CN117987311A - Lactobacillus paracasei YYS-EN2 for producing cellulase, pectase, tannase and phytase and application thereof - Google Patents
Lactobacillus paracasei YYS-EN2 for producing cellulase, pectase, tannase and phytase and application thereof Download PDFInfo
- Publication number
- CN117987311A CN117987311A CN202410139976.0A CN202410139976A CN117987311A CN 117987311 A CN117987311 A CN 117987311A CN 202410139976 A CN202410139976 A CN 202410139976A CN 117987311 A CN117987311 A CN 117987311A
- Authority
- CN
- China
- Prior art keywords
- yys
- lactobacillus paracasei
- fermented
- powder
- tannase
- 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
- 241000186605 Lactobacillus paracasei Species 0.000 title claims abstract description 96
- 108010011619 6-Phytase Proteins 0.000 title claims abstract description 38
- 108010059892 Cellulase Proteins 0.000 title claims abstract description 38
- 229940106157 cellulase Drugs 0.000 title claims abstract description 38
- 229940085127 phytase Drugs 0.000 title claims abstract description 36
- 108010038851 tannase Proteins 0.000 title claims abstract description 35
- -1 pectase Proteins 0.000 title abstract description 21
- 239000000843 powder Substances 0.000 claims abstract description 60
- 238000000855 fermentation Methods 0.000 claims abstract description 34
- 230000004151 fermentation Effects 0.000 claims abstract description 34
- 239000001814 pectin Substances 0.000 claims abstract description 29
- 229920001277 pectin Polymers 0.000 claims abstract description 29
- 235000010987 pectin Nutrition 0.000 claims abstract description 29
- 235000016709 nutrition Nutrition 0.000 claims abstract description 28
- 229920001864 tannin Polymers 0.000 claims abstract description 21
- 235000018553 tannin Nutrition 0.000 claims abstract description 21
- 239000001648 tannin Substances 0.000 claims abstract description 21
- IMQLKJBTEOYOSI-GPIVLXJGSA-N Inositol-hexakisphosphate Chemical compound OP(O)(=O)O[C@H]1[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O IMQLKJBTEOYOSI-GPIVLXJGSA-N 0.000 claims abstract description 20
- IMQLKJBTEOYOSI-UHFFFAOYSA-N Phytic acid Natural products OP(O)(=O)OC1C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C1OP(O)(O)=O IMQLKJBTEOYOSI-UHFFFAOYSA-N 0.000 claims abstract description 20
- 230000035764 nutrition Effects 0.000 claims abstract description 20
- 229940068041 phytic acid Drugs 0.000 claims abstract description 20
- 235000002949 phytic acid Nutrition 0.000 claims abstract description 20
- 239000000467 phytic acid Substances 0.000 claims abstract description 20
- 229940126680 traditional chinese medicines Drugs 0.000 claims abstract description 17
- 238000004519 manufacturing process Methods 0.000 claims abstract description 14
- 229920001184 polypeptide Polymers 0.000 claims abstract description 11
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 11
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 11
- 239000006041 probiotic Substances 0.000 claims abstract description 10
- 235000018291 probiotics Nutrition 0.000 claims abstract description 10
- 230000000529 probiotic effect Effects 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 6
- 108090000790 Enzymes Proteins 0.000 claims description 91
- 102000004190 Enzymes Human genes 0.000 claims description 91
- 229940088598 enzyme Drugs 0.000 claims description 91
- 239000000047 product Substances 0.000 claims description 55
- 239000001963 growth medium Substances 0.000 claims description 28
- 238000002360 preparation method Methods 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 19
- 238000012258 culturing Methods 0.000 claims description 18
- 229920002678 cellulose Polymers 0.000 claims description 17
- 239000001913 cellulose Substances 0.000 claims description 17
- 235000010980 cellulose Nutrition 0.000 claims description 16
- 238000009630 liquid culture Methods 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 230000015556 catabolic process Effects 0.000 claims description 9
- 238000006731 degradation reaction Methods 0.000 claims description 9
- 230000000593 degrading effect Effects 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 239000004460 silage Substances 0.000 claims description 6
- 235000021118 plant-derived protein Nutrition 0.000 claims description 5
- 235000018102 proteins Nutrition 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 108010059820 Polygalacturonase Proteins 0.000 claims description 4
- 108010093305 exopolygalacturonase Proteins 0.000 claims description 4
- 239000002068 microbial inoculum Substances 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 108010084185 Cellulases Proteins 0.000 claims 2
- 102000005575 Cellulases Human genes 0.000 claims 2
- 230000000694 effects Effects 0.000 abstract description 49
- 239000008104 plant cellulose Substances 0.000 abstract description 5
- 238000009629 microbiological culture Methods 0.000 abstract description 4
- 244000005700 microbiome Species 0.000 abstract description 4
- 238000010521 absorption reaction Methods 0.000 abstract description 3
- 235000019658 bitter taste Nutrition 0.000 abstract description 3
- 230000029087 digestion Effects 0.000 abstract description 2
- 235000013376 functional food Nutrition 0.000 abstract description 2
- 230000001737 promoting effect Effects 0.000 abstract description 2
- 230000000968 intestinal effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 92
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 31
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 30
- 238000000034 method Methods 0.000 description 23
- 240000008042 Zea mays Species 0.000 description 21
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 21
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 21
- 235000005822 corn Nutrition 0.000 description 21
- 238000012216 screening Methods 0.000 description 20
- 241000196324 Embryophyta Species 0.000 description 17
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 16
- 239000012153 distilled water Substances 0.000 description 16
- 230000001580 bacterial effect Effects 0.000 description 15
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 12
- 238000012360 testing method Methods 0.000 description 12
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 11
- 238000011161 development Methods 0.000 description 11
- 239000003814 drug Substances 0.000 description 11
- 235000013305 food Nutrition 0.000 description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 10
- 239000008103 glucose Substances 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 238000005303 weighing Methods 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 9
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- ZTHYODDOHIVTJV-UHFFFAOYSA-N gallic acid propyl ester Natural products CCCOC(=O)C1=CC(O)=C(O)C(O)=C1 ZTHYODDOHIVTJV-UHFFFAOYSA-N 0.000 description 9
- 238000002156 mixing Methods 0.000 description 9
- 230000008569 process Effects 0.000 description 9
- 235000014101 wine Nutrition 0.000 description 9
- 238000002835 absorbance Methods 0.000 description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 8
- 235000019341 magnesium sulphate Nutrition 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 239000001632 sodium acetate Substances 0.000 description 8
- 235000017281 sodium acetate Nutrition 0.000 description 8
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 7
- 239000001768 carboxy methyl cellulose Substances 0.000 description 7
- 229940074391 gallic acid Drugs 0.000 description 7
- 235000004515 gallic acid Nutrition 0.000 description 7
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 7
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 6
- FENRSEGZMITUEF-ATTCVCFYSA-E [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].OP(=O)([O-])O[C@@H]1[C@@H](OP(=O)([O-])[O-])[C@H](OP(=O)(O)[O-])[C@H](OP(=O)([O-])[O-])[C@H](OP(=O)(O)[O-])[C@H]1OP(=O)([O-])[O-] Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].OP(=O)([O-])O[C@@H]1[C@@H](OP(=O)([O-])[O-])[C@H](OP(=O)(O)[O-])[C@H](OP(=O)([O-])[O-])[C@H](OP(=O)(O)[O-])[C@H]1OP(=O)([O-])[O-] FENRSEGZMITUEF-ATTCVCFYSA-E 0.000 description 6
- 239000000839 emulsion Substances 0.000 description 6
- 238000001952 enzyme assay Methods 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 239000011734 sodium Substances 0.000 description 6
- 229940083982 sodium phytate Drugs 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 239000001888 Peptone Substances 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 5
- 238000003028 enzyme activity measurement method Methods 0.000 description 5
- 239000000796 flavoring agent Substances 0.000 description 5
- 238000004108 freeze drying Methods 0.000 description 5
- 239000010903 husk Substances 0.000 description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 description 5
- 235000019319 peptone Nutrition 0.000 description 5
- 239000000473 propyl gallate Substances 0.000 description 5
- 229940075579 propyl gallate Drugs 0.000 description 5
- 235000010388 propyl gallate Nutrition 0.000 description 5
