=C(O)C=C1 FJPHHBGPPJXISY-KBPBESRZSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 244000060011 Cocos nucifera Species 0.000 description 1
- 235000013162 Cocos nucifera Nutrition 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 244000147058 Derris elliptica Species 0.000 description 1
- 240000003133 Elaeis guineensis Species 0.000 description 1
- 235000001950 Elaeis guineensis Nutrition 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-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
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-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
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000007817 Olea europaea Species 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 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
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000002155 anti-virotic effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 235000019784 crude fat Nutrition 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- HEILIGJNYTWOHU-UHFFFAOYSA-N ethanol 2-hydroxybenzoic acid Chemical compound CCO.OC(=O)C1=CC=CC=C1O HEILIGJNYTWOHU-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 108010065713 glycyl-glycyl-tyrosyl-arginine Proteins 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000003895 organic fertilizer Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 235000021067 refined food Nutrition 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 238000010563 solid-state fermentation Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000001648 tannin Substances 0.000 description 1
- 235000018553 tannin Nutrition 0.000 description 1
- 229920001864 tannin Polymers 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention provides a method for preparing active polypeptide by camellia seed meal liquid fermentation, which comprises the following steps: respectively inoculating strains of bacillus natto and lactobacillus casei to a solid culture medium for activation, and transferring the strains to a corresponding liquid culture medium for amplification culture to prepare seed liquid; preparing a liquid fermentation culture medium by taking camellia seed meal as a raw material; inoculating seed solutions of bacillus natto and lactobacillus casei into a fermentation culture medium at the same time, performing fermentation culture, and applying a low-intensity magnetic field in the whole fermentation culture process; and after the fermentation is finished, centrifuging the fermentation liquor to obtain the camellia seed polypeptide crude extract. The content of the camellia seed polypeptide prepared by the method reaches 12.63mg/mL, the polypeptide yield reaches 10.53%, and meanwhile, the camellia seed polypeptide has strong antioxidant activity and high application value.
Description
Technical Field
The invention relates to the technical field of polypeptide preparation, in particular to a method for preparing active polypeptide by camellia seed meal liquid fermentation.
Background
Oil tea (tea-oil camellia)Camellia OleiferaAbel) is a perennial woody plant of the camellia family, is a woody oil tree species which is specific in China, is also one of four woody oil plants which are famous with oil palm, olive and coconut, and is mainly distributed in provinces such as Hunan, Jiangxi, Guangxi, Zhejiang, Fujian, Anhui, Guizhou and the like in the south of China. The camellia seed meal is a byproduct of camellia seed after camellia oil is extracted, and contains rich nutrient substances. It is reported that camellia seed mealThe tea saponin contains 30-40% of saccharides, 12-15% of crude protein, 1-5% of crude fat, 6-39% of crude fiber, 2-6% of ash, 10-15% of tea saponin, 1-2% of tannin, and various inorganic trace elements such as iron, copper, manganese, zinc, magnesium, calcium and the like. Along with the continuous increase of the demand and output of the tea oil, the processing by-product camellia oleifera seed meal is also continuously increased, but most camellia oleifera seed meal is only used as organic fertilizer or fuel at present, the development and utilization degree of the camellia oleifera seed meal is far insufficient, and the waste of a large amount of resources is caused, so that the comprehensive processing and utilization research of the camellia oleifera seed meal has important significance.
The oil-tea camellia seed meal has comprehensive amino acid composition of protein and is rich in 17 amino acids, wherein 8 amino acids of threonine, phenylalanine, leucine, lysine, tryptophan, valine, isoleucine and methionine are essential amino acids for human bodies, and the composition and the content of the amino acids meet the recommended values of food organization and world health organization of the United nations, so that the oil-tea camellia seed meal is a high-quality protein resource. The camellia seed meal is used as a raw material to directly extract protein, and can be used as a protein source of animal feed, processed food and the like. The protein is hydrolyzed by various methods to obtain a compound hydrolysate of polypeptide, amino acid and a small amount of protein, wherein the polypeptide is not only beneficial to the digestion and absorption of organisms, but also has wide biological activity, such as immunoregulation, hormone regulation, antivirus, antibiosis, antioxidation, blood pressure reduction, blood fat reduction, cholesterol reduction and the like. Therefore, the protein in the camellia seed meal is prepared into the active polypeptide, and a new way can be provided for the development and utilization of the camellia seed meal protein resource.
The production of polypeptides is technically very diverse, and the quality and biochemical properties of the polypeptides obtained vary widely, both with different methods and with different techniques and with the choice of different conditions in the same technique. At present, the methods for preparing polypeptides mainly include chemical hydrolysis, enzymatic hydrolysis and microbial fermentation. The chemical hydrolysis method has the advantages of difficult control of the process, uneven distribution of produced polypeptide molecules, unstable product quality, large physiological function difference of products, easy damage of amino acid, generation of harmful products, and great environmental pollution, and is rarely adopted at present. The enzymolysis method is a mild and effective method, and has the advantages of shortening the hydrolysis time, better controlling the hydrolysis degree and easily separating the products due to good enzyme specificity and high efficiency, thereby being very suitable for the requirement of industrial production. However, as the enzymatic reaction is studied in depth, the limitations of the enzymatic method emerge. For example, the enzymolysis method firstly extracts the protein in the raw material, and the extraction process is complex; the extracted protein is subjected to enzymolysis to obtain polypeptide, and an enzyme preparation is expensive, so that the production cost is high, and anti-nutritional factors in the raw materials cannot be eliminated. The microbial fermentation method can directly utilize raw materials for fermentation, and the microorganisms can produce a plurality of enzymes including partial enzymes which can not be synthesized by human beings, and the enzymes can carry out enzymolysis on protein into polypeptide and amino acid under specific fermentation conditions, so that the anti-nutritional factors in the raw materials can be degraded, the production steps can be simplified, and compared with a chemical hydrolysis method, the method has low pollution and almost no toxic and harmful byproducts are generated; compared with the enzymolysis method, the method has the advantages of low cost, strong applicability and wider application prospect.
