CN113249359B - 一种利用酶提取桑叶功能成分的方法 - Google Patents
一种利用酶提取桑叶功能成分的方法 Download PDFInfo
- Publication number
- CN113249359B CN113249359B CN202110563357.0A CN202110563357A CN113249359B CN 113249359 B CN113249359 B CN 113249359B CN 202110563357 A CN202110563357 A CN 202110563357A CN 113249359 B CN113249359 B CN 113249359B
- Authority
- CN
- China
- Prior art keywords
- pectase
- cellulase
- crude enzyme
- preparing
- enzyme 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.)
- Active
Links
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 106
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 105
- 240000000249 Morus alba Species 0.000 title claims abstract description 101
- 235000008708 Morus alba Nutrition 0.000 title claims abstract description 101
- 238000000034 method Methods 0.000 title claims abstract description 26
- 239000004615 ingredient Substances 0.000 title description 5
- 229940088598 enzyme Drugs 0.000 claims abstract description 105
- 108010059892 Cellulase Proteins 0.000 claims abstract description 71
- 229940106157 cellulase Drugs 0.000 claims abstract description 71
- 239000007788 liquid Substances 0.000 claims abstract description 64
- 238000000855 fermentation Methods 0.000 claims abstract description 44
- 230000004151 fermentation Effects 0.000 claims abstract description 44
- 239000002609 medium Substances 0.000 claims abstract description 24
- 238000002360 preparation method Methods 0.000 claims abstract description 19
- 239000001963 growth medium Substances 0.000 claims abstract description 17
- 239000007787 solid Substances 0.000 claims abstract description 7
- 238000004519 manufacturing process Methods 0.000 claims abstract description 4
- 230000009849 deactivation Effects 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 37
- 229910001868 water Inorganic materials 0.000 claims description 31
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 20
- 239000000843 powder Substances 0.000 claims description 19
- 239000012141 concentrate Substances 0.000 claims description 13
- 238000002156 mixing Methods 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 12
- 238000001914 filtration Methods 0.000 claims description 11
- 230000001954 sterilising effect Effects 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 11
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 10
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 10
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 10
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 10
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 10
- 239000002002 slurry Substances 0.000 claims description 8
- 238000004537 pulping Methods 0.000 claims description 7
- 239000006187 pill Substances 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- 239000001110 calcium chloride Substances 0.000 claims description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 5
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 5
- 239000003826 tablet Substances 0.000 claims description 5