- 210000003296 saliva Anatomy 0.000 description 5
- 239000007974 sodium acetate buffer Substances 0.000 description 5
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 239000012086 standard solution Substances 0.000 description 5
- 239000012089 stop solution Substances 0.000 description 5
- 235000013311 vegetables Nutrition 0.000 description 5
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 description 4
- 239000001263 FEMA 3042 Substances 0.000 description 4
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 4
- 240000006024 Lactobacillus plantarum Species 0.000 description 4
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 4
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 4
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 4
- IAJILQKETJEXLJ-RSJOWCBRSA-N aldehydo-D-galacturonic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-RSJOWCBRSA-N 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000010227 cup method (microbiological evaluation) Methods 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- 235000019634 flavors Nutrition 0.000 description 4
- 235000015203 fruit juice Nutrition 0.000 description 4
- 229940072205 lactobacillus plantarum Drugs 0.000 description 4
- 239000008176 lyophilized powder Substances 0.000 description 4
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 4
- 229910017604 nitric acid Inorganic materials 0.000 description 4
- 239000011574 phosphorus Substances 0.000 description 4
- 229910052698 phosphorus Inorganic materials 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 239000003223 protective agent Substances 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 4
- 229940033123 tannic acid Drugs 0.000 description 4
- 235000015523 tannic acid Nutrition 0.000 description 4
- 229920002258 tannic acid Polymers 0.000 description 4
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 4
- AEMOLEFTQBMNLQ-DTEWXJGMSA-N D-Galacturonic acid Natural products O[C@@H]1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-DTEWXJGMSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 244000068988 Glycine max Species 0.000 description 3
- 235000010469 Glycine max Nutrition 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000011609 ammonium molybdate Substances 0.000 description 3
- 235000018660 ammonium molybdate Nutrition 0.000 description 3
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 description 3
- 229940010552 ammonium molybdate Drugs 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 229940040526 anhydrous sodium acetate Drugs 0.000 description 3
- UNTBPXHCXVWYOI-UHFFFAOYSA-O azanium;oxido(dioxo)vanadium Chemical compound [NH4+].[O-][V](=O)=O UNTBPXHCXVWYOI-UHFFFAOYSA-O 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 description 3
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 210000002257 embryonic structure Anatomy 0.000 description 3
- 230000001804 emulsifying effect Effects 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 235000013312 flour Nutrition 0.000 description 3
- 229940099596 manganese sulfate Drugs 0.000 description 3
- 239000011702 manganese sulphate Substances 0.000 description 3
- 235000007079 manganese sulphate Nutrition 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000001103 potassium chloride Substances 0.000 description 3
- 235000011164 potassium chloride Nutrition 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
- 238000010298 pulverizing process Methods 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 235000019640 taste Nutrition 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 108090001060 Lipase Proteins 0.000 description 2
- 102000004882 Lipase Human genes 0.000 description 2
- 239000004367 Lipase Substances 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 239000011790 ferrous sulphate Substances 0.000 description 2
- 235000003891 ferrous sulphate Nutrition 0.000 description 2
- 239000002657 fibrous material Substances 0.000 description 2
- 235000012055 fruits and vegetables Nutrition 0.000 description 2
- WPEXVRDUEAJUGY-UHFFFAOYSA-B hexacalcium;(2,3,4,5,6-pentaphosphonatooxycyclohexyl) phosphate Chemical compound [Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])(=O)OC1C(OP([O-])([O-])=O)C(OP([O-])([O-])=O)C(OP([O-])([O-])=O)C(OP([O-])([O-])=O)C1OP([O-])([O-])=O WPEXVRDUEAJUGY-UHFFFAOYSA-B 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- 235000019421 lipase Nutrition 0.000 description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 2
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 235000012054 meals Nutrition 0.000 description 2
- 210000000214 mouth Anatomy 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- KIWUVOGUEXMXSV-UHFFFAOYSA-N rhodanine Chemical compound O=C1CSC(=S)N1 KIWUVOGUEXMXSV-UHFFFAOYSA-N 0.000 description 2
- 239000010802 sludge Substances 0.000 description 2
- 239000004317 sodium nitrate Substances 0.000 description 2
- 235000010344 sodium nitrate Nutrition 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- RNKMOGIPOMVCHO-SJMVAQJGSA-N 1,3,6-trigalloyl glucose Chemical compound C([C@@H]1[C@H]([C@@H]([C@@H](O)[C@H](OC(=O)C=2C=C(O)C(O)=C(O)C=2)O1)OC(=O)C=1C=C(O)C(O)=C(O)C=1)O)OC(=O)C1=CC(O)=C(O)C(O)=C1 RNKMOGIPOMVCHO-SJMVAQJGSA-N 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- USWNUPFIMWECBM-UHFFFAOYSA-N 5-(dimethylamino)naphthalene-1-sulfonic acid;hydrate Chemical compound O.C1=CC=C2C(N(C)C)=CC=CC2=C1S(O)(=O)=O USWNUPFIMWECBM-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000002144 chemical decomposition reaction Methods 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000019985 fermented beverage Nutrition 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 239000002417 nutraceutical Substances 0.000 description 1
- 235000021436 nutraceutical agent Nutrition 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 235000013406 prebiotics Nutrition 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229940073490 sodium glutamate Drugs 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus paracasei YYS-EN2 for producing cellulase, pectase, tannase and phytase and application thereof. The Lactobacillus paracasei (Lactobacillus paracasei) YYS-EN2 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.28531. The lactobacillus paracasei YYS-EN2 provided by the invention has the capability of producing cellulase, pectase, tannase and phytase, can degrade plant cellulose and pectin, decompose tannin and phytic acid, can reduce bitter taste and improve clarity of products when being applied to fermentation products, can be widely applied to production of fermentation products such as fermented traditional Chinese medicines, fermented plant-derived nutrition powder, fermented plant-derived polypeptide powder and the like, can also be applied to probiotic products and functional foods, and has the effects of improving flora structure and promoting digestion and absorption when being subjected to intestinal field planting in human bodies.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus paracasei YYS-EN2 for producing cellulase, pectase, tannase and phytase and application thereof.
Background
Crude fiber materials such as cellulose, pectin, tannin and the like are abundant in traditional Chinese medicines, grains, bran, fruits and vegetables, are not easy to hydrolyze, and the crystalline rigid structure of the crude fiber materials causes great production difficulty; in the product processing process, the processing steps of the product can be increased, and the yield is reduced; even to some extent damaging the nutritional value of the reduced product. The physical and chemical degradation conditions are extreme, the period is long, and secondary pollution is easy to bring; therefore, the microbial degradation method becomes the most effective, healthy and environment-friendly method. The cellulase and the pectase can hydrolyze crude plant fibers, are favorable for dissolving and releasing substances such as starch, protein and the like, are favorable for nutrition absorption, improve the value of the product, and are widely applied to extraction and clarification of traditional Chinese medicines, fruits and vegetables and fermentation of tea and coffee. Tannase and phytase can degrade tannins and phytic acid, and can improve the squeezing and color of fruit juice and vegetables; enhancing the flavor and color of wine.
In conclusion, the cellulase, the pectase, the tannase and the phytase have wider application prospects in fermentation of traditional Chinese medicines, plant-based fermented beverages, fermentation of plant-derived nutrition powder, fermentation of plant-derived polypeptide powder and fermentation of plant-derived protein powder.
The Chinese patent of application No. CN202110864302.3 and publication No. 20230203 discloses a Streptomyces strain GZUI RF-Y1, a method for preparing cellulase fermentation broth and application thereof, wherein the cellulase activity in the fermentation broth of the strain reaches 111.0 U.mL -1. In this technique, the strain degrading the cellulose function is a mold.