The preparation of polypeptide by microbial fermentation can be divided into solid fermentation and liquid fermentation. The former has simple operation, less water content in the culture medium, low requirement on aseptic condition, simple post-treatment and less pollutant. However, the polypeptide prepared by solid state fermentation has low yield and long fermentation period. The liquid fermentation method has the advantages of rapid microbial growth and propagation, full protein degradation, easy control of the fermentation process, relatively high requirements on sterile conditions, and more advantages in consideration of polypeptide content and polypeptide yield.
At present, most of preparation methods of camellia seed polypeptide are enzymolysis methods, related patents and documents are more, research reports for preparing camellia seed polypeptide by adopting a fermentation method are few, and the conventional microbial fermentation method generally has the defects of low fermentation efficiency, low protein utilization rate, low polypeptide content, low yield and the like.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a method for preparing active polypeptide by camellia seed meal liquid fermentation.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing active polypeptide by camellia seed meal liquid fermentation is characterized by comprising the following steps:
1) strain activation and enlarged culture: respectively inoculating strains of bacillus natto and lactobacillus casei in an LB solid culture medium and an MRS solid culture medium, and culturing at 37 ℃ for 24 hours to activate the strains; then picking 2-3 ring strains from the activated strain slant in an aseptic operation mode, transferring the strains to a corresponding liquid culture medium for amplification culture to enable the concentration of the strains to reach 107~108CFU/mL, preparing seed liquid for fermentation;
2) preparing a liquid fermentation culture medium: after the camellia seed meal is pretreated, a fermentation medium is prepared according to the following formula: 100-150 g/L of camellia seed meal, 5-15 g/L of fructose, 6-14 g/L of sodium chloride, 0.5-1.0 g/L of magnesium sulfate, 0.02-0.06 g/L of manganese sulfate, 3.5-6.5 g/L of potassium dihydrogen phosphate, 0.8-2.0 g/L of disodium hydrogen phosphate, 2.0-4.0 g/L of calcium carbonate, and 0-1.2 g/L of polyethylene glycol-2000.8, wherein the initial pH is 6.5-7.0, and the camellia seed meal is subjected to sterilization treatment;
3) magnetic field assisted fermentation culture: simultaneously inoculating seed liquids of bacillus natto and lactobacillus casei into a liquid fermentation culture medium, performing fermentation culture, and applying a low-intensity magnetic field in the whole fermentation culture process; the fermentation temperature is 30-38 ℃, the rotating speed of a shaking table is 150-250 r/min, and the fermentation time is 24-48 h;
4) and (3) treating fermentation liquor: after the fermentation is finished, centrifuging the fermentation liquor for 10min at the speed of 5000r/min, and taking the supernatant fluid, namely the crude extract of the camellia seed polypeptide.
In the step 1), the specific method for the expanded culture comprises the following steps: transferring the bacillus natto into an LB liquid culture medium, and performing shake cultivation for 12-18 h at 37 ℃ and 150-200 r/min; transferring lactobacillus casei into MRS liquid culture, and performing shake culture at 37 ℃ and 150-200 r/min for 18-24 h.
In the step 2), the method for pretreating the camellia seed meal comprises the following steps: airing and drying the mechanically squeezed or low-temperature cold-pressed camellia oleifera seed meal, crushing the camellia oleifera seed meal by using a crusher, and sieving the camellia oleifera seed meal by using a 60-80-mesh sieve.
In the step 2), the sterilization treatment condition is sterilization at 115 ℃ for 20-30 min.
In the step 3), the total inoculation amount of the bacillus natto and the lactobacillus casei is 8-12%, and the inoculation ratio is 1: 1.
In step 3), the fermentation temperature is preferably 34 ℃, the rotating speed of the shaking table is preferably 200r/min, and the fermentation time is preferably 36 h.
In the step 3), the low-intensity magnetic field refers to a magnetic field with the magnetic field intensity of 20-30 kA/m.
In the step 4), the crude extract of the camellia seed polypeptide is subjected to the steps of filtering, vacuum concentration, freeze drying, screening and the like to obtain a camellia seed polypeptide product.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, bacillus natto and lactobacillus casei are selected as composite strains, both the two strains can generate protease with stronger activity, the produced enzyme systems are different, complementation can be formed, and compared with single strain fermentation, macromolecular protein in camellia seed meal can be fully degraded, and the polypeptide with biological activity is generated.