- 239000010902 straw Substances 0.000 claims description 4
- 235000011148 calcium chloride Nutrition 0.000 claims description 2
- 235000013305 food Nutrition 0.000 abstract description 9
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 239000002994 raw material Substances 0.000 abstract description 4
- 230000036541 health Effects 0.000 abstract description 3
- 238000012545 processing Methods 0.000 abstract description 3
- 239000002537 cosmetic Substances 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 46
- 230000000694 effects Effects 0.000 description 43
- 229930003935 flavonoid Natural products 0.000 description 19
- 150000002215 flavonoids Chemical class 0.000 description 19
- 235000017173 flavonoids Nutrition 0.000 description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 15
- 238000002835 absorbance Methods 0.000 description 15
- 238000000605 extraction Methods 0.000 description 14
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 13
- 229930003944 flavone Natural products 0.000 description 13
- 150000002212 flavone derivatives Chemical class 0.000 description 13
- 235000011949 flavones Nutrition 0.000 description 13
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 13
- 241000233866 Fungi Species 0.000 description 9
- 239000001913 cellulose Substances 0.000 description 9
- 229920002678 cellulose Polymers 0.000 description 9
- 230000005764 inhibitory process Effects 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 238000001035 drying Methods 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 239000000706 filtrate Substances 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 238000005303 weighing Methods 0.000 description 7
- QIAFMBKCNZACKA-UHFFFAOYSA-N N-benzoylglycine Chemical compound OC(=O)CNC(=O)C1=CC=CC=C1 QIAFMBKCNZACKA-UHFFFAOYSA-N 0.000 description 6
- 239000000919 ceramic Substances 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 102100030988 Angiotensin-converting enzyme Human genes 0.000 description 5
- 240000005710 Auricularia polytricha Species 0.000 description 5
- 235000000024 Auricularia polytricha Nutrition 0.000 description 5
- 108010059820 Polygalacturonase Proteins 0.000 description 5
- 108010093305 exopolygalacturonase Proteins 0.000 description 5
- 150000004676 glycans Chemical class 0.000 description 5
- 229920001282 polysaccharide Polymers 0.000 description 5
- 239000005017 polysaccharide Substances 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 4
- 241000228143 Penicillium Species 0.000 description 4
- 241000223259 Trichoderma Species 0.000 description 4
- 229930013930 alkaloid Natural products 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 235000013325 dietary fiber Nutrition 0.000 description 4
- 235000019441 ethanol Nutrition 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 150000008442 polyphenolic compounds Chemical class 0.000 description 4
- 235000013824 polyphenols Nutrition 0.000 description 4
- 230000001502 supplementing effect Effects 0.000 description 4