The invention patent of China with the application number of CN202111637611.3 and the publication date of 20220412 discloses an aspergillus oryzae strain with the cellulase activity of 18.6U.g -1 and the acid pectase activity of 33.1U.g -1 in high-salt and high-nitrogen fermented foods. In this technique, the strain degrading the cellulose function is a mold.
The mould can produce cellulase and/or pectase, and does not have the functions of producing cellulase, pectase, tannase and phytase. Also, at present, most of bacterial strains degrading cellulose, pectin, tannin and phytic acid are mould or bacillus, and mould or bacillus are less beneficial bacteria and cannot be applied to food fermentation.
In summary, how to develop natural probiotics with cellulase, pectase, tannase and phytase production is the technical problem in the field to be solved.
Disclosure of Invention
In order to solve the defects of the prior art mentioned in the background art, the invention provides lactobacillus paracasei YYS-EN2, wherein the lactobacillus paracasei YYS-EN2 is a natural strain, has the capability of producing cellulase, pectase, tannase and phytase, can degrade plant cellulose and pectin, decompose tannin and phytic acid, can reduce bitter taste and improve the clarity of products, and is widely applied to the production of fermented products such as fermented traditional Chinese medicines, fermented plant-derived nutrition powder, fermented plant-derived polypeptide powder and the like.
The lactobacillus paracasei YYS-EN2 provided by the invention, latin Wen Xueming: lactobacillus paracasei, and is preserved in China general microbiological culture Collection center (CGMCC) at 25/09/2023 with the preservation number of CGMCC No.28531.
The Lactobacillus paracasei YYS-EN2 is isolated from saliva of the oral cavity of a healthy adult, and is subjected to sequencing analysis, and is aligned to be highly homologous with the Lactobacillus paracasei through Blast sequences, and is named as Lactobacillus paracasei YYS-EN2.
Among them, lactobacillus paracasei YYS-EN2 has the ability to produce cellulase, pectinase, tannase and phytase, and has the ability to degrade cellulose, pectin, tannin and phytic acid.
The invention also provides an application of lactobacillus paracasei YYS-EN2 in preparing a fermentation product. The lactobacillus paracasei YYS-EN2 is applied to the preparation of a fermentation product, and can obviously reduce the content of cellulose and pectin in the product.
In some embodiments, the fermentation product includes, but is not limited to, fermented traditional Chinese medicine, fermented nutritional powder, fermented polypeptide powder, fermented protein powder, ferment, silage.
In some embodiments, the fermented nutritional powder comprises a fermented plant-derived nutritional powder; the fermented polypeptide powder comprises fermented plant-derived polypeptide powder; the fermented protein powder comprises fermented plant-derived protein powder.
In some embodiments, the fermented plant-derived nutritional powder comprises a fermented corn nutritional powder. Lactobacillus paracasei YYS-EN2 is applied to a corn nutrition powder product, and can ferment corn husks and corn embryos, decompose cellulose and pectin, reduce tannin and phytic acid, and strengthen the flavor and taste of the corn nutrition powder.
The invention also provides a method for producing cellulase, pectase, tannase and phytase: inoculating lactobacillus paracasei YYS-EN2 into an enzyme-producing liquid culture medium, culturing at the temperature of between 30 and 40 ℃ for 48 to 72 hours, and carrying out solid-liquid separation on the enzyme-producing liquid culture medium after fermentation culture to obtain a supernatant; the supernatant is an enzyme solution containing cellulase and/or pectase and/or tannase and/or phytase.
The invention also provides a freeze-dried product, the components of which comprise lactobacillus paracasei YYS-EN2 as described above.
In some embodiments, the viable count of lactobacillus paracasei YYS-EN2 within the lyophilized product is (2-8) ×10 1CFU·g-1.
The invention also provides a preparation method of the freeze-dried product, which comprises the following preparation steps:
1) Preparing lactobacillus paracasei YYS-EN2 seed liquid;
2) Performing seed liquid expansion culture;
3) Fermenting and culturing the seed liquid to obtain fermentation liquid;
4) Centrifuging, and discarding supernatant to obtain bacterial sludge;
5) Uniformly mixing bacterial mud and a freeze-drying protective agent, and then emulsifying and embedding to obtain emulsion;
6) Freeze-drying the emulsion, and pulverizing to obtain freeze-dried powder of Lactobacillus paracasei YYS-EN 2.
The invention also provides a microbial inoculum, the components of which comprise the freeze-dried powder and/or the components of which comprise lactobacillus paracasei YYS-EN2.
In some embodiments, the microbial agent further comprises other ingredients that are currently available for microbial agents, such as at least one of prebiotics, fillers, acidulants, solvents, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, stabilizers, glidants, flavoring agents, preservatives, coating materials, fragrances, anti-binding agents, integration agents, thickening agents, and inclusion agents.
The invention also provides application of lactobacillus paracasei YYS-EN2 in preparation of a probiotic product.
The invention also provides the use of lactobacillus paracasei YYS-EN2 as described above for the preparation of a functional product comprising at least one of the following effects:
(1) Has cellulose decomposing ability;
(2) Has pectin decomposing ability;
(3) Has the ability of decomposing tannin;
(4) Has the capability of decomposing phytic acid.
In some embodiments, the functional product comprises a food, a nutraceutical; lactobacillus paracasei YYS-EN2 can be used for preparing food or health product.
Based on the characteristics, the lactobacillus paracasei YYS-EN2 provided by the invention has the following beneficial effects:
the lactobacillus paracasei YYS-EN2 provided by the invention has the capability of producing cellulase, pectase, tannase and phytase, and can degrade plant cellulose, pectin, tannin and phytic acid.
The lactobacillus paracasei YYS-EN2 provided by the invention has the capability of producing cellulase, can degrade cellulose, and can be applied to development of fermented traditional Chinese medicines, fermented plant-derived nutrition powder and silage products.
The pectin has the pectase degradation capability, can be widely applied to the production of products such as fermented traditional Chinese medicines, fruit juice, vegetables, wines, enzymes and the like, can obviously reduce the pectin content in raw materials, and improves the clarity and the yield of the products.
The product has the capability of producing the tannase, can be widely applied to the production of products such as traditional Chinese medicines, wines, enzymes and the like, can reduce the content of the tannase in the wine, and improves the mouthfeel of the products.
The phytase-producing enzyme has the capability of producing phytase, can be widely applied to flour and soybean food processing, improves the degradation rate of phytic acid, and improves the nutrition and commodity value of the phytic acid.
In conclusion, the lactobacillus paracasei YYS-EN2 provided by the invention has the functions of producing cellulase, pectase, tannase and phytase, can be applied to various probiotic fermentation products, and lays a foundation for providing green healthy, high-nutrition-value and high-utilization-rate plant-source crop products.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments or the description of the prior art will be briefly described below, it will be obvious that the drawings in the following description are some embodiments of the present invention, and that other drawings can be obtained according to these drawings without inventive effort to a person skilled in the art.
FIG. 1 is a graph of the colony of Lactobacillus paracasei YYS-EN2 on MRS plate medium;
FIG. 2 is a graph showing a glucose standard curve;
FIG. 3 is a graph showing a D-galacturonic acid standard curve;
FIG. 4 is a gallic acid standard curve display diagram;
FIG. 5 is a graph showing a standard curve of sodium phytate.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The scheme of the invention is as follows:
The invention provides lactobacillus paracasei (Lactobacillus paracasei) YYS-EN2:
Lactobacillus paracasei (Lactobacillus paracasei) YYS-EN2 was deposited with the China general microbiological culture Collection center, with accession number No. 3, the west way 1, north Star, the korean region, of beijing, at year 2023, and month 09, 25: CGMCC No.28531;
The source is as follows: the lactobacillus paracasei is isolated from saliva of oral cavity of a healthy adult, and the strain is subjected to sequencing analysis, and is highly homologous to the lactobacillus paracasei through Blast sequence alignment, and is named as lactobacillus paracasei YYS-EN2.