(2) The optimal fermentation process for preparing the polypeptide by the liquid fermentation of the camellia seed meal is determined by optimizing the components of the fermentation medium and the fermentation conditions, and a low-strength magnetic field is applied in the whole fermentation process, so that the growth and metabolism of the strain can be effectively promoted and the enzyme production capacity of the strain can be improved under the auxiliary action of the magnetic field, the conversion efficiency of the strain on the medium is improved, the content of the polypeptide in the camellia seed after fermentation reaches 12.63mg/mL, and the yield of the polypeptide reaches 10.53%.
(3) The camellia seed polypeptide prepared by the invention has small molecular weight and strong antioxidant activity, and the clearance rate of DPPH free radicals reaches 77.40%, and the clearance rate of OH free radicals reaches 89.25%. The technical scheme of the invention has the advantages of no pollution, low cost, safe product, no toxicity, no side effect and higher popularization value.
Drawings
In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,
FIG. 1 shows the effect of the addition of camellia seed meal on the content of polypeptides;
FIG. 2 shows the effect of the amount of PEG-200 added on the amount of polypeptide;
FIG. 3 is a graph showing the effect of inoculum size on polypeptide content;
FIG. 4 is a graph of the effect of fermentation temperature on polypeptide content;
FIG. 5 is a graph showing the effect of fermentation time on polypeptide content.
Detailed Description
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available.
Bacillus natto (Bacillus natto) ((Bacillus natto) Provided by China agricultural microorganism strain preservation management center, with the number ACCC 19833; lactobacillus casei (A), (B), (C), (Lactobacillus casei) Provided by China center for culture Collection of Industrial microorganisms, with the number CICC 20277; the low-temperature cold-pressed tea seed meal is purchased from Tianfu grease Co., Ltd. of Fujian province, wherein the content of crude protein is 14.37%.
Example 1
A method for preparing active polypeptide by camellia seed meal liquid fermentation comprises the following steps:
1) strain activation and enlarged culture: respectively inoculating strains of bacillus natto and lactobacillus casei in an LB solid culture medium and an MRS solid culture medium, and culturing at 37 ℃ for 24 hours to activate the strains; picking 2-3 ring strains from the activated strain inclined plane in an aseptic operation mode, transferring the bacillus natto into an LB liquid culture medium, and carrying out shake cultivation for 18h at 37 ℃ and 180 r/min; transferring Lactobacillus casei to MRS liquid culture, shake culturing at 37 deg.C and 180r/min for 24 hr to make thallus concentration reach 107~108CFU/mL, preparing seed liquid for fermentation;
2) preparing a liquid fermentation culture medium: drying and drying the low-temperature cold-pressed tea seed meal, crushing the tea seed meal by using a crusher, screening the crushed tea seed meal by using a 60-80-mesh screen, and preparing a fermentation culture medium according to the following formula: 120g/L of camellia seed meal, 10g/L of fructose, 10g/L of sodium chloride, 0.75g/L of magnesium sulfate, 0.04g/L of manganese sulfate, 5.0g/L of monopotassium phosphate, 1.4g/L of disodium hydrogen phosphate, 3.0g/L of calcium carbonate, 2001.0 g/L of polyethylene glycol, the initial pH value of 6.8, and sterilizing at 115 ℃ for 20 min;
3) magnetic field assisted fermentation culture: simultaneously inoculating seed liquids of bacillus natto and lactobacillus casei into a liquid fermentation culture medium, wherein the total inoculation amount is 10%, the inoculation ratio is 1:1, performing fermentation culture, and applying a low-strength magnetic field with the magnetic field intensity of 20-30 kA/m in the whole fermentation culture process; the fermentation temperature is 34 ℃, the rotating speed of a shaking table is 200r/min, and the fermentation time is 36 h;
4) and (3) treating fermentation liquor: after the fermentation is finished, centrifuging the fermentation liquor for 10min at the speed of 5000r/min, and taking the supernatant fluid, namely the crude extract of the camellia seed polypeptide.
Example 2 optimization test of camellia seed meal liquid state fermentation process
Determination of polypeptide content
Drawing a standard curve: using 5% TCA (trichloroacetic acid) solution to prepare Gly-Gly-Tyr-Arg tetrapeptide standard solutions with mass concentrations of 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4mg/mL in sequence, then respectively taking 6.0mL of the standard solutions, adding 4.0mL of biuret reagent, uniformly mixing on a vortex mixer, standing for 10min, centrifuging at 2000r/min for 10min, taking supernatant, and measuring OD value at the wavelength of 540 nm. A standard curve was prepared with the mass concentration of the tetrapeptide as X (mg/mL) on the abscissa and the OD value as Y on the ordinate.
And (3) sample determination: taking 2mL of sample solution, adding 2mL of 10% TCA solution, uniformly mixing on a vortex mixer, standing for 30min, centrifuging for 10min at 10000r/min, transferring all supernate into a 50mL volumetric flask, metering volume to a scale with 5% TCA solution, and shaking up. Putting 6mL of the solution into a test tube, adding 4mL of biuret reagent, uniformly mixing on a vortex mixer, standing for 10min, centrifuging at 2000r/min for 10min, and taking supernatant to measure OD value at 540 nm. And comparing with a standard curve to obtain the polypeptide content in the sample solution.