- 240000004507 Abelmoschus esculentus Species 0.000 description 3
- 241000228212 Aspergillus Species 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 108010084185 Cellulases Proteins 0.000 description 3
- 102000005575 Cellulases Human genes 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 230000002292 Radical scavenging effect Effects 0.000 description 3
- 102000003425 Tyrosinase Human genes 0.000 description 3
- 108060008724 Tyrosinase Proteins 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 150000003797 alkaloid derivatives Chemical class 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- AMTWCFIAVKBGOD-UHFFFAOYSA-N dioxosilane;methoxy-dimethyl-trimethylsilyloxysilane Chemical compound O=[Si]=O.CO[Si](C)(C)O[Si](C)(C)C AMTWCFIAVKBGOD-UHFFFAOYSA-N 0.000 description 3
- 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 3
- 239000000411 inducer Substances 0.000 description 3
- 235000019359 magnesium stearate Nutrition 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 229920001542 oligosaccharide Polymers 0.000 description 3
- 150000002482 oligosaccharides Chemical class 0.000 description 3
- 230000000630 rising effect Effects 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 238000007873 sieving Methods 0.000 description 3
- 229940083037 simethicone Drugs 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- -1 DPPH free radical Chemical class 0.000 description 2
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 101710138460 Leaf protein Proteins 0.000 description 2
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 108010028144 alpha-Glucosidases Proteins 0.000 description 2
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 description 2
- 230000036772 blood pressure Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 229910001448 ferrous ion Inorganic materials 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000005714 functional activity Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 235000012015 potatoes Nutrition 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000007711 solidification Methods 0.000 description 2
- 230000008023 solidification Effects 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 108700012359 toxins Proteins 0.000 description 2
- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 description 1
- 206010000234 Abortion spontaneous Diseases 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 206010039424 Salivary hypersecretion Diseases 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 235000018927 edible plant Nutrition 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000004438 eyesight Effects 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 208000015994 miscarriage Diseases 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- 239000000419 plant extract Substances 0.000 description 1
- 229940057838 polyethylene glycol 4000 Drugs 0.000 description 1