Colony morphology: colonies in MRS solid culture medium are milky white, round and smooth and neat in edge.
The functions are as follows: has the capability of producing cellulase and pectase, can degrade cellulose and pectin, has the capability of producing tannase and phytase, and can decompose tannin and phytic acid.
The invention also provides an operation example of a method for producing cellulase and/or pectinase and/or tannase and/or phytase:
Inoculating lactobacillus paracasei YYS-EN2 into an enzyme-producing liquid culture medium, culturing at the temperature of between 30 and 40 ℃ for 48 to 72 hours, and carrying out solid-liquid separation on the enzyme-producing liquid culture medium after fermentation culture to obtain a supernatant; the supernatant is an enzyme solution containing cellulase and/or pectase and/or tannase and/or phytase.
The invention also provides a preparation method of the freeze-dried product, which comprises the following preparation steps:
1) Preparing lactobacillus paracasei YYS-EN2 seed liquid;
2) Performing seed liquid expansion culture;
3) Fermenting and culturing the seed liquid to obtain fermentation liquid;
4) Centrifuging the fermentation liquor to obtain bacterial sludge;
5) Mixing bacterial mud with a freeze-drying protective agent, and then emulsifying and embedding to obtain emulsion;
6) Freeze-drying the emulsion, and pulverizing to obtain lactobacillus paracasei YYS-EN2 freeze-dried powder.
The invention also provides an operation example of an application method in the preparation of the corn nutrition powder:
Crushing corn embryo and corn husk, treating with lipase and amylase, sterilizing at high temperature, adding lactobacillus paracasei YYS-EN2 bacterial powder and/or lyophilized powder, mixing, fermenting, sterilizing at high temperature, and drying.
The technical solutions provided by the present invention are described in detail below in conjunction with examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
EXAMPLE 1 isolation and characterization of Lactobacillus paracasei YYS-EN2
Separating:
Sterile sampling from oral saliva of a healthy adult, dipping the oral saliva sample of the healthy adult into a sterile homogenizing bag by using a sterile cotton swab, adding 45mL of 0.9% physiological saline, and homogenizing and uniformly mixing to obtain a sample. 100. Mu.L of the sample was pipetted for 10-fold serial gradient dilutions, 100. Mu.L of the sample with dilution factor of 10 -2、10-3、10-4 was pipetted respectively, and the samples were spread on MRS solid plate medium containing 2.5% CaCO 3 and incubated for 24h at 37℃in an inverted position. Selecting colony with good growth vigor and large calcium dissolving ring, repeatedly separating and purifying by plate streak separation method until single colony is obtained, screening the separated bacteria to obtain high-yield cellulase, pectase, tannase and phytase, named YYS-EN2, and adding glycerol for preservation in a fungus bank at-80deg.C.
The colony morphology of the isolated and purified Lactobacillus paracasei YYS-EN2 is as follows: colonies in MRS solid culture medium are milky white, round and smooth and neat in edge.
Wherein, the bacteria for obtaining the high-yield cellulase, pectase, tannase and phytase are obtained by screening, and the process comprises the following steps:
screening cellulase-producing strains:
1 screening Medium (g.L-1): 20g of sodium carboxymethylcellulose (CMC-Na), 10g of peptone, 5g of yeast powder, 5g of sodium chloride, 1g of monopotassium phosphate and 0.3g of magnesium sulfate; congo red 0.4g;
2 screening of strains: inoculating bacteria into MRS liquid culture medium from-80deg.C refrigerator, and culturing at 37deg.C for 24 hr; then 100 mu L of culture solution is placed on a screening culture medium plate for culturing for 48 hours at 37 ℃ by a oxford cup method, the diameter of a transparent circle is observed, a strain with high-yield cellulase is selected according to the diameter of the transparent circle, and the enzyme activity of the cellulase is measured, so that the strain with high-yield cellulase is screened.
Similarly, bacterial screening of high-yield pectase, tannase and phytase is sequentially carried out, and the process is specifically as follows:
Screening pectase-producing strains:
1. screening media (g.L-1): 10g of pectin, 0.2g of Congo red, 1.5g of peptone, 4g of dipotassium hydrogen phosphate, 2g of monopotassium phosphate, 4g of ammonium sulfate and 0.5g of magnesium sulfate heptahydrate.
2 Screening of strains: inoculating bacteria into MRS liquid culture medium from-80deg.C refrigerator, and culturing at 37deg.C for 24 hr; then 100 mu L of culture solution is placed on a screening culture medium plate for culturing for 48 hours at 37 ℃ by a oxford cup method, the diameter of a transparent circle is observed, strains with high pectase yield are selected according to the diameter of the transparent circle, enzyme activity determination of pectase is carried out, and bacteria with high pectase yield are screened out.
Screening of tannase-producing Strain
1. Screening media (g.L-1): 3g of sodium nitrate, 1g of dipotassium hydrogen phosphate, 0.5g of potassium chloride and 0.5g of magnesium sulfate; 0.04g of bromophenol blue and 10g of tannic acid, and sterilizing separately.
2 Screening of strains: inoculating bacteria into MRS liquid culture medium from-80deg.C refrigerator, and culturing at 37deg.C for 24 hr; then 100 mu L of culture solution is placed on a screening culture medium flat plate for culturing for 48 hours at 37 ℃, tannic acid and bromophenol blue indicator are added into the screening culture medium, if the strain produces the tannin enzyme, tannic acid in the culture medium is hydrolyzed to produce gallic acid, and the bromophenol blue indicator around the colony is changed from blue-purple to yellow, so that an obvious color-changing ring is formed around the colony. And selecting a strain with high tannase yield according to the color depth and the diameter of the color-changing ring, and carrying out tannase activity measurement to screen out the strain with high tannase yield.
Phytase producing strain selection
1. Screening medium (g.L -1): 15g of glucose, 5.0g of ammonium nitrate, 0.5g of potassium chloride, 0.5g of magnesium sulfate, 0.3g of ferrous sulfate, 0.3g of manganese sulfate and 2g of calcium phytate.
2. Strain screening: inoculating bacteria into MRS liquid culture medium from-80 refrigerator, and culturing at 37deg.C for 24 hr; then 100 mu L of culture solution is placed on a screening culture medium plate for culturing for 48 hours at 37 ℃ by adopting an oxford cup method, and the strain producing phytase can hydrolyze calcium phytate to form transparent rings around bacterial colonies. And selecting a strain with high phytase yield according to the diameter of the transparent ring, measuring the enzyme activity of the phytase, and screening out the strain with high phytase yield.
The formula of the MRS liquid culture medium is as follows: 10.0g of beef extract, 20.0g of glucose, 10.0g of tryptone, 5.0g of yeast extract powder, 1.0mL of Tween 80, 2.0g of dipotassium hydrogen phosphate, 2.0g of ammonium citrate, 5.0g of anhydrous sodium acetate, 0.5g of magnesium sulfate, 0.25g of manganese sulfate monohydrate, 1.0L of deionized water and pH6.5 (2% agar is added as MRS solid culture medium).
And (3) strain identification:
(1) Morphological observation of bacteria
Gram staining and catalase test are carried out on the screened and purified strain YYS-EN2, physiological and biochemical indexes are measured, and the test result is compared with the eighth edition of Bojie's system bacteriology handbook to carry out preliminary judgment on strains. As shown in FIG. 1, the test shows that the screened strain YYS-EN2 is gram-stained purple and positive. The shape of the material is rod-shaped, the contact enzyme is negative, and no spore is formed.