2.1 selection of the amount of Camellia seed meal added
According to the method in the embodiment 1, by taking the addition amount of the camellia oleifera seed meal in a fermentation medium as a factor to be considered, fermentation media with the addition amounts of the camellia oleifera seed meal of 40g/L, 80g/L, 120g/L, 160g/L and 200g/L are respectively prepared, seed liquids of bacillus natto and lactobacillus casei are inoculated for fermentation culture, the total inoculation amount is 10%, the inoculation ratio is 1:1, the fermentation temperature is 34 ℃, the rotating speed of a shaking table is 200r/min, the fermentation time is 36 hours, and a low-intensity magnetic field with the magnetic field intensity of 20-30 kA/m is applied in the whole fermentation culture process. After fermentation, the supernatant was taken to determine the polypeptide content, and the results are shown in FIG. 1.
The camellia oleifera seed meal is a main fermentation substrate and is also a nitrogen source of bacillus natto and lactobacillus casei. As can be seen from FIG. 1, with the increase of the addition amount of the camellia seed meal, the content of the polypeptide in the crude extract increases firstly and then decreases, and when the addition amount of the camellia seed meal is 120g/L, the content of the polypeptide reaches the maximum value. When the addition amount of the camellia seed meal is 40-120 g/L, the polypeptide content is gradually increased, because the protein content is increased along with the increase of the substrate concentration, sufficient nutrition is provided for the thalli, and the growth and metabolism of the thalli are facilitated; when the addition amount of the camellia seed meal is further increased, the moisture content is reduced, the fermentation liquor becomes viscous, the dissolved oxygen is reduced, and bacillus natto and lactobacillus casei are aerobic bacteria, so that certain negative effects are exerted on the growth and metabolism of bacteria, and the polypeptide content is reduced.
2.2 selection of the amount of polyethylene glycol-200 added
According to the method in the embodiment 1, by taking the addition amount of polyethylene glycol-200 in the fermentation medium as a factor to be considered, fermentation media with the addition amounts of PEG-200 of 0, 0.5g/L, 1.0g/L, 1.5g/L, 2.0g/L and 2.5g/L are respectively prepared, seed solutions of bacillus natto and lactobacillus casei are inoculated for fermentation culture, the total inoculation amount is 10%, the inoculation ratio is 1:1, the fermentation temperature is 34 ℃, the rotation speed of a shaking table is 200r/min, the fermentation time is 36h, and a low-intensity magnetic field with the magnetic field intensity of 20-30 kA/m is applied in the whole process of the fermentation culture. After fermentation, the supernatant was taken to determine the polypeptide content, and the results are shown in FIG. 2.
As can be seen from FIG. 2, when the amount of PEG-200 added was 1.0g/L, the amount of the polypeptide in the crude extract reached the maximum, and the amount of the polypeptide decreased below or above this amount. PEG-200 is a non-ionic surfactant, i.e., it does not ionize in aqueous solutions and does not generate ions. The proper amount of PEG-200 can increase the permeability of the cell membrane of the strain, promote the secretion of protease of the strain and is beneficial to improving the conversion efficiency of the strain to fermentation substrates.
2.3 selection of inoculum size
According to the method in the embodiment 1, taking the total inoculation amount as a factor for investigation, taking the liquid fermentation medium, simultaneously inoculating the seed liquid of bacillus natto and lactobacillus casei, wherein the inoculation ratio is 1:1, the total inoculation amount is respectively 4%, 6%, 8%, 10%, 12% and 14%, the fermentation temperature is 34 ℃, the rotating speed of a shaking table is 200r/min, the fermentation time is 36h, and a low-intensity magnetic field with the magnetic field intensity of 20-30 kA/m is applied in the whole fermentation and culture process. After fermentation, the supernatant was taken to determine the polypeptide content, and the results are shown in FIG. 3.
As can be seen from FIG. 3, with the increase of the inoculation amount, the polypeptide content in the crude extract is in an increasing trend, reaches the maximum value when the inoculation amount is 10%, and is in a decreasing trend when the inoculation amount is between 10% and 14%, because when the inoculation amount is too large, a large amount of microorganisms grow and reproduce, so that the nutrition in the culture medium at the later stage of fermentation is insufficient, and part of microorganisms die or small molecular polypeptides liberated by using enzymes are used for self growth and development. Therefore, the inoculation amount is preferably 10%.
2.4 selection of fermentation temperature
According to the method in the embodiment 1, taking the fermentation temperature as a factor to be considered, taking the liquid fermentation medium, inoculating the seed liquid of bacillus natto and lactobacillus casei at the same time, wherein the inoculation ratio is 1:1, the total inoculation amount is 10%, the fermentation temperatures are respectively 28 ℃, 31 ℃, 34 ℃, 37 ℃ and 40 ℃, the rotating speed of a shaking table is 200r/min, the fermentation time is 36h, and a low-intensity magnetic field with the magnetic field intensity of 20-30 kA/m is applied in the whole process of fermentation culture. After fermentation, the supernatant was taken to determine the polypeptide content, and the results are shown in FIG. 4.
In the liquid fermentation process, the temperature is one of the main factors of microorganism growth and enzyme production, and the control of the temperature is very important. As can be seen from FIG. 4, the polypeptide content tends to increase first and then decrease with increasing fermentation temperature, and the fermentation effect is optimal at 34 ℃. When the fermentation temperature is lower, the metabolic activity of the strain is weaker, and the enzyme activity is lower; the over-high fermentation temperature can inhibit the growth of the strain, and the enzyme activity is not high as well. Therefore, the fermentation temperature is preferably 34 ℃.