- 229940093429 polyethylene glycol 6000 Drugs 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 208000026451 salivation Diseases 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 208000000995 spontaneous abortion Diseases 0.000 description 1
- 210000004243 sweat Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000002087 whitening effect Effects 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/60—Moraceae (Mulberry family), e.g. breadfruit or fig
- A61K36/605—Morus (mulberry)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/51—Concentration
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- 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
- C12N1/14—Fungi; 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- 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
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
-
- 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
-
- 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
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01011—Pectinesterase (3.1.1.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01015—Polygalacturonase (3.2.1.15)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02002—Pectate lyase (4.2.2.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/0201—Pectin lyase (4.2.2.10)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
本发明公开了一种利用酶提取桑叶功能成分的方法,属于酶技术领域。本发明公开了一种制备纤维素酶粗酶液以及果胶酶粗酶液的方法,包括以下步骤:制备PDA斜面培养基;制备产纤维素酶或果胶酶发酵培养基;固体菌种的制备;发酵;纤维素酶粗酶液及果胶酶粗酶液的制备。本发明还公开了一种桑叶浓缩液的制备方法,包括以下步骤:酶解、灭酶、浓缩。本发明还公开了将桑叶浓缩液制备成桑叶产品的技术方案。本发明通过木耳母种制得的纤维素酶和果胶酶更加安全可靠;本发明提高了桑叶中蛋白及其他功能性成分的含量和得率;本发明所制得的桑叶产品可直接应用于食品、保健产品和化妆品的加工生产,提高了桑叶原料的综合利用率。
Description
技术领域
本发明涉及酶技术领域,特别是涉及一种利用酶提取桑叶功能成分的方法。
背景技术
桑叶既可药用亦可食用,《本草新编》中写道:桑叶之功,更佳于桑皮,最善补骨中之髓、添肾中之精,止身中之汗,填脑明目,活血生津,种子安胎,调和血脉,通利关节。近年来,对桑叶的研究发现,桑叶中富含多种功能成分,如多糖、生物碱和黄酮等,具有降血糖、降血压、降血脂、增强免疫力、去黑色素以及美白等多种功能。桑叶作为药食同源的植物,具有突出的保健功能,然而目前开发利用十分有限。现有技术对于桑叶功能成分提取的含量和得率较低,比如朱天明等人(纤维素酶辅助提取桑叶中叶蛋白的工艺)研究了利用纤维素酶辅助提取桑叶中的蛋白,其桑叶蛋白的提取率为仅6.5g/100g,因此,需要研发一种新的提取工艺来提高桑叶产品中功能成分的含量和得率。
同时,现有技术中用于桑叶成分提取的纤维素酶与果胶酶主要来自于真菌,比较典型的有木霉属、曲霉属和青霉属。不可避免的是木霉属和青霉属在发酵生产酶的过程中会伴随产生毒素,即使是安全性比较高的曲霉属在生产酶的过程中也时常会伴随微量不确定的有害物质的产生,后期分离脱毒工艺复杂,难度很大;同时,在后期酶处理桑叶得到的浆液时,需要经过浓缩过程,导致即使是微量的有害物质也会因浓缩使含量增加。因此,如何确保在食品及药品生产中使用的纤维素酶和果胶酶的食用安全性,是目前亟需解决的技术难题。
发明内容
本发明的目的是提供一种利用酶提取桑叶功能成分的方法,以解决上述现有技术存在的问题,一方面使纤维素酶和果胶酶整个制备过程不产生任何毒素及有害物质,更加安全可靠,保证人们食用的安全性;另一方面利用纤维素酶和果胶酶对桑叶中功能成分进行提取,来提高桑叶产品中功能成分的含量和得率。
为实现上述目的,本发明提供了如下方案:
本发明目的之一是提供一种制备纤维素酶粗酶液的方法,包括以下步骤:
步骤1,制备PDA斜面培养基;
步骤2,制备产纤维素酶发酵培养基:将麸皮、秸秆粉、硫酸铵、磷酸二氢钾、硫酸镁、氯化钙和水混合均匀,灭菌,得到产纤维素酶发酵培养基;
步骤3,固体菌种的制备:将木耳母种接入PDA斜面培养基培养,得到菌丝;
步骤4,发酵:取所述菌丝接入到所述产纤维素酶发酵培养基中拌匀,培养;
步骤5,纤维素酶粗酶液的制备:取步骤4中培养好的产纤维素酶发酵培养基,加水震荡、静置、过滤、离心,得到纤维素酶粗酶液。
进一步地,步骤2中麸皮、秸秆粉、硫酸铵、磷酸二氢钾、硫酸镁、氯化钙和水混合均匀后的pH值为3.5-5。
进一步地,步骤2中麸皮、秸秆粉、硫酸铵、磷酸二氢钾、硫酸镁、氯化钙和水的质量比为562.5:437.5:40:3:1:1:1500。
进一步地,步骤4中所述培养的温度为25-32℃,时间为3-4天。
本发明目的之二是提供一种制备果胶酶粗酶液的方法,包括以下步骤:
步骤1,制备PDA斜面培养基;
步骤2,制备产果胶酶发酵培养基:将麸皮、橘子皮粉、硫酸铵、硫酸镁、磷酸氢二钾、和水混合均匀,灭菌,得到产果胶酶发酵培养基;
步骤3,固体菌种的制备:将木耳母种接入PDA斜面培养基培养,得到菌丝;
步骤4,发酵:取所述菌丝接入到所述产果胶酶发酵培养基中拌匀,培养;
步骤5,果胶酶粗酶液的制备:取步骤4中培养好的产果胶酶发酵培养基,加水震荡、静置、过滤、离心,得到果胶酶粗酶液。
进一步地,步骤2中麸皮、橘子皮粉、硫酸铵、硫酸镁、磷酸氢二钾、和水混合均匀后的pH值为4-5。