Extracting YYS-EN2 DNA according to the instruction of the bacterial gene DNA extraction kit, and performing PCR amplification:
PCR amplification process:
Amplifying the 16S rDNA gene sequence by using a primer 27F (5'-AGAGTT TGATCCTGGCTCAG-3') 1492R (5'-GGTTACCTTGTTACGACTT-3'); PCR reaction system: 27F (10 uM) 1 uL, 1492R (10 uM) 1 uL, 10XEasyTag@R Buffer (2.5 mM) 5U L, dNTPs (2.5 mM) 4U L, DNA template 1 uL, easy tag@R DNA Polymerase (5U/L) 0.3U L, ddH 2 0.37.7 uL. PCR amplification procedure: 94℃for 5min, 94℃for 30s, 55℃for 30s, 72℃for 90s, 72℃for 10min, and 32 cycles from the second to the fourth step, 4 ℃.
③ PCR product detection and sequencing analysis, namely, the amplified product is sent to the Guangzhou Qingzhou biological engineering Co Ltd for sequencing, and the strain YYS-EN2 is identified as lactobacillus paracasei.
Wherein, the 16S rDNA gene sequence is as follows:
GCAGTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGG
GTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCC
AACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCC
GGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAA
CCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAG
CGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGAC
GCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGAT
GAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTG
GGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATG
TGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGAT
CAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGAT
GTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCT
AGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGA
CCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATC
TCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGT
AATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAT
GAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTTAGGGAGCGAGCCGTCTAA。
EXAMPLE 2 preparation of Lactobacillus paracasei YYS-EN2 lyophilized powder
Inoculating activated Lactobacillus paracasei YYS-EN2 into a culture medium sterilized at 121deg.C for 15min according to 2% (v/v), culturing at 37deg.C for 24 hr, centrifuging at 4deg.C for 10min at 6000r/min, discarding supernatant to obtain bacterial mud, emulsifying with protective agent for 15min to obtain emulsion with bacterial solution concentration of 5× 10CFU·mL-1, pre-freezing the emulsion in a refrigerator at-40deg.C for 4 hr, and freeze-drying at-35deg.C for 35 hr to obtain lyophilized powder of active Lactobacillus paracasei YYS-EN2 with living bacterial count of 5100 hundred million CFU.g -1.
Wherein the culture medium comprises the following components in percentage by mass: 2.5% glucose, 2.5% soy peptone, 1% yeast extract, 1% beef extract, 0.6% anhydrous sodium acetate, 0.05% magnesium sulfate, 0.03% manganese sulfate, 0.1% tween 80 and the balance water. The protective agent consists of 50 g.L -1 skimmed milk powder, 10 g.L -1 sucrose, 10 g.L -1 trehalose and 10 g.L -1 L-sodium glutamate.
It should be noted that: the protectant may employ existing protectant components or formulations, including but not limited to the embodiments described above.
EXAMPLE 3 Lactobacillus paracasei YYS-EN2 cellulase Activity
1 Enzyme-producing medium (g.L -1): 20g of sodium carboxymethylcellulose (CMC-Na), 3g of peptone, 0.5g of yeast extract, 2g of ammonium sulfate, 4g of monopotassium phosphate, 0.3g of magnesium sulfate and 0.3g of calcium chloride;
2 measurement of cellulase Activity
(1) Definition of enzyme activity:
the amount of enzyme required to decompose 10 g.L -1 sodium carboxymethylcellulose (CMC-Na) substrate to 1mg glucose per minute at 50℃and pH4.5 was defined as one enzyme activity unit (U.mL -1).
(2) And (3) preparation of a reagent:
0.1 mol.L -1 acetic acid-sodium acetate buffer (pH 4.6): 0.1 mol.L -1 sodium acetate solution and 0.1 mol.L -1 acetic acid solution were mixed and the pH was adjusted to 4.5. Wherein, 0.1 mol.L -1 sodium acetate solution, accurately weighing 8.20g anhydrous sodium acetate, adding distilled water and fixing the volume to a 1000mL volumetric flask; 0.1 mol.L -1 acetic acid solution, accurately weighing 6g acetic acid, adding distilled water and fixing the volume to a 1000mL volumetric flask;
Sodium carboxymethyl cellulose (CMC-Na) solution (10 g·l -1): 1g of sodium carboxymethylcellulose (CMC-Na) was accurately weighed and added to 100mL of acetic acid-sodium acetate buffer.
(3) Enzyme activity determination: the cellulase activity was measured by DNS.
1% Sodium carboxymethylcellulose (CMC-Na) solution is placed in a water bath kettle at 40 ℃ in advance for preheating, 1mL of enzyme solution is added into 1mL of sodium carboxymethylcellulose solution, 1mL of 0.4 mol.L - 1 NaOH is added for stopping the reaction after the reaction is carried out for 30min at 40 ℃, and a stop solution is added before the enzyme solution is added into a blank group.
After the reaction, 1mL of the reaction solution is taken and mixed with 1mL of DNS (5- (dimethylamino) -1-naphthalene sulfonic acid hydrate) solution, and the mixture is reacted in a boiling water bath for 5min for color development. Diluting the reaction solution after color development by 5 times, zeroing by taking distilled water as a reference, measuring the absorbance of a sample at the wavelength of 540nm by using a spectrophotometer, and calculating the enzyme activity according to a formula.
The crude enzyme solution obtaining process in this embodiment is as follows: inoculating Lactobacillus paracasei YYS-EN2 into enzyme production culture medium, culturing at 37deg.C for 48 hr, centrifuging at 4deg.C, and collecting supernatant to obtain crude enzyme solution; the crude enzyme solution was diluted to an enzyme solution at a multiple to perform an enzyme activity assay, wherein Lactobacillus paracasei YYS-EN2 was inoculated in an amount of 2% (v/v) of the medium.
The result measuring and calculating mode is as follows:
The enzyme activity measurement formula is: enzyme activity = OD 540 x n x 1/k x 1/30;
where n is the dilution factor in enzyme solution testing (dilution factor from crude enzyme solution to test enzyme solution), k is the slope of the fitted standard curve, and 30 is the enzymatic reaction time.
Wherein, the standard curve is drawn: standard solutions (0 g.L -1、0.2g·L-1、0.4g·L-1、0.6g·L-1、0.8g·L-1、1.0g·L-1) were prepared in different concentration gradients from 1 g.L -1 glucose solutions, and absorbance at 540nm was measured according to the enzyme activity assay method. A linear standard curve was plotted with glucose concentration on the abscissa and OD on the ordinate (see FIG. 2).
The measurement result shows that the cellulase activity of Lactobacillus paracasei YYS-EN2 is 47.34+ -0.06 U.multidot.mL -1.
EXAMPLE 4 Lactobacillus paracasei YYS-EN2 pectase Activity
1. Enzyme-producing medium (g.L -1): 10g of pectin, 10g of glucose, 4g of peptone, 4g of dipotassium hydrogen phosphate, 2g of potassium dihydrogen phosphate and 0.5g of magnesium sulfate heptahydrate;
2. Pectase activity assay
(1) Definition of enzyme activity:
the amount of enzyme required to decompose 10 g.L -1 of pectin substrate to 1mg of galacturonic acid per minute at 50℃and pH 5.0 is defined as one enzyme activity unit (U.mL -1);
(2) Preparing a solution:
0.2 mol.L -1 acetic acid-sodium acetate buffer (pH 4.8): 0.2 mol.L -1 sodium acetate solution and 0.2 mol.L -1 acetic acid solution were mixed and the pH was adjusted to 5.0. Wherein, 0.2 mol.L -1 of acetic acid solution: accurately weighing 12g of acetic acid, adding distilled water and fixing the volume to a 1000mL volumetric flask; 0.2 mol.L -1 of sodium acetate solution, accurately weighing 16.41g of sodium acetate, adding distilled water and fixing the volume to a 1000mL volumetric flask. The solution was mixed and the pH was adjusted to 5.0.