2.5 selection of fermentation time
According to the method in the embodiment 1, taking fermentation time as a factor to be considered, taking a liquid fermentation medium, inoculating seed liquid of bacillus natto and lactobacillus casei at the same time, carrying out fermentation culture, wherein the inoculation ratio is 1:1, the total inoculation amount is 10%, the fermentation temperature is 34 ℃, the rotating speed of a shaking table is 200r/min, applying a low-intensity magnetic field with the magnetic field intensity of 20-30 kA/m in the whole fermentation culture process, carrying out fermentation for 72h, taking supernatant liquid every 12h, and measuring the polypeptide content, wherein the result is shown in figure 5.
As can be seen from FIG. 5, in the range of 12h to 36h, the polypeptide content gradually increases with the increase of the fermentation time, and the polypeptide content reaches the maximum value in 36h of fermentation. The reason why the polypeptide content is decreased as the fermentation time is prolonged is that when the number of the bacteria is accumulated to a certain degree, the nutrient in the culture solution is insufficient, and the microorganisms need to use the small molecular polypeptide decomposed by the enzyme for growth and reproduction, so that the product yield is reduced. Therefore, the fermentation time is preferably 36 hours.
Comparative example 1
A method for preparing active polypeptide by camellia seed meal liquid fermentation comprises the following steps:
1) strain activation and enlarged culture: inoculating the bacillus natto into an LB solid culture medium, and culturing at 37 ℃ for 24h to activate the strains; then picking 2-3 ring strains from the activated strain slant in an aseptic operation mode, transferring the strains into an LB liquid culture medium, and carrying out shake cultivation at 37 ℃ and 180r/min for 18h to ensure that the concentration of the strains reaches 107~108CFU/mL, preparing seed liquid for fermentation;
2) preparing a liquid fermentation culture medium: drying and drying the low-temperature cold-pressed tea seed meal, crushing the tea seed meal by using a crusher, screening the crushed tea seed meal by using a 60-80-mesh screen, and preparing a fermentation culture medium according to the following formula: 120g/L of camellia seed meal, 10g/L of fructose, 10g/L of sodium chloride, 0.75g/L of magnesium sulfate, 0.04g/L of manganese sulfate, 5.0g/L of monopotassium phosphate, 1.4g/L of disodium hydrogen phosphate, 3.0g/L of calcium carbonate, 2001.0 g/L of polyethylene glycol, the initial pH value of 6.8, and sterilizing at 115 ℃ for 20 min;
3) fermentation culture: inoculating a liquid fermentation culture medium into a seed solution of bacillus natto for fermentation culture, wherein the inoculation amount is 10%, the fermentation temperature is 34 ℃, the rotating speed of a shaking table is 200r/min, the fermentation time is 36h, and a low-intensity magnetic field is applied in the whole fermentation culture process, and the magnetic field intensity is 20-30 kA/m;
4) and (3) treating fermentation liquor: after the fermentation is finished, centrifuging the fermentation liquor for 10min at the speed of 5000r/min, and taking the supernatant fluid, namely the crude extract of the camellia seed polypeptide.
Comparative example 2
A method for preparing active polypeptide by camellia seed meal liquid fermentation comprises the following steps:
1) strain activation and enlarged culture: inoculating lactobacillus casei strain in MRS solid culture medium, and culturing at 37 deg.C for 24 hr to activate the strain; selecting 2-3 ring strains from the activated strain slant in an aseptic operation mode, transferring the strains to MRS liquid culture, and performing shake culture at 37 ℃ and 180r/min for 24h to ensure that the concentration of the strains reaches 107~108CFU/mL, preparing seed liquid for fermentation;
2) preparing a liquid fermentation culture medium: drying and drying the low-temperature cold-pressed tea seed meal, crushing the tea seed meal by using a crusher, screening the crushed tea seed meal by using a 60-80-mesh screen, and preparing a fermentation culture medium according to the following formula: 120g/L of camellia seed meal, 10g/L of fructose, 10g/L of sodium chloride, 0.75g/L of magnesium sulfate, 0.04g/L of manganese sulfate, 5.0g/L of monopotassium phosphate, 1.4g/L of disodium hydrogen phosphate, 3.0g/L of calcium carbonate, 2001.0 g/L of polyethylene glycol, the initial pH value of 6.8, and sterilizing at 115 ℃ for 20 min;
3) fermentation culture: inoculating a liquid fermentation culture medium into a seed solution of lactobacillus casei for fermentation culture, wherein the inoculation amount is 10%, the fermentation temperature is 34 ℃, the rotating speed of a shaking table is 200r/min, the fermentation time is 36h, and a low-strength magnetic field is applied in the whole fermentation culture process, and the magnetic field strength is 20-30 kA/m;
4) and (3) treating fermentation liquor: after the fermentation is finished, centrifuging the fermentation liquor for 10min at the speed of 5000r/min, and taking the supernatant fluid, namely the crude extract of the camellia seed polypeptide.