进一步地,步骤2中麸皮、橘子皮粉、硫酸铵、硫酸镁、磷酸氢二钾、和水的质量比为2000:264:46:1:3:1270。
进一步地,步骤4中所述培养的温度为25-32℃,时间为3-4天。
本发明目的之三是提供一种桑叶浓缩液的制备方法,包括以下步骤:
步骤1,将桑叶与水混合,打浆,制得浆液,之后加入上述的纤维素酶粗酶液和果胶酶粗酶液进行酶解;
步骤2,将步骤1酶解后的浆液进行灭酶处理,之后浓缩,得到桑叶浓缩液。
进一步地,在步骤2之前还包括对浆液进行过滤的步骤。
进一步地,所述纤维素酶粗酶液和果胶酶粗酶液的质量比为3:2。
本发明目的之四是提供一种桑叶产品,包括上述的制备方法制备得到的桑叶浓缩液;所述桑叶产品的剂型为滴丸、粉剂或片剂。
本发明公开了以下技术效果:
(1)本发明中的纤维素酶和果胶酶粗酶液通过木耳母种发酵制得,整个制备过程安全无毒素产生(木耳作为一种可食用菌,其安全性已经经过了成百上千年的验证),所制得的纤维素酶和果胶酶粗酶液安全可靠,保证了食用的安全性,明显优于目前市场使用青霉、木霉、黑曲霉等生产的纤维素酶和果胶酶;
(2)本发明通过在产果胶酶发酵培养基中添加橘子皮粉作为诱导物,诱导木耳菌丝产生果胶酶(果胶酶属于诱导酶,诱导物的存在可以明显诱导相应酶基因的转录和表达);
(3)本发明利用由木耳母种发酵制得的纤维素酶和果胶酶粗酶液对桑叶进行功能性成分提取,在保证了制备过程安全性的同时,提高了桑叶中蛋白及其他功能性成分的含量和得率;
(4)本发明所制得的桑叶浓缩液功能活性强,通过将桑叶浓缩液制成滴丸、粉剂或片剂,可作为辅料或直接应用于食品、保健产品(抗氧化、降血糖、降血压等)和化妆品(抑制酪氨酸酶的活性,从而抑制机体黑色素生成)等的加工生产,提高了桑叶原料的综合利用率,市场潜力巨大,具有很好的经济效益和社会效益。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单的介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为发酵时间对菌种产纤维素酶与果胶酶酶活的影响图;
图2为发酵温度对菌种产纤维素酶与果胶酶酶活的影响图;
图3为发酵pH对菌种产纤维素酶与果胶酶酶活的影响图;
图4为纤维素酶粗酶液和果胶酶粗酶液添加量对桑叶总黄酮含量的影响图;
图5为纤维素酶粗酶液与果胶酶粗酶液配比对桑叶总黄酮含量的影响图;
图6为pH值对桑叶总黄酮含量的影响图;
图7为提取温度对桑叶总黄酮含量的影响图;
图8为提取时间对桑叶总黄酮含量的影响图。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
本发明所用木耳为白背毛木耳,购自武汉市周玉麟食用菌研究所。
本发明实施例如无特殊说明,均在无菌条件下进行。
实施例1纤维素酶粗酶液的制备
步骤1,制备PDA斜面培养基:称取去皮土豆200g,切成1cm左右的小方块,倒入陶瓷缸中,并向其加入800mL水,煮沸15min,将煮沸液用两层纱布过滤到烧杯中,然后补充水分至1L,将滤液重新倒入清洗干净的瓷缸中,加入葡萄糖20g,琼脂20g,用玻璃棒不断搅拌至充分溶解,将上述液体倒入到1L的烧杯中,补充水分至1L,然后分装于18(mm)×180(mm)的试管,分装完毕后在试管口塞上棉塞或橡胶塞,用报纸包扎,放入高压灭菌锅,121℃下灭菌20min,灭菌完成后,趁热摆成斜面,凝固后得到PDA斜面培养基备用;
步骤2,制备产纤维素酶发酵培养基:准确称取麸皮4.5g,玉米秸秆粉3.5g,硫酸铵0.32g,磷酸二氢钾0.024g,硫酸镁0.008g,氯化钙0.008g,置入250mL的三角瓶中,加入蒸馏水12mL,初始pH为4,用玻璃棒搅拌均匀,然后用8层纱布包扎好锥形瓶口,外再用两层报纸包住,放入高压灭菌锅,121℃下灭菌30min,冷却后备用;
步骤3,固体菌种的制备:将白背毛木耳母种在无菌操作下接入步骤1制得的PDA斜面培养基,然后置于培养箱中,30℃培养7d,并将生长状态良好、无杂菌污染的斜面放入冰箱中,在4℃条件下保存备用;
步骤4,发酵:将步骤3中的PDA斜面培养基中的菌种用灭过菌的接种钩切取0.5cm2左右菌丝体接入到产纤维素酶发酵培养基中拌匀,在30℃条件下培养4d,培养过程中要确保无杂菌污染,这也是产品安全性的保障;
步骤5:纤维素酶粗酶液的制备:称取10g已培养好的产纤维素酶发酵培养基,加入50mL蒸馏水,然后在200r/min条件下摇床震荡10min,静置两小时后四层纱布过滤,将滤液3500r/min速度下离心10min,上清液为纤维素酶粗酶液。
结果:所制得纤维素酶粗酶液的酶活为157.35U/mL。
实施例2纤维素酶粗酶液的制备
与实施例1不同之处在于,步骤2中初始pH值为3.5,培养温度为28℃,时间为3.5天。
结果:所制得纤维素酶粗酶液的酶活为145.36U/mL。
实施例3纤维素酶粗酶液的制备
与实施例1不同之处在于,步骤2中初始pH值为5,培养温度为32℃,时间为4天。
结果:所制得纤维素酶粗酶液的酶活为140.27U/mL。
实施例4果胶酶粗酶液的制备
步骤1,制备PDA斜面培养基:称取去皮土豆200g,切成1cm左右的小方块,倒入陶瓷缸中,并向其加入800mL水,煮沸15min,将煮沸液用两层纱布过滤到烧杯中,然后补充水分至1L,将滤液重新倒入清洗干净的瓷缸中,加入葡萄糖20g,琼脂20g,用玻璃棒不断搅拌至充分溶解,将上述液体倒入到1L的烧杯中,补充水分至1L,然后分装于18(mm)×180(mm)的试管,分装完毕后在试管口塞上棉塞或橡胶塞,用报纸包扎,放入高压灭菌锅,121℃下灭菌20min,灭菌完成后,趁热摆成斜面,凝固后得到PDA斜面培养基备用;
步骤2,制备产果胶酶发酵培养基:准确称取麸皮10g,橘子皮粉1.32g,硫酸铵0.23g,硫酸镁0.005g,磷酸氢二钾0.015g,置入250mL的三角瓶中,加入蒸馏水6.35mL,初始pH为4,用玻璃棒搅拌均匀,然后用8层纱布包扎好锥形瓶口,外再用两层报纸包住,放入高压灭菌锅,121℃下灭菌30min,灭菌完成后冷却备用;
步骤3,固体菌种的制备:将白背毛木耳母种在无菌操作下接入步骤1制得的PDA斜面培养基,然后置于培养箱中,30℃培养7d,并将生长状态良好、无杂菌污染的斜面放入冰箱中,在4℃条件下保存备用;
步骤4,发酵:将步骤3中的PDA斜面培养基中的菌种用灭过菌的接种钩切取0.5cm2左右菌丝体接入到产果胶酶发酵培养基中拌匀,在30℃条件下培养4d,培养过程中要确保无杂菌污染,这也是产品安全性的保障;
步骤5:果胶酶粗酶液的制备:称取10g已培养好的产果胶酶发酵培养基,加入50mL蒸馏水,然后在200r/min条件下摇床震荡10min,静置两小时后四层纱布过滤,将滤液3500r/min速度下离心10min,上清液为果胶酶粗酶液。
结果:所制得果胶酶粗酶液的酶活为101.77U/mL。
实施例5果胶酶粗酶液的制备
与实施例4不同之处在于,步骤2中初始pH值为3.5,培养温度为28℃,时间为3.5天。