Pectin substrate (10 g.L -1): 1g pectin is added to 100mL acetic acid-sodium acetate buffer
(3) Enzyme activity determination:
Placing pectin substrate in a water bath kettle at 50 ℃ in advance for preheating; 1mL of enzyme solution is taken, 1mL of preheated pectin substrate is added, the mixture is shaken uniformly, the mixture is reacted in a water bath at 50 ℃ for 30min, 1mL of 10% trichloroacetic acid solution is added for stopping the reaction, and a stop solution is added before the enzyme solution is added in a blank group. After the reaction is finished, 1mL of reaction solution is taken and mixed with 1mLDNS solution uniformly, and the mixture is reacted in boiling water bath for 5min for color development. Diluting the reaction solution after color development by 5 times, zeroing by taking distilled water as a reference, measuring the absorbance of a sample at the wavelength of 540nm by using a spectrophotometer, and calculating the enzyme activity according to a formula.
The crude enzyme solution obtaining process in this embodiment is as follows: inoculating Lactobacillus paracasei YYS-EN2 into enzyme production culture medium, culturing at 37deg.C for 48 hr, centrifuging at 4deg.C, and collecting supernatant to obtain crude enzyme solution; the crude enzyme solution was diluted to an enzyme solution at a multiple to perform an enzyme activity assay, wherein Lactobacillus paracasei YYS-EN2 was inoculated in an amount of 2% (v/v) of the medium.
The result measuring and calculating mode is as follows:
The enzyme activity measurement formula is: enzyme activity = OD 540 x n x 1/k x 1/30;
where n is the dilution factor in enzyme solution testing (dilution factor from crude enzyme solution to test enzyme solution), k is the slope of the fitted standard curve, and 30 is the enzymatic reaction time.
Wherein, the standard curve is drawn: standard solutions (0 g.L -1、0.2g·L-1、0.4g·L-1、0.6g·L-1、0.8g·L-1、1.0g·L-1) were prepared in different concentration gradients from 1 g.L -1 of D-galacturonic acid solution, and absorbance at 540nm was measured according to the enzyme activity assay method. A linear standard curve was plotted with D-galacturonic acid concentration on the abscissa and OD on the ordinate (see FIG. 3).
The measurement result shows that the pectase activity of Lactobacillus paracasei YYS-EN2 is 47.81 + -0.07U/mL.
Example 5 Lactobacillus paracasei YYS-EN2 tannin enzyme Activity
1. Enzyme-producing medium (g.L -1): 10g of glucose, 3g of sodium nitrate, 1g of dipotassium hydrogen phosphate, 0.5g of potassium chloride, 0.5g of magnesium sulfate and 20g of tannic acid.
2. Tannin enzyme activity assay
(1) Definition of enzyme activity:
The amount of enzyme required to decompose the substrate 0.01 mol.L -1 propyl gallate per minute to produce 1mg of gallic acid at 30℃and pH 5.0 was defined as one enzyme activity unit (U.mL -1);
(2) Preparing a solution:
0.1 mol.L -1 citric acid-sodium citrate buffer (pH 5.0): 0.1 mol.L -1 of citric acid solution and 0.1 mol.L -1 of sodium citrate solution were mixed and the pH was adjusted to 5.0. Wherein, 0.1 mol.L -1 citric acid solution: accurately weighing 19.21g of citric acid, and fixing the volume to a 1000mL volumetric flask by using distilled water; sodium citrate solution of 0.1mol·l -1: 29.41g sodium citrate was accurately weighed, dissolved in distilled water and fixed to a 1000mL volumetric flask.
Propyl gallate solution (0.01 mol·l -1): accurately weighing 0.21g propyl gallate, and fixing the volume to a 100mL volumetric flask by using a citric acid buffer solution.
Methanol rhodamine solution: accurately weighing 0.667g of rhodanine, and fixing the volume to a 100mL volumetric flask by using methanol.
Gallic acid standard solution (5 mg. L -1): accurately weighing 0.5g gallic acid, and fixing the volume to a 100mL volumetric flask by using methanol.
(3) Enzyme activity determination:
Preheating propyl gallate solution in a water bath kettle at 30 ℃ in advance; 0.5mL of the enzyme solution is taken, 0.5mL of preheated propyl gallate solution is added, the mixture is shaken uniformly, the mixture is reacted in a water bath at 30 ℃ for 5min, 0.6mL of methanol rhodanine is added, the water bath at 30 ℃ for 5min, 0.8mL of KOH is added, the water bath at 30 ℃ for 5min, 7.6mL of distilled water is added, and the water bath at 30 ℃ for 10min. Zeroing by taking distilled water as a reference, measuring the absorbance of a sample at a wavelength of 520nm by using a spectrophotometer, and calculating the enzyme activity according to a formula. The blank group is added with a stop solution KOH before the enzyme solution is added.
The crude enzyme solution obtaining process in this embodiment is as follows: inoculating Lactobacillus paracasei YYS-EN2 into an enzyme production culture medium, culturing at 37deg.C for 48 hr, centrifuging at 4deg.C, collecting supernatant to obtain crude enzyme solution, and diluting the crude enzyme solution to enzyme solution for testing according to a certain multiple to perform enzyme activity measurement; wherein, the inoculation amount of lactobacillus paracasei YYS-EN2 is 2% (v/v) of the culture medium.
The result measuring and calculating mode is as follows:
The enzyme activity measurement formula is: enzyme activity = OD 520 x n x 1/k x 1/5 x 1/0.5;
Wherein n is the dilution factor (dilution factor of crude enzyme solution to that of enzyme solution for test or that of reaction solution after color development) in enzyme solution test, k is the slope of a fitted standard curve, 5 is the enzymatic reaction time, and 1/0.5 is converted into 1mL of enzyme solution.
Wherein, the standard curve is drawn: standard solutions (0mg.L -1、1mg·L-1、2mg·L-1、3mg·L-1、4mg·L-1、5mg·L-1) with different concentration gradients were prepared from 5 mg.L -1 gallic acid solution, and absorbance at 520nm was measured according to the enzyme activity assay method. Drawing a linear standard curve (see FIG. 4) with gallic acid concentration on the abscissa and OD value on the ordinate
The measurement result showed that the tannase activity of Lactobacillus paracasei YYS-EN2 was 29.85.+ -. 0.03 U.mL -1.
EXAMPLE 6 Lactobacillus paracasei YYS-EN2 Phytase Activity
1. Enzyme-producing medium (g.L -1): 3g of peptone, 15g of glucose, 0.5g of magnesium sulfate, 0.03g of manganese sulfate and 0.03g of ferrous sulfate.
2. Phytase Activity assay
(1) Definition of enzyme activity:
1mL of liquid enzyme, at 37 degrees C, pH 5.5 conditions per minute to decompose 50 mmol L -1 sodium phytate substrate, to produce 1U mo L inorganic phosphorus, namely a phytase activity unit, to represent U.mL -1.
(2) And (3) preparation of a reagent:
0.2 mol.L -1 acetic acid-sodium acetate buffer (pH 5.5): 0.2 mol.L -1 acetic acid solution and 0.2 mol.L -1 sodium acetate solution were mixed and the pH was adjusted to 5.5. Wherein, 0.2 mol.L -1 of acetic acid solution: 12g acetic acid is fixed to volume by distilled water into a 1000mL volumetric flask; 0.2 mol.L -1 sodium acetate solution; 16.406g of sodium acetate are dissolved in distilled water and are taken up in 1000mL volumetric flasks.
Sodium phytate solution (75 mmol. L -1): accurately weighing 0.69g of sodium phytate, dissolving the sodium phytate by using a buffer solution, and fixing the volume into a 100mL volumetric flask.
Nitric acid solution: nitric acid: water (v/v) =1: 2
Ammonium molybdate solution (100 g/L) 10g ammonium molybdate was accurately weighed, added to 1mL 25% (v/v) ammonia water and distilled water was used to volume to a 100mL volumetric flask.
The ammonium metavanadate solution (2.35 g/L) is prepared by accurately weighing 0.235g of ammonium metavanadate, dissolving in distilled water, adding 2mL of nitric acid solution, and using distilled water to fix the volume to a 100mL volumetric flask, and storing in a dark place for use.