Comparative example 3
A method for preparing active polypeptide by camellia seed meal liquid fermentation comprises the following steps:
1) strain activation and enlarged culture: respectively inoculating strains of bacillus natto and lactobacillus casei in an LB solid culture medium and an MRS solid culture medium, and culturing at 37 ℃ for 24 hours to activate the strains; picking 2-3 ring strains from the activated strain inclined plane in an aseptic operation mode, transferring the bacillus natto into an LB liquid culture medium, and carrying out shake cultivation for 18h at 37 ℃ and 180 r/min; transferring Lactobacillus casei to MRS liquid culture, shake culturing at 37 deg.C and 180r/min for 24 hr to make thallus concentration reach 107~108CFU/mL, preparing seed liquid for fermentation;
2) preparing a liquid fermentation culture medium: drying and drying the low-temperature cold-pressed tea seed meal, crushing the tea seed meal by using a crusher, screening the crushed tea seed meal by using a 60-80-mesh screen, and preparing a fermentation culture medium according to the following formula: 120g/L of camellia seed meal, 10g/L of fructose, 10g/L of sodium chloride, 0.75g/L of magnesium sulfate, 0.04g/L of manganese sulfate, 5.0g/L of monopotassium phosphate, 1.4g/L of disodium hydrogen phosphate, 3.0g/L of calcium carbonate, 2001.0 g/L of polyethylene glycol, the initial pH value of 6.8, and sterilizing at 115 ℃ for 20 min;
3) fermentation culture: inoculating a liquid fermentation culture medium into seed liquid of bacillus natto and lactobacillus casei for fermentation culture, wherein the total inoculation amount is 10%, the inoculation ratio is 1:1, the fermentation temperature is 34 ℃, the rotating speed of a shaking table is 200r/min, and the fermentation time is 36 h;
4) and (3) treating fermentation liquor: after the fermentation is finished, centrifuging the fermentation liquor for 10min at the speed of 5000r/min, and taking the supernatant fluid, namely the crude extract of the camellia seed polypeptide.
Example 3
1. Determination of polypeptide content and polypeptide yield
The polypeptide content was determined according to the method in example 2; the polypeptide yield was calculated according to the following formula:
polypeptide content = (C × V)/M × 100%
In the formula: c is the content of polypeptide in the fermentation liquor, mg/mL; v is the total volume of the fermentation liquor, mL; m is the mass of the camellia seed meal, mg.
2. Determination of the relative molecular weight distribution of the Polypeptides
And (3) measuring the molecular weight distribution of the sample solution by adopting a high-efficiency gel filtration chromatography, and analyzing and calculating the mass fraction of the polypeptide with the relative molecular weight of less than 1000 Da.
3. Determination of antioxidant Activity of Polypeptides
DPPH clearance measurement: 2mL of the sample solution is added with 2mL of 0.1mmol/L DPPH 95% ethanol solution, the mixture is shaken evenly, the reaction is carried out for 30min in a dark place at room temperature, and the absorbance is measured at the wavelength of 517 nm by an ultraviolet spectrophotometer. Each experiment was repeated 3 times and the average was taken. The formula for the calculation of DPPH.clear capacity of the sample is as follows:
dpph.clearance (%) = [1- (a)1-A2)/A3]×100%
In the formula: a. the1Is the absorbance of the sample; a. the2Absorbance for blank (equal amount of 95% ethanol solution instead of DPPH solution); a. the3Absorbance was taken as the control (the sample was replaced with an equal amount of 95% ethanol solution).
Determination of OH scavenging Capacity: 1.0mL of sample solution is added with 2mL of 1.8mmol/L FeSO41.5mL of 1.8mmol/L salicylic acid-ethanol and 0.1mL of 8.8mmol/LH2O2And carrying out water bath at 37 ℃ for 30min, and measuring the absorbance at the wavelength of 510nm by using an ultraviolet spectrophotometer after the reaction is finished. Each experiment was repeated 3 times and the average was taken. The sample has the following calculation formula for OH scavenging capacity:
OH clearance (%) = (a)0-Ai)/A0×100%
In the formula: a. the0Absorbance for control (deionized water instead of sample solution); a. theiIs the absorbance of the sample.
The crude extracts of camellia seed polypeptides obtained in example 1, comparative example 2 and comparative example 3 were measured according to the above methods, and the results are shown in table 1.