结果:所制得果胶酶粗酶液的酶活为98.43U/mL。
实施例6果胶酶粗酶液的制备
与实施例4不同之处在于,步骤2中初始pH值为5,培养温度为32℃,时间为4天。
结果:所制得果胶酶粗酶液的酶活为92.26U/mL。
木耳(由木耳菌丝分化生长而成)是可以直接作为食材经过加工后大量食用的,而无论是青霉、木霉,还是曲霉的菌丝即使是少量的都不能直接作为食材加工食用的,这也证明了利用木耳产生的纤维素酶和果胶酶的食用安全性是有保障的。
发酵时间对菌种产纤维素酶与果胶酶酶活的影响:
本发明经过试验发现,纤维素酶酶活与果胶酶酶活随发酵时间的延长呈现先上升后降低的趋势,在发酵为3d的时候,纤维素酶酶活与果胶酶酶活达到最高,分别为156.32U/mL、103.05U/mL。在发酵后期,发酵体系中营养物质消耗,有害代谢产物积累,体系中酶的生成速度变慢,部分酶可能失活,纤维素酶活与果胶酶酶活呈下降趋势。发酵时间对菌种产纤维素酶与果胶酶酶活的影响如图1所示。
发酵温度对菌种产纤维素酶与果胶酶酶活的影响:
本发明经过试验发现,当发酵温度为30℃时,纤维素酶与果胶酶酶活最高,分别为152.26U/mL、96.08U/mL。纤维素酶与果胶酶的本质为蛋白质,当温度过高或过低,酶的活性会降低甚至失活,因此随温度的升高,纤维素酶与果胶酶的酶活力逐渐降低。发酵温度对菌种产纤维素酶与果胶酶酶活的影响如图2所示。
发酵pH对菌种产纤维素酶与果胶酶酶活的影响:
发明人经过试验发现,纤维素酶与果胶酶酶活随发酵pH的升高呈现先上升后降低的趋势,发酵初始pH为4的时候,纤维素酶酶活与果胶酶酶活达到最高,分别为147.55U/mL、107.95U/mL。pH过高或过低,同样会使酶的活性降低。发酵pH对菌种产纤维素酶与果胶酶酶活的影响如图3所示。
实施例7桑叶浓缩液的制备
步骤1,选取完整无病虫害的时至秋季的新鲜桑叶,用清水清洗叶片后,除去表面水分,摘除叶梗,按料水比为1:7,即取10g桑叶,置于打浆机中,加入70mL水打浆,打浆时间2.5min,打浆结束后,按纤维素酶粗酶液与果胶酶粗酶液质量比3:2加入实施例1制得的纤维素酶粗酶液和实施例4制得的果胶酶粗酶液,纤维素酶粗酶液和果胶酶粗酶液按3:2配比混合,占桑叶与水混合打浆得到的浆液质量的15%,于pH 4.5、温度50℃的条件下酶解两个小时;
步骤2,用双层40目滤布过滤得到滤液;
步骤3,将滤液置于80℃水浴锅中灭酶15min,然后将滤液转至旋转蒸发仪,60℃、-0.1MPa的条件下浓缩至10mL,得到桑叶浓缩液。
实施例8桑叶浓缩液的制备
与实施例7不同之处在于,省略步骤2。
纤维素酶粗酶液和果胶酶粗酶液添加量对桑叶总黄酮含量的影响:
本发明经过试验发现,在酶解温度为50℃,酶解时间为2h,初始pH为4.5,纤维素酶粗酶液和果胶酶粗酶液配比为3:2时,随着纤维素酶和果胶酶添加量的增加桑叶总黄酮的含量显著增加,说明纤维素酶与果胶酶有破坏植物细胞壁的作用,破壁后细胞内的活性成分更容易释放出来,当纤维素酶和果胶酶的添加量为桑叶与水混合打浆得到的浆液质量的15%时,总黄酮的含量最高,为5.52mg/mL;当纤维素酶和果胶酶的添加量大于15%时,总黄酮的含量没有明显变化。纤维素酶粗酶液和果胶酶粗酶液添加量对桑叶总黄酮含量的影响如图4所示。
纤维素酶粗酶液与果胶酶粗酶液配比对桑叶总黄酮含量的影响:
本发明经过试验发现,当纤维素酶粗酶液与果胶酶粗酶液配比为3:2时,桑叶总黄酮含量最高为5.232mg/mL,说明纤维素酶与果胶酶起协同作用破坏桑叶细胞壁,利于桑叶活性成分的溶出。当纤维素酶粗酶液与果胶酶粗酶液配比为0:5时,桑叶总黄酮含量最低,说明当只有果胶酶存在时,对于桑叶中活性成分的溶出帮助不大。纤维素酶粗酶液与果胶酶粗酶液配比对桑叶总黄酮含量的影响如图5所示。
pH值对桑叶总黄酮含量的影响:
本发明经过试验发现,桑叶总黄酮含量随pH值的升高呈现先升高后降低的趋势,当pH为4.5时,桑叶黄酮的含量达到最大为5.87mg/mL,这是由于纤维素酶与果胶酶在一定值范围内发挥作用,pH值过低或过高均会抑制其活性,从而影响纤维素酶与果胶酶对桑叶细胞壁的作用。pH值对桑叶总黄酮含量的影响如图6所示。
提取温度对桑叶总黄酮含量的影响:
本发明经过试验发现,当温度为50℃时,桑叶黄酮的含量最大为4.827mg/mL,而当温度超55℃时,由于温度过高,致使纤维素酶与果胶酶部分失活,因此桑叶黄酮的含量则呈现下降趋势。提取温度对桑叶总黄酮含量的影响如图7所示。
提取时间对桑叶总黄酮含量的影响:
本发明经过试验发现,随着时间的增加,桑叶总黄酮的含量逐渐增大,当大于2h时,桑叶总黄酮的含量没有明显的增加。提取时间对桑叶总黄酮含量的影响如图8所示。
实施例9桑叶滴丸的制备
取适量基质药用聚乙二醇6000与药用聚乙二醇4000按照1:1的比例置于烧杯中,于水浴锅中加热至液体状态,按照基质与桑叶浓缩液2:1的比例加入实施例7所制备的桑叶浓缩液,搅拌混合均匀,在水浴中静置20分钟排除气泡,在80℃保温条件下,滴入二甲基硅油中,其中滴距为5cm,二甲基硅油温度为5℃,冷却收缩成丸,擦掉附着在桑叶滴丸表面的二甲基硅油,室温(25℃)下干燥,得到形状、大小、色泽优良的桑叶滴丸。
实施例10桑叶粉剂的制备
将实施例8所制备的桑叶浓缩液在40~45℃恒温干燥箱中干燥,使含水量小于5%,然后进行粉碎,粉碎后的过100目筛,得到桑叶粉剂。
实施例11桑叶片剂的制备
步骤1,原辅料预处理:先将实施例7所制备的桑叶浓缩液、秋葵粉、低聚糖与硬脂酸镁在40~45℃恒温干燥箱中干燥,使含水量小于5%;
步骤2,粉碎过筛:分别将干燥后的桑叶浓缩物、秋葵粉、低聚糖、硬脂酸镁进行粉碎,粉碎后的原辅料过100目筛,以免颗粒较大引起口感粗糙;
步骤3,混合:将桑叶浓缩物与秋葵粉以10:1的比例混合均匀,加入占桑叶浓缩物质量2%的低聚糖、1%的硬脂酸镁混合均匀;
步骤4,压片:压片操作要求在空气相对湿度50%~60%的干燥室内进行,压片前后需要用75%的酒精对压片机压片部位进行仔细清洗消毒;
步骤5,成品包装:在空气相对湿度50%~60%的环境条件下密封包装。
对比例1
与实施例7不同之处在于,省略步骤1中纤维素酶粗酶液与果胶酶粗酶液的添加。
对比例2
与实施例8不同之处在于,省略步骤1中纤维素酶粗酶液与果胶酶粗酶液的添加。
对实施例7、实施例8以及对比例1和对比例2所制得的桑叶浓缩液进行功能成分检测,检测方法如下:
主要试剂与仪器
水分的测定:参照GB 5009.3-2016《食品中水分的测定》测定桑叶浓缩液的水分;
膳食纤维的测定:参照GB 5009.88-2014《食品中膳食纤维的测定》测定桑叶浓缩液的可溶膳食纤维(SDF)、不溶膳食纤维(IDF);
蛋白的测定:参照SN/T39-2014《出口乳、蛋、豆类食品中蛋白质含量的测定考马斯亮蓝法》测定桑叶浓缩液蛋白的含量;