Color development liquid (stop liquid): nitric acid solution: ammonium molybdate solution: ammonium metavanadate solution (v/v) =2: 1:1, storing in a dark place, and preparing at present.
(3) Enzyme activity determination:
firstly, 1.8mL of acetic acid buffer solution is mixed with 0.2mL of to-be-reacted liquid (namely enzyme liquid), the mixture is preheated for 5min at 37 ℃ in a water bath kettle, then 4mL of sodium phytate is added for uniform mixing, water bath is carried out for 30min at 37 ℃, and finally 4mL of chromogenic liquid (stop solution) is added for uniform mixing. Zeroing by taking distilled water as a reference, measuring the absorbance of a sample at 415nm by using a spectrophotometer, and calculating the enzyme activity according to a formula. The blank group is added with stop solution before enzyme solution is added.
The crude enzyme solution obtaining process in this embodiment is as follows: inoculating Lactobacillus paracasei YYS-EN2 into an enzyme production culture medium, culturing at 37deg.C for 48 hr, centrifuging at 4deg.C, collecting supernatant to obtain crude enzyme solution, and diluting the crude enzyme solution to enzyme solution according to a certain multiple for enzyme activity determination; wherein, the inoculation amount of lactobacillus paracasei YYS-EN2 is 2% (v/v) of the culture medium.
The result measuring and calculating mode is as follows:
The enzyme activity measurement formula is: enzyme activity = OD 415×n×1/k×1/30×1/0.2×103;
Wherein n is the dilution factor (from crude enzyme to enzyme for test) in enzyme solution test, k is the slope of a fitted standard curve, 30 is the enzymatic reaction time, 1/0.2 is converted into 1mL of enzyme solution, and 10 3 is the conversion factor of inorganic phosphorus ion concentration.
Wherein, the standard curve is drawn: inorganic phosphorus standard solutions (0mmo l·L-1、10mmo l·L-1、20mmo l·L-1、30mmo l·L-1、40mmo l·L-1、50mmo l·L-1), with different concentrations are prepared from potassium dihydrogen phosphate mother solution, the light absorption value of OD 415 is measured according to an enzyme activity measuring method, the inorganic phosphorus ion concentration is taken as an abscissa, the OD value is taken as an ordinate, and a linear standard curve is drawn (see FIG. 5).
The measurement result showed that the phytase activity of Lactobacillus paracasei YYS-EN2 was 18.67.+ -. 0.04U/mL.
Example 7 use of Lactobacillus paracasei YYS-EN2 in the preparation of fermented corn nutritional powder
Crushing corn embryo and corn husk, treating with lipase and amylase, sterilizing at high temperature, adding lactobacillus paracasei YYS-EN2 bacterial powder and/or freeze-dried powder 3% (by mass), mixing, fermenting at 37deg.C for 72 hr, sterilizing at high temperature after fermentation, and drying at 60deg.C to obtain corn nutrition powder. Lactobacillus paracasei YYS-EN2 ferments corn husks and corn embryos, breaks down cellulose and pectin and reduces tannins and phytic acid, enhancing the flavor and mouthfeel of the corn nutritional powder. The flavor results before and after the specific fermentation are shown in Table 1 below:
TABLE 1
Example 12 use of Lactobacillus paracasei YYS-EN2 in preparation of fermented traditional Chinese medicine
Pulverizing rhizoma Polygonati, ginseng radix (artificially cultivated), fructus Lycii, mori fructus and fructus Jujubae, and adding at a ratio of 0.5%, 0.3%, 1%, 1.5% and 2% by mass respectively; mixing uniformly, boiling, and keeping micro boiling for 1h; cooling, adding lactobacillus paracasei YYS-EN2 powder and/or lyophilized powder 0.03% and lactobacillus plantarum BXM2 powder 0.02%, mixing, fermenting at 37deg.C for 48 hr, and sterilizing at high temperature. Lactobacillus paracasei YYS-EN2 ferments the Chinese medicine, decomposes cellulose and pectin, reduces tannin and phytic acid, reduces bitter taste and enhances the mouthfeel.
The Chinese medicine is not limited to the specific Chinese medicine in the embodiment, and comprises the types in the medicine-food homology list. The probiotic combination comprises but is not limited to lactobacillus paracasei YYS-EN2 bacterial powder and lactobacillus plantarum BXM2 bacterial powder, and can also be independently used by lactobacillus paracasei YYS-EN2 bacterial powder or used together with other probiotic powder.
Wherein, the lactobacillus plantarum (Lactobacillus plantarum) BMX2 is adopted and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) in 9 and 6 days of 2018, the preservation address is North Star Xiyu No.1 and 3 in the Korean area of Beijing city, and the preservation number is CGMCC No.16436.
According to the results of the above examples, lactobacillus paracasei YYS-EN2 provided by the present invention has the following properties and effects:
1. Has the capability of producing cellulase, and the cellulase activity is 47.34+/-0.06 U.mL -1; can decompose plant cellulose and can be widely applied to the development of fermented traditional Chinese medicine and silage products;
2. Has pectase capability, and pectase activity is 47.81 +/-0.07 U.mL -1; can decompose pectin in traditional Chinese medicines, fruits, vegetables and the like, and can be widely applied to the development of products such as fermented traditional Chinese medicines, fruit juice, vegetables, wines, enzymes and the like;
3. Has tannase capability, and the tannase activity is 29.85+/-0.03 U.mL -1; the tannin content in the fermented traditional Chinese medicine and wine can be reduced, and the method can be widely applied to the development of fermented traditional Chinese medicine, wine and ferment products, and can improve the taste of the products;
4. Has phytase ability, and the phytase activity is 18.67+/-0.04 U.mL -1; can effectively decompose phytic acid, can be widely applied to flour and soybean food processing, and improves the nutrition and commodity value of the flour and soybean food;
5. can be used for preparing corn nutrition powder, degrading cellulose and phytic acid in corn husks and corn embryos, improving the nutrition value of the corn nutrition powder and improving the taste;
6. the strain is derived from oral saliva of healthy adults, is natural probiotics, and can be used as a starter for preparing fermented traditional Chinese medicines, feeds, wine, ferment and the like.
In summary, compared with the prior art, the lactobacillus paracasei YYS-EN2 provided by the invention has the following beneficial effects:
The lactobacillus paracasei YYS-EN2 provided by the invention can be used for probiotic products and functional foods, and can be planted in human intestinal tracts to play roles in improving flora structures and promoting digestion and absorption. The method can produce cellulase, pectase, tannase and phytase, and can be applied to probiotic fermentation products (fermentation products comprise but are not limited to fermentation traditional Chinese medicines, beverages, foods, feeds and the like), wherein the fermentation products comprise but are not limited to preparation of fermentation traditional Chinese medicines, fermentation plant-derived nutrition powder, fermentation plant-derived polypeptide powder, fermentation plant-derived protein powder, ferment, silage and the like;
The lactobacillus paracasei YYS-EN2 provided by the invention can produce cellulase and pectase, can degrade plant cellulose and pectin, and can be applied to development of products such as fermented traditional Chinese medicines, fermented plant-derived nutrition powder, fruit juice, vegetable feed and the like; meanwhile, the strain has high pectase and tannase capabilities, can be widely applied to the production of fermented products such as fermented traditional Chinese medicines, wines, enzymes and the like, and can obviously reduce the content of tannin in the products.
In summary, it is particularly applicable in functional products comprising at least one of the following actions:
(1) Has cellulose degradation capability;
(2) Has pectin degradation capability;
(3) Has tannin degradation capability;
(4) Has the capability of degrading phytic acid;
The products with the functions of (1) - (4) comprise, but are not limited to, the functions of decomposing plant fibers and pectin, improving the clarity of the products and the like, and can also be products with other obvious effects based on the functions of decomposing plant fibers and pectin and degrading tannins or phytic acid.
It should be noted that:
(1) Definition:
the term "food" as used herein is broad and includes human foods and drinks. In certain embodiments, the food product is suitable and designed for human feeding. The application can be used for preparing solid preparations such as powder, tablets, gel and the like, and also can be dispersed in liquid to prepare liquid preparations, including but not limited to the embodiment.
The traditional Chinese medicine in the "fermented traditional Chinese medicine product" described herein comprises the species contained in the catalogue of homology of medicine and food.
(2) Related prior art means or prior art terms to which the present application relates:
The "OD" value is an abbreviation of optical density (also called absorbance), and the difference between the energy of light passing through the object to be measured and the energy of light passing through the object to be measured is the energy absorbed by the object to be measured. "OD X" is an optical density value measured when the wavelength is set to Xnm, and is a standard indicator for tracking the density of microorganisms in a liquid culture, and is generally used to indicate the cell density of a cell. Among these, the "OD" value determination method is prior art, and the principle and method thereof will not be described here.
The oxford cup method is used in the prior art, and the process is not tired. (3) Strain application:
The examples illustrate the application of lactobacillus paracasei YYS-EN2 to corn meal products, which can be applied to various types of products containing cellulose, pectin, tannin and phytic acid according to the design concept described above, and can be applied to the preparation of fermented products including fermented traditional Chinese medicines, fermented plant-derived nutrition powder, fermented plant-derived polypeptide powder, fermented plant-derived protein powder, ferment, silage and the like, including but not limited to corn meal in the examples.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. Lactobacillus paracasei YYS-EN2 is characterized in that the preservation number is CGMCC No.28531.
2. Lactobacillus paracasei YYS-EN2 according to claim 1, characterized in that: it has the ability to produce cellulases, pectinases, tannase and phytases.
3. Lactobacillus paracasei YYS-EN2 according to claim 1, characterized in that: it has the ability to degrade cellulose, pectin, tannins and phytic acid.
4. A method for producing cellulases, pectinases, tannase and phytases, characterized by: inoculating lactobacillus paracasei YYS-EN2 into an enzyme-producing liquid culture medium, culturing at the temperature of between 30 and 40 ℃ for 48 to 72 hours, and carrying out solid-liquid separation on the enzyme-producing liquid culture medium after fermentation culture to obtain a supernatant; the supernatant is an enzyme solution containing cellulase and/or pectase and/or tannase and/or phytase;
Wherein the Lactobacillus paracasei YYS-EN2 is Lactobacillus paracasei YYS-EN2 according to any of claims 1-3.
5. The application of lactobacillus paracasei YYS-EN2 in preparing a fermentation product is characterized in that: the lactobacillus paracasei YYS-EN2 employs the lactobacillus paracasei YYS-EN2 as claimed in any of claims 1-3.
6. Use of lactobacillus paracasei YYS-EN2 according to claim 5 for the preparation of a fermented product, characterized in that: the fermentation product comprises fermented traditional Chinese medicines, fermented nutrition powder, fermented polypeptide powder, fermented protein powder, ferment and silage;
The fermentation nutrition powder comprises fermentation plant source nutrition powder; the fermented polypeptide powder comprises fermented plant-derived polypeptide powder; the fermented protein powder comprises fermented plant-derived protein powder.
7. A lyophilized product, characterized in that: a component comprising lactobacillus paracasei YYS-EN2 as claimed in any of claims 1 to 3.
8. A microbial inoculum, characterized in that: the components of the microbial inoculum comprise the freeze-dried powder of claim 7; and/or the components of the microbial inoculum comprise lactobacillus paracasei YYS-EN2 as claimed in any one of claims 1-3.
9. The application of lactobacillus paracasei YYS-EN2 in the preparation of a probiotic product is characterized in that: the lactobacillus paracasei YYS-EN2 employs the lactobacillus paracasei YYS-EN2 as claimed in any of claims 1-3.
10. The application of lactobacillus paracasei YYS-EN2 in the preparation of functional products is characterized in that: the functional product comprises at least one of the following functions:
(1) Has cellulose degradation capability;
(2) Has pectin degradation capability;
(3) Has tannin degradation capability;
(4) Has the capability of degrading phytic acid;
Wherein the Lactobacillus paracasei YYS-EN2 is Lactobacillus paracasei YYS-EN2 according to any of claims 1-3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410139976.0A CN117987311A (en) | 2024-01-31 | 2024-01-31 | Lactobacillus paracasei YYS-EN2 for producing cellulase, pectase, tannase and phytase and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410139976.0A CN117987311A (en) | 2024-01-31 | 2024-01-31 | Lactobacillus paracasei YYS-EN2 for producing cellulase, pectase, tannase and phytase and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117987311A true CN117987311A (en) | 2024-05-07 |
Family
ID=90898886
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410139976.0A Pending CN117987311A (en) | 2024-01-31 | 2024-01-31 | Lactobacillus paracasei YYS-EN2 for producing cellulase, pectase, tannase and phytase and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117987311A (en) |
-
2024
- 2024-01-31 CN CN202410139976.0A patent/CN117987311A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101904164B1 (en) | Saccharomyces cerevisae SRCM100936 strain for manufacturing the wine using various berries and not producing biogenic amine and uses thereof | |
| KR20110031324A (en) | Yeast extract with high glutamic acid content and producing method thereof | |
| CN113413351A (en) | Fermentation liquor with whitening and anti-aging effects, fermented polypeptide, and preparation method and application thereof | |
| CN101691551A (en) | Lactobacillus plantarum for food fermentation and applications thereof | |
| CN109554265A (en) | A kind of fermented glutinous rice low alcohol beverage and preparation method thereof | |
| KR20140051543A (en) | Method for producing fermented herb extract with high gaba content using lactobacillus plantarum k154 | |
| KR20200145254A (en) | Manufacturing method for a composition rich in ginsenosides Rg3, Rg5 and Rk1, which is excellent in improving brain function and cognitive function using ginseng | |
| CN113969242B (en) | Saccharomyces cerevisiae for high yield of gamma-aminobutyric acid and application thereof in preparation of gamma-aminobutyric acid products | |
| CN114107113B (en) | Method for reducing ethyl carbamate in fermented food by using synthetic starter | |
| CN109852558B (en) | Bacillus subtilis GZU03 for producing beta-glucosidase and nattokinase and application method thereof | |
| CN102899275A (en) | Method for preparing directed vat starter for making steamed breads | |
| CN104342372B (en) | Method for producing yeast autolysate by probiotic fermentation | |
| CN112111434B (en) | Excellent lactic acid bacteria, screening method and application of excellent lactic acid bacteria in preparation of Xiaoqu | |
| CN113564061A (en) | Saccharomyces cerevisiae SG35, fermentation inoculant containing Saccharomyces cerevisiae SG35 and application of fermentation inoculant | |
| CN111449239B (en) | Functional food additive of ganoderma lucidum fermented sea buckthorn seed meal and preparation method thereof | |
| CN108949629A (en) | A kind of composite ferment and the preparation method and application thereof | |
| CN111067081B (en) | Soybean paste based on bacillus natto mutant strain as dominant bacterial system and preparation method thereof | |
| CN114437990B (en) | Pediococcus pentosaceus, microbial inoculum, application thereof and method for preparing low glycemic index food | |
| CN109566995A (en) | A kind of Semen Coicis canned sweet fermented glatinous rice and preparation method thereof | |
| CN117987311A (en) | Lactobacillus paracasei YYS-EN2 for producing cellulase, pectase, tannase and phytase and application thereof | |
| CN117946924A (en) | Lactobacillus plantarum YYS-EN1 for producing cellulase, pectase, tannase and phytase and application thereof | |
| CN100406551C (en) | Leaven for fermenting meat product and its special-purpose strain | |
| CN111518703A (en) | Mucor for producing fermented soya beans and application thereof | |
| CN115462427B (en) | Preparation method of Kangpu tea | |
| CN116064280B (en) | Siamese bacillus 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 |