As can be seen from Table 1, in example 1, compared with comparative examples 1, 2 and 3, the content of polypeptide, the yield of polypeptide, DPPH clearance and OH clearance are all obviously improved, and the proportion of high-activity polypeptide fragments (the relative molecular weight is less than 1000 Da) is also obviously increased, which shows that the composite strain fermentation adopted by the invention has better fermentation effect than single strain fermentation, and the low-frequency magnetic field is applied in the fermentation process, so that the improvement of the fermentation effect is also obviously realized. The bacillus natto and the lactobacillus casei are aerobic bacteria, the growth temperature is approximate to the proper growth temperature, the bacillus natto and the lactobacillus casei can be symbiotic in one system, both the bacillus natto and the lactobacillus casei can produce protease with stronger activity, the produced enzyme systems are different and can form complementation, so that the macromolecular protein in the camellia seed meal can be fully degraded through composite fermentation, and the polypeptide with biological activity is generated. The magnetic field has great influence on the growth and metabolism of microorganisms, and the low-frequency magnetic field with the magnetic field intensity of 20-30 kA/m is applied in the fermentation process, so that the growth and the propagation of bacillus natto and lactobacillus casei can be promoted, the enzyme production activity of the bacillus natto and lactobacillus casei is improved, the polypeptide content and the yield of the fermentation product are improved, and the antioxidant activity of the fermentation product polypeptide is positively influenced.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Claims (8)
1. A method for preparing active polypeptide by camellia seed meal liquid fermentation is characterized by comprising the following steps:
1) strain activation and enlarged culture: respectively inoculating strains of bacillus natto and lactobacillus casei in an LB solid culture medium and an MRS solid culture medium, and culturing at 37 ℃ for 24 hours to activate the strains; then picking 2-3 ring strains from the activated strain slant in an aseptic operation mode, transferring the strains to a corresponding liquid culture medium for amplification culture to enable the concentration of the strains to reach 107~108CFU/mL, preparing seed liquid for fermentation;
2) preparing a liquid fermentation culture medium: after the camellia seed meal is pretreated, a fermentation medium is prepared according to the following formula: 100-150 g/L of camellia seed meal, 5-15 g/L of fructose, 6-14 g/L of sodium chloride, 0.5-1.0 g/L of magnesium sulfate, 0.02-0.06 g/L of manganese sulfate, 3.5-6.5 g/L of potassium dihydrogen phosphate, 0.8-2.0 g/L of disodium hydrogen phosphate, 2.0-4.0 g/L of calcium carbonate, and 0-1.2 g/L of polyethylene glycol-2000.8, wherein the initial pH is 6.5-7.0, and the camellia seed meal is subjected to sterilization treatment;
3) magnetic field assisted fermentation culture: simultaneously inoculating seed liquids of bacillus natto and lactobacillus casei into a liquid fermentation culture medium, performing fermentation culture, and applying a low-intensity magnetic field in the whole fermentation culture process; the fermentation temperature is 30-38 ℃, the rotating speed of a shaking table is 150-250 r/min, and the fermentation time is 24-48 h;
4) and (3) treating fermentation liquor: after the fermentation is finished, centrifuging the fermentation liquor for 10min at the speed of 5000r/min, and taking the supernatant fluid, namely the crude extract of the camellia seed polypeptide.
2. The method for preparing active polypeptide by liquid fermentation of camellia seed meal according to claim 1, wherein in the step 1), the specific method for enlarged culture is as follows: transferring the bacillus natto into an LB liquid culture medium, and performing shake cultivation for 12-18 h at 37 ℃ and 150-200 r/min; transferring lactobacillus casei into MRS liquid culture, and performing shake culture at 37 ℃ and 150-200 r/min for 18-24 h.
3. The method for preparing active polypeptide by liquid fermentation of camellia seed meal according to claim 1, wherein in the step 2), the method for pretreating camellia seed meal comprises the following steps: airing and drying the mechanically squeezed or low-temperature cold-pressed camellia oleifera seed meal, crushing the camellia oleifera seed meal by using a crusher, and sieving the camellia oleifera seed meal by using a 60-80-mesh sieve.
4. The method for preparing active polypeptide by liquid fermentation of camellia seed meal according to claim 1, wherein in the step 2), the sterilization treatment condition is sterilization at 115 ℃ for 20-30 min.
5. The method for preparing active polypeptide by oil-tea camellia seed meal liquid fermentation according to claim 1, wherein in the step 3), the total inoculation amount of bacillus natto and lactobacillus casei is 8% -12%, and the inoculation ratio is 1: 1.
6. The method for preparing active polypeptide by liquid fermentation of camellia seed meal according to claim 1, wherein in the step 3), the fermentation temperature is preferably 34 ℃, the rotating speed of a shaking table is preferably 200r/min, and the fermentation time is preferably 36 h.
7. The method for preparing active polypeptide by liquid fermentation of camellia seed meal according to claim 1, wherein in the step 3), the low-intensity magnetic field is a magnetic field with the magnetic field intensity of 20-30 kA/m.
8. The method for preparing active polypeptide by liquid fermentation of camellia seed meal according to claim 1, wherein in the step 4), the crude camellia seed polypeptide extract is subjected to the steps of filtering, vacuum concentration, freeze drying, screening and the like to obtain a camellia seed polypeptide product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010056581.6A CN111019996A (en) | 2020-01-18 | 2020-01-18 | Method for preparing active polypeptide by liquid fermentation of camellia seed meal |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010056581.6A CN111019996A (en) | 2020-01-18 | 2020-01-18 | Method for preparing active polypeptide by liquid fermentation of camellia seed meal |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111019996A true CN111019996A (en) | 2020-04-17 |
Family
ID=70199239
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010056581.6A Pending CN111019996A (en) | 2020-01-18 | 2020-01-18 | Method for preparing active polypeptide by liquid fermentation of camellia seed meal |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111019996A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112553069A (en) * | 2020-11-18 | 2021-03-26 | 江苏大学 | Solid state fermentation method based on combination of multiple technologies of pulse magnetic field, pulse intense light and near infrared spectrum online detection |
| CN115029385A (en) * | 2022-07-25 | 2022-09-09 | 华南理工大学 | Method for extracting tea saponin from tea seed meal by microbial fermentation |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101773203A (en) * | 2010-03-05 | 2010-07-14 | 中国农业科学院油料作物研究所 | Method for biologically improving oilseed residue |
| JP2012056902A (en) * | 2010-09-10 | 2012-03-22 | Makoto Shigeta | Production method for camellia fermented extract exhibiting ceramide increasing action |
| CN102965301A (en) * | 2012-09-24 | 2013-03-13 | 上海交通大学 | Lactobacillus crustorum strain and its application |
| CN103039710A (en) * | 2013-01-15 | 2013-04-17 | 上海交通大学 | Method for producing camellia oil cake feed through mixed strain fermentation |
| CN103211084A (en) * | 2013-04-17 | 2013-07-24 | 安徽农业大学 | Method for preparing biological enzyme-enriched camellia seed meal fermented feed |
-
2020
- 2020-01-18 CN CN202010056581.6A patent/CN111019996A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101773203A (en) * | 2010-03-05 | 2010-07-14 | 中国农业科学院油料作物研究所 | Method for biologically improving oilseed residue |
| JP2012056902A (en) * | 2010-09-10 | 2012-03-22 | Makoto Shigeta | Production method for camellia fermented extract exhibiting ceramide increasing action |
| CN102965301A (en) * | 2012-09-24 | 2013-03-13 | 上海交通大学 | Lactobacillus crustorum strain and its application |
| CN103039710A (en) * | 2013-01-15 | 2013-04-17 | 上海交通大学 | Method for producing camellia oil cake feed through mixed strain fermentation |
| CN103211084A (en) * | 2013-04-17 | 2013-07-24 | 安徽农业大学 | Method for preparing biological enzyme-enriched camellia seed meal fermented feed |
Non-Patent Citations (1)
| Title |
|---|
| 刘学高等: "《广东省生物工程会1990-1992年学术研讨论文集》", 30 November 1992, 暨南大学出版社 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112553069A (en) * | 2020-11-18 | 2021-03-26 | 江苏大学 | Solid state fermentation method based on combination of multiple technologies of pulse magnetic field, pulse intense light and near infrared spectrum online detection |
| CN112553069B (en) * | 2020-11-18 | 2024-05-14 | 江苏大学 | Solid state fermentation method based on combination of pulse magnetic field, pulse strong light and near infrared spectrum on-line detection multiple technologies |
| CN115029385A (en) * | 2022-07-25 | 2022-09-09 | 华南理工大学 | Method for extracting tea saponin from tea seed meal by microbial fermentation |
| CN115029385B (en) * | 2022-07-25 | 2024-05-24 | 华南理工大学 | Method for extracting tea saponin from tea seed cake by microbial fermentation |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103549130B (en) | Method for producing seaweed protein feed through synergistic effect of enzymolysis and fermentation | |
| CN103710292A (en) | Pseudomonas aeruginosa and application of pseudomonas aeruginosa in aspect of degrading aflatoxin | |
| CN111019996A (en) | Method for preparing active polypeptide by liquid fermentation of camellia seed meal | |
| CN110093281B (en) | Phellinus igniarius liquid submerged fermentation culture process | |
| CN114107403B (en) | Method for co-producing ellagic acid and biological feed by fermenting pericarpium Granati with microbial community | |
| CN106348887A (en) | Method for preparing amino acid liquid water-soluble fertilizer through compound stepped fermentation of seaweed | |
| CN102511650B (en) | Method for preparing protein feed by using Jerusalem artichoke residues | |
| CN110713939B (en) | Strain for degrading lignocellulose source inhibitor under extremely low pH condition and application | |
| CN1699585A (en) | Process for preparing biological antibacterial agent | |
| CN110396483B (en) | High-temperature straw degradation bacterium B-8, and microbial inoculum and application thereof | |
| CN112195172A (en) | Composite microbial inoculum for kitchen waste water treatment and method for recycling kitchen waste water | |
| CN108796027B (en) | Method for producing carotenoid | |
| CN105767508A (en) | Protein feed production method by mixing kitchen waste and vinegar residue | |
| CN102586115A (en) | Method for aerobically producing yeast extract by using tetracycline fungi residues | |
| CN110583867B (en) | Method for producing protein feed from waste potato residues in potato starch processing | |
| CN112746088B (en) | Method for co-producing xylitol and fuel ethanol by fermenting lignocellulose serving as raw material | |
| CN113430152A (en) | Glutamic acid fermentation method for improving thallus utilization rate | |
| CN103865865B (en) | A kind of sea cucumber enteron aisle produces Sumizyme MP bacterial strain and application thereof | |
| CN116354520A (en) | Application of monascus comosus in treatment of ultra-high concentration white spirit wastewater | |
| WO2022104772A1 (en) | Cyanobacteria composting composite microbial agent and preparation method therefor | |
| CN103749957B (en) | Preparation method for blue-green algae single-cell protein feed | |
| CN111333469A (en) | Organic fertilizer prepared by utilizing white spirit solid waste lees through two-step method and preparation method thereof | |
| AU2020102037A4 (en) | A method of efficiently increasing the alpha-glucosidase inhibitor content in fresh mulberry leaves by the solid-state fermentation | |
| CN112280103B (en) | Antibacterial packaging film and preparation method thereof | |
| CN114084967B (en) | Lucid ganoderma fermented Shuanghuanglian mushroom dreg for repairing polycyclic aromatic hydrocarbon benzo [ a ] pyrene polluted water environment and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20200417 |