总多糖的测定:参照NY/T1676-2008《食用菌中粗多糖含量的测定》,结合硫酸-苯酚法测定桑叶浓缩液中多糖的含量,测定前对桑叶浓缩液进行处理:取1mL桑叶浓缩液,加入30mL无水乙醇,然后置于4℃冰箱,48h后取出,4000r/min离心15min,弃去上清液,沉淀用75%的乙醇5mL洗2次,干燥后用5mL蒸馏水溶解,4℃冰箱冷藏备用;
总黄酮的测定:参照DB43/T 476-2009《植物源性食品中总黄酮的测定》,结合AlCl3法测定桑叶浓缩液中总黄酮的含量。测定前对桑叶浓缩液进行处理:吸取浓缩液2mL,用无水乙醇定容至50mL容量瓶中,4℃冰箱冷藏备用;
总多酚的测定:参照T/AHFIA 005—2018《植物提取物及其制品中总多酚含量的测定分光光度法》结合Folin-Denis试剂法测定桑叶浓缩液中多酚含量。测定前对桑叶浓缩液进行处理:取1mL桑叶浓缩液,蒸馏水定容至50mL容量瓶中,4℃冰箱冷藏备用;
总生物碱的测定:分别取0.1、0.4、0.8、1.2、1.6mL哌啶酶标准溶液,用0.05mol/L盐酸定容至2mL,配制成质量浓度为0.002、0.008、0.016、0.024、0.032mol/L的标准溶液。加入3mL雷氏铵盐溶液,混匀,冰浴2h,然后4000r/min离心10min,除上清,加入99%乙醚1.0mL,4000r/min离心10min,除上清液,自然挥发,加入5.0mL70%丙酮溶液溶解沉淀,在525nm波长处测定吸光度值,绘制标准曲线。准确称取2g浓缩液加入40mL30%酸性乙醇溶液(取0.05mol/L盐酸溶液350mL,用无水乙醇定容至500mL),涡旋震荡30s,超声波50℃提取30min,5000r/min离心15min,取上清液,残渣加入40mL 30%酸性乙醇溶液重复提取1次,合并2次提取液,减压浓缩定容至25mL,大孔吸附树脂HPD100吸附30min,纯水洗脱,洗脱液减压浓缩,定容至10mL待测。取2mL桑叶浓缩液替代哌啶酶标准溶液,按上述步骤测定桑叶浓缩液中的生物碱含量。
检测结果如表1所示:
表1
由表1能够看出,实施例7对桑叶中蛋白的提取率达到了14.7mg/1g,多糖的提取率达到了6.58mg/1g,黄酮的提取率达到了7.14mg/1g,多酚的提取率达到了13.95mg/1g、生物碱的提取率达到了3.213mg/1g。
对实施例7、实施例8以及对比例1和对比例2所制得的桑叶浓缩液的功能活性进行检测,其中,DPPH自由基清除率计算公式如下:
DPPH自由基清除率=1–(A1-A2)/A0×100%
式中A1:样品溶液与DPPH溶液反应后的吸光度值;A2:样品溶液与无水乙醇代替DPPH溶液反应后的吸光度值;A0:无水乙醇与DPPH反应后的吸光度值。
羟自由基清除率计算公式如下:
S/%=(A0-A1+A2)/A0×100
式中:S,羟自由基清除率,%;A0,不加样品液测得的吸光度值;A1,样品液测得的吸光度值;A2,不加H2O2测得的吸光度值。
亚铁离子螯合能力计算公式如下:
S/%=(A0-A1)/A0×100
式中:S,Fe2+螯合能力,%;A0,不加样品液测得的吸光度值;A1,样品液测得的吸光度值。
各物质对α-葡萄糖苷酶的抑制率计算公式如下:
式中:A0为空白组吸光度值;A1为样品背景组吸光度值;A2为样品组吸光度值。
酪氨酸酶抑制率计算公式如下:
I(%)=1-(C-D)/(A-B)×100
其中A、B、C、D为表各溶剂体系在475nm下的吸光度值。
ACE抑制率由以下公式计算:
ACE抑制率(%)=(A-B)/A×100
式中:A表示ACE与HHL完全反应后生成马尿酸的吸光值;B表示提取液与ACE和HHL反应后生成马尿酸的吸光值;(A-B)表示由于加入提取液使马尿酸减少的量。
检测结果如表2所示:
表2
由表2能够看出,本发明所制备的桑叶浓缩液对DPPH自由基清除率达到了93.47%,对羟基自由基清除率达到了90.36%,对亚铁离子螯合能力达到了84.75%,对α-葡萄糖苷酶的抑制率达到了88.09%,对血管紧张素转化酶抑制能力达到了81.9%,对酪氨酸酶抑制率达到了78.77%。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (3)
1.一种桑叶浓缩液的制备方法,其特征在于,包括以下步骤:
步骤1,将桑叶与水混合,打浆,制得浆液,之后加入纤维素酶粗酶液和果胶酶粗酶液于pH 4.5、温度50℃的条件下进行酶解两个小时;所述纤维素酶粗酶液和果胶酶粗酶液的质量比为3:2;所述纤维素酶粗酶液和果胶酶粗酶液的加入量为所述浆液质量的15%;
步骤2,将步骤1酶解后的浆液进行灭酶处理,之后浓缩,得到桑叶浓缩液;
所述纤维素酶粗酶液的制备方法包括以下步骤:
步骤(1),制备PDA斜面培养基;
步骤(2),制备产纤维素酶发酵培养基:将麸皮、秸秆粉、硫酸铵、磷酸二氢钾、硫酸镁、氯化钙和水混合均匀,pH值为4,灭菌,得到产纤维素酶发酵培养基;
步骤(3),固体菌种的制备:将木耳母种接入PDA斜面培养基培养,得到菌丝;
步骤(4),发酵:取所述菌丝接入到所述产纤维素酶发酵培养基中拌匀,30℃培养3天;
步骤(5),纤维素酶粗酶液的制备:取步骤4中培养好的产纤维素酶发酵培养基,加水震荡、静置、过滤、离心,得到纤维素酶粗酶液;
所述果胶酶粗酶液的方法包括以下步骤:
步骤1),制备PDA斜面培养基;
步骤2),制备产果胶酶发酵培养基:将麸皮、橘子皮粉、硫酸铵、硫酸镁、磷酸氢二钾和水混合均匀,pH值为4,灭菌,得到产果胶酶发酵培养基;
步骤3),固体菌种的制备:将木耳母种接入PDA斜面培养基培养,得到菌丝;
步骤4),发酵:取所述菌丝接入到所述产果胶酶发酵培养基中拌匀,30℃培养3天;
步骤5),果胶酶粗酶液的制备:取步骤4中培养好的产果胶酶发酵培养基,加水震荡、静置、过滤、离心,得到果胶酶粗酶液。
2.根据权利要求1所述的一种桑叶浓缩液的制备方法,其特征在于,在步骤2之前还包括对浆液进行过滤的步骤。
3.一种桑叶产品,其特征在于,包括权利要求1或2所述的制备方法制备得到的桑叶浓缩液;所述桑叶产品的剂型为滴丸、粉剂或片剂。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110563357.0A CN113249359B (zh) | 2021-05-24 | 2021-05-24 | 一种利用酶提取桑叶功能成分的方法 |
US17/551,221 US20220370538A1 (en) | 2021-05-24 | 2021-12-15 | Method for extracting functional ingredients of mulberry leaves using enzymes |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110563357.0A CN113249359B (zh) | 2021-05-24 | 2021-05-24 | 一种利用酶提取桑叶功能成分的方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113249359A CN113249359A (zh) | 2021-08-13 |
CN113249359B true CN113249359B (zh) | 2023-05-30 |
Family
ID=77183949
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110563357.0A Active CN113249359B (zh) | 2021-05-24 | 2021-05-24 | 一种利用酶提取桑叶功能成分的方法 |
Country Status (2)
Country | Link |
---|---|
US (1) | US20220370538A1 (zh) |
CN (1) | CN113249359B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115400054B (zh) * | 2022-09-01 | 2023-06-27 | 广东省农业科学院蚕业与农产品加工研究所 | 一种基于桑资源的美白抗氧化组合物的制备方法和应用 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102304501A (zh) * | 2011-09-01 | 2012-01-04 | 天津滨海诺奥酶工程技术有限公司 | 一种复合酶制剂及其用于提取植物多糖的应用和方法 |
CN103387599A (zh) * | 2013-08-08 | 2013-11-13 | 河北考力森生物科技有限公司 | 一种桑叶蛋白的提取工艺 |
CN105154511A (zh) * | 2015-10-21 | 2015-12-16 | 重庆都好生物科技有限公司 | 一种从桑叶提取氨基酸的方法 |
CN107699549A (zh) * | 2017-11-21 | 2018-02-16 | 广西农业职业技术学院 | 一种果胶酶及其制备方法 |
CN111304183A (zh) * | 2020-04-22 | 2020-06-19 | 湖南农业大学 | 一种纤维素酶的发酵方法 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111449182A (zh) * | 2020-05-07 | 2020-07-28 | 广东省农业科学院蚕业与农产品加工研究所 | 桑叶蛋白水解物及其制备方法和用其制备的酵素饮料 |
-
2021
- 2021-05-24 CN CN202110563357.0A patent/CN113249359B/zh active Active
- 2021-12-15 US US17/551,221 patent/US20220370538A1/en active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102304501A (zh) * | 2011-09-01 | 2012-01-04 | 天津滨海诺奥酶工程技术有限公司 | 一种复合酶制剂及其用于提取植物多糖的应用和方法 |
CN103387599A (zh) * | 2013-08-08 | 2013-11-13 | 河北考力森生物科技有限公司 | 一种桑叶蛋白的提取工艺 |
CN105154511A (zh) * | 2015-10-21 | 2015-12-16 | 重庆都好生物科技有限公司 | 一种从桑叶提取氨基酸的方法 |
CN107699549A (zh) * | 2017-11-21 | 2018-02-16 | 广西农业职业技术学院 | 一种果胶酶及其制备方法 |
CN111304183A (zh) * | 2020-04-22 | 2020-06-19 | 湖南农业大学 | 一种纤维素酶的发酵方法 |
Non-Patent Citations (1)
Title |
---|
张贵会等新疆药桑叶中国黄酮类化合物的分离与鉴定.新疆药桑叶中国黄酮类化合物的分离与鉴定.《食品工业科技》.2016,全文. * |
Also Published As
Publication number | Publication date |
---|---|
CN113249359A (zh) | 2021-08-13 |
US20220370538A1 (en) | 2022-11-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107557224B (zh) | 一种拐枣枸杞保健酒及其的酿造方法 | |
CN103271951B (zh) | 一种制备高腺苷含量的虫草制剂的方法 | |
CN100389687C (zh) | 山楂醋酸发酵饮料及其酿造方法 | |
CN105942061A (zh) | 一种复合酶解制备马蹄果汁的方法 | |
CN109486604A (zh) | 一种八月瓜果酒的制备方法 | |
CN106497723B (zh) | 一种由核桃花、核桃叶、分心木和核桃壳混合酿制的果酒 | |
CN105754809B (zh) | 一种工业化生产有机富硒藏虫草原浆酒的方法及所得产物 | |
CN113249359B (zh) | 一种利用酶提取桑叶功能成分的方法 | |
CN106666007A (zh) | 一种海棠茶饮料的制备方法及其制备的海棠茶 | |
CN106947680A (zh) | 一种富硒梨渣的发酵方法 | |
CN113647490A (zh) | 一种黑玉米芯代茶加工方法 | |
CN111587974B (zh) | 一种饮料 | |
CN110607218B (zh) | 一种醒酒醋及其制备方法与用途 | |
CN111743065A (zh) | 一种天冬芦笋饮料及其制备方法 | |
CN107279639A (zh) | 一种黑老虎保健复合饮料 | |
CN106010922A (zh) | 菊花茶醋饮料的生产方法 | |
CN105661248A (zh) | 一种富含黄酮的固体饮料及其制备方法 | |
CN105942111A (zh) | 玉米茶醋饮料的生产方法 | |
CN112430516B (zh) | 一种发酵莓子酒及其制备方法 | |
CN105441277B (zh) | 一种红景天樱桃酒及其制备方法 | |
CN105918547B (zh) | 一种双孢菇沙棘速溶茶及其制备方法 | |
CN108713747A (zh) | 一种具有补铁功效的综合酵素液及其制备方法 | |
CN108504537A (zh) | 一种安梨玫瑰醋酿造工艺 | |
CN109328858B (zh) | 使用蛹虫草制备袋装茶、糖果、饮品、和酒的方法及其制备的袋装茶、糖果、饮品、和酒 | |
CN107897766A (zh) | 一种解酒复方制剂及其制备方法 |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |