CN115477708B - Antibacterial yeast active polysaccharide, preparation method, identification method and application - Google Patents
Antibacterial yeast active polysaccharide, preparation method, identification method and application Download PDFInfo
- Publication number
- CN115477708B CN115477708B CN202110598906.8A CN202110598906A CN115477708B CN 115477708 B CN115477708 B CN 115477708B CN 202110598906 A CN202110598906 A CN 202110598906A CN 115477708 B CN115477708 B CN 115477708B
- Authority
- CN
- China
- Prior art keywords
- yeast
- bacteriostatic
- active polysaccharide
- polysaccharide
- hours
- 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
- 150000004676 glycans Chemical class 0.000 title claims abstract description 167
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 165
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 165
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 149
- 238000000034 method Methods 0.000 title claims abstract description 87
- 238000002360 preparation method Methods 0.000 title claims abstract description 29
- 230000000844 anti-bacterial effect Effects 0.000 title description 13
- 230000003385 bacteriostatic effect Effects 0.000 claims abstract description 124
- 210000005253 yeast cell Anatomy 0.000 claims abstract description 50
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 145
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 46
- 239000000203 mixture Substances 0.000 claims description 41
- 150000002772 monosaccharides Chemical class 0.000 claims description 33
- 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 claims description 29
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 29
- 239000008103 glucose Substances 0.000 claims description 29
- 229960001031 glucose Drugs 0.000 claims description 29
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims description 27
- CBOJBBMQJBVCMW-BTVCFUMJSA-N (2r,3r,4s,5r)-2-amino-3,4,5,6-tetrahydroxyhexanal;hydrochloride Chemical compound Cl.O=C[C@H](N)[C@@H](O)[C@H](O)[C@H](O)CO CBOJBBMQJBVCMW-BTVCFUMJSA-N 0.000 claims description 26
- 239000003153 chemical reaction reagent Substances 0.000 claims description 26
- 229960001911 glucosamine hydrochloride Drugs 0.000 claims description 26
- 229940041290 mannose Drugs 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 229920000392 Zymosan Polymers 0.000 claims description 15
- 238000004255 ion exchange chromatography Methods 0.000 claims description 15
- 238000000926 separation method Methods 0.000 claims description 15
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 claims description 13
- 239000006228 supernatant Substances 0.000 claims description 13
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 12
- 238000004108 freeze drying Methods 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 12
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 claims description 11
- 229940097043 glucuronic acid Drugs 0.000 claims description 11
- 238000012869 ethanol precipitation Methods 0.000 claims description 10
- 239000012141 concentrate Substances 0.000 claims description 9
- 238000002329 infrared spectrum Methods 0.000 claims description 9
- 239000000463 material Substances 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 7
- 230000003544 deproteinization Effects 0.000 claims description 7
- 238000000605 extraction Methods 0.000 claims description 7
- 244000144972 livestock Species 0.000 claims description 7
- 239000002994 raw material Substances 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 6
- 238000001704 evaporation Methods 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- 244000144977 poultry Species 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- 125000004309 pyranyl group Chemical group O1C(C=CC=C1)* 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- 238000004440 column chromatography Methods 0.000 claims description 4
- 230000008020 evaporation Effects 0.000 claims description 3
- 238000005057 refrigeration Methods 0.000 claims 24
- 239000003242 anti bacterial agent Substances 0.000 abstract description 6
- 229940088710 antibiotic agent Drugs 0.000 abstract description 6
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 238000011156 evaluation Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 38
- 238000010521 absorption reaction Methods 0.000 description 19
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 229920002307 Dextran Polymers 0.000 description 12
- 208000035404 Autolysis Diseases 0.000 description 10
- 206010057248 Cell death Diseases 0.000 description 10
- 230000028043 self proteolysis Effects 0.000 description 10
- 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 9
- 239000008367 deionised water Substances 0.000 description 9
- 229910021641 deionized water Inorganic materials 0.000 description 9
- 235000013336 milk Nutrition 0.000 description 9
- 239000008267 milk Substances 0.000 description 9
- 210000004080 milk Anatomy 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000010828 elution Methods 0.000 description 8
- 238000004321 preservation Methods 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 230000001276 controlling effect Effects 0.000 description 6
- 235000019441 ethanol Nutrition 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
- 238000004192 high performance gel permeation chromatography Methods 0.000 description 5
- 239000011259 mixed solution Substances 0.000 description 5
- 235000013594 poultry meat Nutrition 0.000 description 5
- 238000001694 spray drying Methods 0.000 description 5
- 239000012086 standard solution Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 108091005658 Basic proteases Proteins 0.000 description 3
- 101710130006 Beta-glucanase Proteins 0.000 description 3
- 102100032487 Beta-mannosidase Human genes 0.000 description 3
- 108010059892 Cellulase Proteins 0.000 description 3
- 108090000526 Papain Proteins 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 108010055059 beta-Mannosidase Proteins 0.000 description 3
- 229940106157 cellulase Drugs 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 229940055729 papain Drugs 0.000 description 3
- 235000019834 papain Nutrition 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 229920000057 Mannan Polymers 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000010668 complexation reaction Methods 0.000 description 2
- 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 2
- 239000003480 eluent Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- CBOJBBMQJBVCMW-NQZVPSPJSA-N (2r,3r,4r,5r)-2-amino-3,4,5,6-tetrahydroxyhexanal;hydrochloride Chemical compound Cl.O=C[C@H](N)[C@@H](O)[C@@H](O)[C@H](O)CO CBOJBBMQJBVCMW-NQZVPSPJSA-N 0.000 description 1
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 1
- 229920002498 Beta-glucan Polymers 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- AEMOLEFTQBMNLQ-BZINKQHNSA-N D-Guluronic Acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@@H](O)[C@H]1O AEMOLEFTQBMNLQ-BZINKQHNSA-N 0.000 description 1
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 description 1
- AEMOLEFTQBMNLQ-VANFPWTGSA-N D-mannopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H]1O AEMOLEFTQBMNLQ-VANFPWTGSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- 244000286779 Hansenula anomala Species 0.000 description 1
- 235000014683 Hansenula anomala Nutrition 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 description 1
- 244000285963 Kluyveromyces fragilis Species 0.000 description 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 229960002737 fructose Drugs 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 229960003082 galactose Drugs 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 244000000074 intestinal pathogen Species 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- LUEWUZLMQUOBSB-GFVSVBBRSA-N mannan Chemical class O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-GFVSVBBRSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000013613 poultry product Nutrition 0.000 description 1
- 150000003214 pyranose derivatives Chemical group 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- MBMQEIFVQACCCH-UHFFFAOYSA-N trans-Zearalenon Natural products O=C1OC(C)CCCC(=O)CCCC=CC2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-UHFFFAOYSA-N 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 229960003487 xylose Drugs 0.000 description 1
- MBMQEIFVQACCCH-QBODLPLBSA-N zearalenone Chemical compound O=C1O[C@@H](C)CCCC(=O)CCC\C=C\C2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-QBODLPLBSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/3563—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing solids; Preparation of samples therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/3563—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing solids; Preparation of samples therefor
- G01N2021/3572—Preparation of samples, e.g. salt matrices
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Physiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Materials Engineering (AREA)
- Sustainable Development (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of microbial application, in particular to bacteriostatic yeast active polysaccharide, a preparation method, an identification method and application. According to the invention, the bacteriostatic yeast active polysaccharide with the total sugar content of 65-85% and the weight average molecular weight of 90000-110000Da is obtained by extracting, separating and purifying the yeast cell wall, SWNP, SWAP-1 and SWAP-2 are obtained by further structural identification, and the bacteriostatic activity evaluation is carried out on the bacteriostatic active polysaccharide, so that the bacteriostatic activity of the bacteriostatic yeast active polysaccharide is improved by more than 4 times compared with that of the yeast cell wall, the bacteriostatic activity of the bacteriostatic active polysaccharide can be greatly improved when the bacteriostatic active polysaccharide is used in the field of feed addition, and the aim of replacing antibiotics is fulfilled.
Description
Technical Field
The invention relates to the technical field of microbial application, in particular to bacteriostatic yeast active polysaccharide, a preparation method, an identification method and application.
Background
The long-term unreasonable and nonstandard use of the feed growth-promoting antibiotics causes the serious safety risks such as bacterial drug resistance, antibiotic residues of livestock and poultry products, and the like, and the research of novel products with the function of replacing the feed growth-promoting antibiotics is continuously emerging under the large background demands of industries such as 'resistance reduction, no resistance' and the like of livestock and poultry cultivation.
The polysaccharide is used as a natural macromolecular substance of a higher animal and plant cell membrane and a microbial cell wall, and is researched and found to have various bioactive functions, such as immunity, blood sugar reduction, tumor resistance, intestinal flora regulation and the like. Research into functional biopolysaccharides is also receiving extensive attention immediately following nucleic acid and protein studies. In the field of animal cultivation, along with the increasing demands of green healthy cultivation, antibiotic-free cultivation and the like, functional biological polysaccharides are also widely focused.
The yeast cell wall is taken as an important component of the yeast source biological feed, and the chemical components of the yeast cell wall are mainly mannans, glucans, proteins, a small amount of chitin, lipids and the like. The current research reports that the components of the yeast cell wall exerting main physiological effects are mainly alpha-mannans with main chains connected by alpha-1, 6 glycosidic bonds, branched chains connected by alpha-1, 2 or alpha-1, 3 glycosidic bonds and beta-glucans connected by beta-1, 3/1,6 glycosidic bonds. The research shows that the traditional yeast cell wall can adsorb intestinal pathogens, promote intestinal beneficial bacteria, regulate body immunity, specifically adsorb zearalenone and other physiological effects, so that the traditional yeast cell wall has been widely applied to livestock and poultry cultivation.
Disclosure of Invention
The invention aims to solve the technical problems: provides a bacteriostatic yeast active polysaccharide with more stable bacteriostatic effect.
Aiming at the defects existing in the prior art, one of the purposes of the invention is to provide a bacteriostatic yeast active polysaccharide, the other one of the purposes of the invention is to provide a preparation method of the bacteriostatic yeast active polysaccharide, the other one of the purposes of the invention is to provide an identification method of the bacteriostatic yeast active polysaccharide, and the fourth one of the purposes of the invention is to provide an application of the bacteriostatic yeast active polysaccharide in livestock and poultry cultivation.
The technical scheme of the invention is as follows:
the invention provides a bacteriostatic yeast active polysaccharide, which comprises 65-85% of total sugar and 2-5% of protein, wherein the total weight of the bacteriostatic yeast active polysaccharide is 100%, and the weight average molecular weight is 90000-110000Da.
Preferably, the bacteriostatic yeast active polysaccharide is a polysaccharide with a pyran ring structure, preferably, the bacteriostatic yeast active polysaccharide has a three-dimensional spiral structure.
Preferably, the monosaccharide composition of the bacteriostatic yeast active polysaccharide is glucosamine hydrochloride, glucose and mannose.
Preferably, the bacteriostatic yeast active polysaccharide is SWAP-1: the monosaccharide composition molar ratio is glucosamine hydrochloride to glucose to mannose=0.109 to 0.422 to 0.469.
Preferably, the bacteriostatic yeast active polysaccharide is SWAP-2: the monosaccharide composition molar ratio is glucosamine hydrochloride to glucose to mannose=0.090 to 0.551 to 0.349.
Preferably, the bacteriostatic yeast active polysaccharide is SWNP, the monosaccharide composition of the bacteriostatic yeast active polysaccharide also comprises glucuronic acid, and the molar ratio of the monosaccharide composition is glucosamine hydrochloride, glucose, mannose and glucuronic acid=0.086:0.552:0.344:0.018.
Preferably, the bacteriostatic yeast active polysaccharide comprises two or more of the following 3 polysaccharides: (1) SWAP-1: the monosaccharide composition molar ratio is glucosamine hydrochloride to glucose to mannose=0.109:0.422:0.469; (2) SWAP-2: the monosaccharide composition molar ratio is glucosamine hydrochloride to glucose to mannose=0.090 to 0.551 to 0.349; (3) SWNP: the monosaccharide composition molar ratio is glucosamine hydrochloride to glucose to mannose to glucuronic acid=0.086 to 0.552 to 0.344 to 0.018.
The invention also provides a preparation method of the bacteriostatic yeast active polysaccharide, which comprises the following steps:
(1) And (3) heat extraction: extracting yeast cell walls with hot water, and centrifuging to obtain supernatant;
(2) And (3) evaporation: evaporating the supernatant obtained in the step (1) to obtain a concentrated solution;
(3) Ethanol precipitation: adding ethanol into the concentrated solution obtained in the step (2), and performing centrifugal separation to obtain solid crude zymosan;
(4) Deproteinization: preparing the crude yeast active polysaccharide obtained in the step (3) into a solution, adding a sevag reagent for repeated deproteinization for 1-10 times, centrifugally separating and collecting filtrate, and repeating the step (3) on the filtrate to obtain deproteinized yeast active polysaccharide.
(5) And (3) separating and purifying: and (3) sequentially carrying out ion exchange chromatography and gel column chromatography on the deproteinized polysaccharide obtained in the step (4) to obtain the antibacterial yeast active polysaccharide.
Preferably, in the preparation method of the bacteriostatic yeast active polysaccharide, the preparation method of the yeast cell wall is as follows:
A. autolysis wall breaking
Autolysis treatment is carried out on the yeast raw material, and autolysis conditions are as follows: the mass concentration of sodium chloride is 5%, the pH is 5.5, the temperature is 75 ℃, after autolysis is carried out for 25 hours, the yeast autolysate at the upper layer and the yeast cell wall milk at the lower layer are obtained by centrifugation at 5000rpm, and the yeast cell wall milk is collected for enzymolysis treatment.
B. Enzymolysis
Adding water into the yeast cell wall milk obtained in the step A, and sequentially adding mannanase (calculated by dry matter of the yeast cell wall, the same applies below), alkaline protease, papain, cellulase and beta-glucanase for enzymolysis treatment;
C. spray drying
And C, heating the solution obtained in the step B to 85 ℃, carrying out heat preservation treatment for 1.5h, and then carrying out spray drying treatment to obtain modified yeast cell wall powder.
Preferably, in the preparation method of the bacteriostatic yeast active polysaccharide, the yeast raw material of the yeast cell wall in the step (1) is derived from Saccharomyces cerevisiae and/or candida.
Preferably, in the preparation method of the bacteriostatic yeast active polysaccharide, the heat extraction temperature in the step (1) is 30-50 ℃, and preferably the weight ratio of feed liquid is 1:5-15.
Preferably, in the preparation method of the bacteriostatic yeast active polysaccharide, the volume ratio of the ethanol to the concentrated solution in the ethanol precipitation in the step (3) is 1:1-10.
Preferably, in the preparation method of the bacteriostatic yeast active polysaccharide, the step (3) further comprises refrigerating the mixed solution of ethanol and concentrated solution before centrifugal separation, preferably, the refrigerating temperature is 2-6 ℃, and more preferably, the refrigerating time is 12-24h.
Preferably, in the preparation method of the bacteriostatic yeast active polysaccharide, the step (3) further comprises freeze-drying the solid obtained by centrifugal separation.
Preferably, in the preparation method of the bacteriostatic yeast active polysaccharide, the volume ratio of the crude polysaccharide solution to the sevag reagent in the step (4) is 3-5:1. The sevag reagent is prepared by adopting chloroform and n-butanol with the volume ratio of 5:1.
The invention also provides an identification method of the antibacterial yeast active polysaccharide or the antibacterial yeast active polysaccharide prepared by the preparation method, which comprises the following steps: taking a bacteriostatic active yeast active polysaccharide sample, and respectively carrying out ion chromatography detection and infrared spectrum detection.
The invention also provides application of the bacteriostatic yeast active polysaccharide or the bacteriostatic yeast active polysaccharide prepared by the preparation method in livestock and poultry cultivation.
The invention has the beneficial effects that: according to the invention, the bacteriostatic yeast active polysaccharide with the total sugar content of 65-85% and the weight average molecular weight of 90000-110000Da is obtained by extracting, separating and purifying the yeast cell wall, the polysaccharide SWNP, the polysaccharide SWAP-1 and the polysaccharide SWAP-2 are further obtained by structural identification, and the bacteriostatic activity evaluation is carried out on the polysaccharide SWNP, the polysaccharide SWAP-1 and the polysaccharide SWAP-2, so that the polysaccharide has bacteriostatic activity, and compared with the yeast cell wall, the antibacterial activity is improved by more than 4 times, and the stability of the bacteriostatic effect can be greatly improved when the polysaccharide is used in the field of feed addition, and the purpose of replacing antibiotics is realized.
Drawings
FIG. 1 is a molecular weight map of the active polysaccharide SWNP of bacteriostatic yeast
FIG. 2 is a molecular weight map of the bacteriostatic yeast active polysaccharide SWAP-1
FIG. 3 is a molecular weight map of the bacteriostatic yeast active polysaccharide SWAP-2
FIG. 4 is a graph showing the variation of the maximum absorption wavelength of SWNP, SWAP-1, and mixed solutions of SWAP-2 and Congo red reagent at different NaOH concentrations
FIG. 5 is an infrared spectrum of the active polysaccharide SWNP of bacteriostatic yeast
FIG. 6 is an infrared spectrum of the bacteriostatic yeast active polysaccharide SWAP-1
FIG. 7 is an infrared spectrum of the bacteriostatic yeast active polysaccharide SWAP-2
FIG. 8 is an ion chromatogram of the bacteriostatic yeast active polysaccharide SWNP
FIG. 9 is an ion chromatogram of the bacteriostatic yeast active polysaccharide SWAP-1
FIG. 10 is an ion chromatogram of the bacteriostatic yeast active polysaccharide SWAP-2
Detailed Description
The invention provides bacteriostatic yeast active polysaccharide which is extracted, separated and purified from a yeast cell wall, and compared with the yeast cell wall, the bacteriostatic activity of the bacteriostatic yeast active polysaccharide is greatly improved. The invention provides a bacteriostatic yeast active polysaccharide, which comprises 65-85% of total sugar and 2-5% of protein, wherein the total weight of the bacteriostatic yeast active polysaccharide is 100%, and the weight average molecular weight is 90000-110000Da. The bacteriostatic yeast active polysaccharide can be applied as a feed additive, improves the stability of bacteriostatic effect and can replace antibiotics.
In still another preferred embodiment of the present invention, the bacteriostatic yeast active polysaccharide is a polysaccharide having a pyran ring structure, preferably, the bacteriostatic yeast active polysaccharide has a three-dimensional helical structure.
In yet another preferred embodiment of the present invention, the monosaccharide composition of the bacteriostatic yeast active polysaccharide is glucosamine hydrochloride, glucose and mannose.
In yet another preferred embodiment of the present invention, the bacteriostatic yeast active polysaccharide is SWAP-1: the monosaccharide composition molar ratio is glucosamine hydrochloride to glucose to mannose=0.109 to 0.422 to 0.469.
In yet another preferred embodiment of the present invention, the bacteriostatic yeast active polysaccharide is SWAP-2: the monosaccharide composition molar ratio is glucosamine hydrochloride to glucose to mannose=0.090 to 0.551 to 0.349.
In yet another preferred embodiment of the present invention, the bacteriostatic yeast active polysaccharide is SWNP: the monosaccharide composition also comprises glucuronic acid, and the molar ratio of the monosaccharide composition is glucosamine hydrochloride, glucose, mannose, glucuronic acid=0.086:0.552:0.344:0.018.
In still another preferred embodiment of the present invention, the bacteriostatic yeast active polysaccharide comprises two or more of the following 3 polysaccharides: (1) SWAP-1: the monosaccharide composition molar ratio is glucosamine hydrochloride to glucose to mannose=0.109:0.422:0.469; (2) SWAP-2: the monosaccharide composition molar ratio is glucosamine hydrochloride to glucose to mannose=0.090 to 0.551 to 0.349; (3) SWNP: the monosaccharide composition molar ratio is glucosamine hydrochloride to glucose to mannose to glucuronic acid=0.086 to 0.552 to 0.344 to 0.018.
Without being limited by theory, the bacteriostatic effect of the bacteriostatic yeast active polysaccharide provided by the invention is related to the monosaccharide composition content of the bacteriostatic yeast active polysaccharide, the structure of the polysaccharide and the like.
The invention also provides a preparation method of the bacteriostatic yeast active polysaccharide, which comprises the following steps:
(1) And (3) heat extraction: extracting yeast cell walls with hot water, and centrifuging to obtain supernatant;
(2) And (3) evaporation: evaporating the supernatant obtained in the step (1) to obtain a concentrated solution;
(3) Ethanol precipitation: adding ethanol into the concentrated solution obtained in the step (2), and performing centrifugal separation to obtain solid crude zymosan;
(4) Deproteinization: preparing the crude zymosan obtained in the step (3) into a solution, adding a sevag reagent for repeated deproteinization for 1-10 times, centrifugally separating and collecting filtrate, and repeating the step (3) on the filtrate to obtain deproteinized zymosan.
(5) And (3) separating and purifying: and (3) sequentially carrying out ion exchange chromatography and gel column chromatography on the deproteinized polysaccharide obtained in the step (4) to obtain the antibacterial yeast active polysaccharide.
In still another preferred embodiment of the present invention, the preparation method of the bacteriostatic yeast active polysaccharide comprises the following steps:
A. autolysis wall breaking
Autolysis treatment is carried out on the yeast raw material, and autolysis conditions are as follows: the mass concentration of sodium chloride is 5%, the pH is 5.5, the temperature is 75 ℃, after autolysis is carried out for 25 hours, the yeast autolysate at the upper layer and the yeast cell wall milk at the lower layer are obtained by centrifugation at 5000rpm, and the yeast cell wall milk is collected for enzymolysis treatment.
B. Enzymolysis
Adding water into the yeast cell wall milk obtained in the step A, and sequentially adding mannanase (calculated by dry matter of the yeast cell wall, the same applies below), alkaline protease, papain, cellulase and beta-glucanase for enzymolysis treatment;
C. spray drying
And C, heating the solution obtained in the step B to 85 ℃, carrying out heat preservation treatment for 1.5h, and then carrying out spray drying treatment to obtain modified yeast cell wall powder.
In still another preferred embodiment of the present invention, in the method for producing a bacteriostatic yeast active polysaccharide, the yeast material of the yeast cell wall in the step (1) is derived from Saccharomyces cerevisiae and/or Candida.
In still another preferred embodiment of the present invention, in the preparation method of the bacteriostatic yeast active polysaccharide, the heat extraction temperature in the step (1) is 30-50 ℃, and preferably, the weight ratio of the feed liquid is 1:5-15.
In still another preferred embodiment of the present invention, in the preparation method of the bacteriostatic yeast active polysaccharide, the volume ratio of the ethanol to the concentrated solution in the ethanol precipitation in the step (3) is 1:1-10.
In still another preferred embodiment of the present invention, the preparation method of the bacteriostatic yeast active polysaccharide according to the above step (3) further comprises refrigerating the mixture of ethanol and the concentrated solution before the centrifugal separation, preferably, the refrigerating temperature is 2-6 ℃, and more preferably, the refrigerating time is 12-24 hours.
In still another preferred embodiment of the present invention, in the method for preparing the bacteriostatic yeast active polysaccharide, the step (3) further comprises freeze-drying the solid obtained by centrifugal separation.
In still another preferred embodiment of the present invention, in the preparation method of the bacteriostatic yeast active polysaccharide, the volume ratio of the crude polysaccharide solution and the sevag reagent in the step (4) is 3-5:1. The sevag reagent is prepared by adopting chloroform and n-butanol with the volume ratio of 5:1.
The invention also provides an identification method of the antibacterial yeast active polysaccharide or the antibacterial yeast active polysaccharide prepared by the preparation method, which comprises the following steps: taking a bacteriostatic yeast active polysaccharide sample, and respectively carrying out ion chromatography detection and infrared spectrum detection.
The invention also provides application of the bacteriostatic yeast active polysaccharide or the bacteriostatic yeast active polysaccharide prepared by the preparation method in livestock and poultry cultivation.
Strain preservation information:
the Saccharomyces cerevisiae FX-2 (Saccharomyces cerevisiae FX-2) adopted by the invention is preserved in China Center for Type Culture Collection (CCTCC) at 8-1-2016, and the preservation number is CCTCC NO: M2016418. This strain is described in the publication No. CN 108220175A.
The candida C1.7 (Wickerhamomyces anomalus C1.7) adopted by the invention is preserved in China Center for Type Culture Collection (CCTCC) at 12 months 11 days 2017, and the preservation number is CCTCCNO: M2017782. This strain is described in the publication No. CN 110959853A.
The bacteriostatic active polysaccharide, the preparation method and the identification method thereof are specifically described by specific examples and experimental examples.
The raw materials and equipment sources used in the inventive examples and comparative examples are shown in Table 1.
Table 1 inventive and comparative examples use raw materials and equipment sources
Example 1
Preparation of Yeast cell wall
1. The saccharomyces cerevisiae FX-2 strain inclined plane is placed into 100mL shake flask liquid culture medium with the inoculation amount of 2 loops, shake cultivation is carried out, the rotating speed is set to 250r/min, the temperature is 30 ℃, and the cultivation is carried out for 18 hours. Wherein, the formula of the fermentation medium is as follows: molasses with a total sugar concentration of 28%, 10L; (NH) 4 ) 2 SO 4 ，500g；NH 4 H 2 PO 4 ,80g；MgSO 4 ,56g；ZnSO 4 ,28g；H 2 O,20L; the pH was 6.0.
2. Autolysis treatment is carried out on the primary yeast raw material, sodium chloride is added to ensure that the mass concentration of the sodium chloride is 5%, the pH is 5.5, the temperature is 75 ℃, after autolysis is carried out for 25 hours, centrifugal treatment is carried out at 5000rpm, thus obtaining an autolysate of the yeast at the upper layer and a yeast cell wall milk at the lower layer, and the yeast cell wall milk is collected for enzymolysis treatment.
3. Enzymolysis
(1) Diluting the yeast cell wall milk obtained in the step 2 into 20% concentration by adding water, adding mannanase with 0.5% mass concentration (calculated by dry matter of the yeast cell wall, the same shall apply hereinafter), controlling the temperature to 45 ℃, controlling the pH to 7.5, and controlling the hydrolysis time to 10 hours;
(2) Continuously adding alkaline protease with the mass concentration of 0.6%, controlling the temperature to 50 ℃, controlling the pH value to 7.5, and controlling the hydrolysis time to 10 hours;
(3) Regulating the temperature of the solution treated in the step (2) to 58 ℃ and the pH value to 7.0, and then continuously adding papain with the mass concentration of 0.03%, and carrying out enzymolysis for 5 hours;
(4) Regulating the temperature of the solution treated in the step (2) to 85 ℃, and carrying out heat preservation treatment for 1.5h to obtain an inactivated solution;
(5) The temperature of the solution after enzyme deactivation is regulated to 50 ℃ and the pH value is regulated to 4.5, and then cellulase with the mass concentration of 0.1% is added for enzymolysis for 5 hours;
(6) Adding 0.1% mass concentration of beta-glucanase into the solution treated in the step (5), and hydrolyzing for 12 hours;
(7) And (3) heating the solution obtained in the step (6) to 85 ℃ for heat preservation treatment for 1.5 hours, and then performing spray drying treatment to obtain yeast cell wall powder.
(II) preparation of bacteriostatic yeast active polysaccharide
1. And (3) heat extraction: accurately weighing 60g of yeast cell wall prepared in the step (I), adding 600g of deionized water, and magnetically stirring for 1h at 50 ℃ to uniformly dissolve a sample; subpackaging the sample into a 100mL centrifuge tube, centrifuging at 6000rpm for 20min, and collecting supernatant; the supernatant was concentrated by rotary evaporation and the volume of the concentrate was 60mL.
2. Ethanol precipitation: adding 4 times volume of absolute ethyl alcohol into the concentrated solution obtained in the step 1, uniformly mixing, refrigerating for 12 hours at 4 ℃, centrifuging at 4500rpm for 10min, collecting precipitate, and freeze-drying to obtain crude polysaccharide.
3. Deproteinization: preparing the crude polysaccharide obtained in the step 2 into 20mg/mL, adding sevag reagent (the volume ratio of the crude polysaccharide solution to the sevag reagent is 3:1), centrifuging at 10000rpm for 1 min after shaking for 0.5h on a shaking table, collecting an upper layer water layer, and repeatedly removing protein with the sevag reagent for 4 times according to the method. Pouring the collected upper water layer into a centrifuge tube, adding 4 times of absolute ethyl alcohol into the centrifuge tube, uniformly mixing, refrigerating in a refrigerator at 4 ℃ for 12h, centrifuging at 4500rpm for 10min, collecting precipitate, and freeze-drying to obtain deproteinized polysaccharide.
4. Ion exchange chromatography: the deproteinized polysaccharide obtained in the step 3 is prepared into a solution of 20mg/mL, and the solution is applied to a DEAE cellulose chromatographic column, the volume is 5mL, the flow rate is 1 mL/min, the column is eluted with 200mL of deionized water, and then gradient elution is carried out with 200mL of 0.1 mol/L, 0.3 mol/L and 0.5 mol/L of NaC solution respectively, and a tube is collected every 10 mL. Every other tube, 100uL of the sample is tracked and detected in the eluent by using a phenol-concentrated sulfuric acid method, an elution curve is drawn, two elution peaks appear, the collected solutions of the same elution peaks are combined, concentrated, dialyzed for desalting and freeze-dried, and polysaccharide SWN and polysaccharide SWA are obtained.
5. Gel column chromatography: and (3) preparing polysaccharide SWN and polysaccharide SWA obtained in the step (4) into 40mg/mL solutions respectively, loading the two polysaccharide solutions onto Sephadex G-100 chromatographic columns respectively, eluting with 200mL deionized water, and collecting 5mL of polysaccharide solutions per test tube. Sampling 100uL, tracking and detecting the content of polysaccharide in the eluent by using a phenol-concentrated sulfuric acid method, drawing an elution curve, wherein a polysaccharide SWN sample shows an elution peak, a polysaccharide SWA sample shows two elution peaks, combining the collected solutions of the same elution peak, and freeze-drying to obtain bacteriostatic yeast active polysaccharide SWNP, bacteriostatic yeast active polysaccharide SWAP-1 and bacteriostatic yeast active polysaccharide SWAP-2.
(III) determination of bacteriostatic Yeast active polysaccharide product
1. Determination of total sugar and protein content
1) Method for measuring total sugar content
The total sugar content was determined using the phenol-concentrated sulfuric acid method. 0.1mg/mL of standard glucose standard solution, 0.1,0.2,0.4,0.6,0.8,1.0mL, was pipetted, respectively, and each was filled with water (deionized water) to 1mL. 0.5mL of 6% phenol solution was added to each tube, 2.5mL of concentrated sulfuric acid was rapidly added dropwise, the mixture was immediately shaken well, and 20min was allowed to stand for blank reference (deionized water 1.0 mL) to measure absorbance at 490 nm. The standard solutions of the same concentration were each repeated 3 times. Regression is performed on the glucose mass concentration (X) with the absorbance value (Y) to obtain a regression equation.
Accurately weighing 10mg of the sample, and fixing the volume in a 100mL volumetric flask to prepare 0.1mg/mL. After 1mL was taken, the procedure was followed as described above, and polysaccharide content was calculated against the standard curve, and repeated three times in parallel. Total sugar content (%) =concentration in glucose (mg/mL) ×0.9/sample concentration (mg/mL).
2) Method for measuring protein content
The protein content was determined using coomassie brilliant blue method. 0.1mg/mL of bovine serum albumin standard solution 0.1,0.2,0.3,0.4 and 0.5mL were aspirated, respectively, and added to the test tube. The absorbance was measured at 595nm with blank reference to 1mL of each of the two-phase aqueous solutions, 5mL of a coomassie brilliant blue solution, and 5-20 min. Regression was performed on the protein mass (X) with absorbance (Y) to obtain a regression equation.
Accurately weighing 10mg of the sample, and fixing the volume in a 100mL volumetric flask to prepare 0.1mg/mL. After 1mL was taken, the procedure was followed as described above, and the protein content was calculated against a standard curve, and repeated in triplicate.
3) Measurement results
The results of the determination of total sugar content and protein content in the bacteriostatic yeast active polysaccharide SWNP, SWAP-1 and SWAP-2 are shown in Table 2,
TABLE 2 determination of total sugar content and protein content of bacteriostatic Yeast active polysaccharide
| Antibacterial yeast active polysaccharide | Total sugar (%) | Protein (%) |
| SWNP | 74.04±1.35 | 2.73±0.37 |
| SWAP-1 | 84.01±1.46 | 2.53±0.12 |
| SWAP-2 | 67.74±1.46 | 2.35±0.15 |
2. Determination of molecular weight of active polysaccharide of bacteriostatic yeast
1) Method for measuring molecular weight of active polysaccharide of bacteriostatic yeast
Determining the molecular weight of the bacteriostatic yeast active polysaccharide by adopting High Performance Gel Permeation Chromatography (HPGPC);
HPGPC chromatographic conditions: chromatographic column: BRT105-104-102 series gel column (8X 300 mm); mobile phase: 0.05mol/LNaCl solution; flow rate: 0.6mL/min, column temperature: 40 ℃; sample injection amount: 20. Mu.L; a detector: differential detector RID-10A.
Drawing a standard curve: the standard solutions were prepared by weighing 5mg/mL of dextran standards (dextran standard 1152, dextran standard 5000, dextran standard 11600, dextran standard 23800, dextran standard 48600, dextran standard 80900, dextran standard 148000, dextran standard 273000, dextran standard 409800, dextran standard 667800, dextran standard 3693000) respectively, separating for 10min at 12000rpm, filtering the supernatant with a microporous filter membrane of 0.22 μm, detecting with HPGPC, and drawing standard curves.
Accurately weighing the sample, preparing the sample into a 5mg/mL solution, separating the sample from 10min at 12000rpm, taking supernatant, filtering the supernatant with a microporous filter membrane of 0.22 mu m, detecting the supernatant by HPGPC, and calculating the molecular weight of the sample according to a standard curve and the concentration.
2) Molecular weight measurement result of bacteriostatic yeast active polysaccharide
The molecular weight patterns of the bacteriostatic yeast active polysaccharides SWNP, SWAP-1, and SWAP-2 are shown in FIGS. 1-3, and the calculated molecular weights of SWNP, SWAP-1, and SWAP-2 are shown in Table 3.
TABLE 3 determination of polysaccharide molecular weight
4. Reaction with Congo Red reagent
1) Measurement method
Accurately weighing 4mg of bacteriostatic yeast active polysaccharide sample, adding 2mL of deionized water and 80umo L/L Congo red reagent, adding a proper amount of 1mo L/LNaOH solution to ensure that the alkali concentration in the solution is 0.0, 0.1,0.2,0.3,0.4 and 0.5mo L/L respectively, fully mixing uniformly, standing for 5min, and then scanning in a wavelength range of 400-600nm by using an ultraviolet-visible spectrophotometer to determine the maximum absorption wavelength. Congo red solution without polysaccharide sample was used as control. And the abscissa represents NaOH concentration, and the ordinate represents the maximum absorption wavelength, and a curve is drawn.
2) Measurement results
The change in maximum absorption wavelength of the mixed solution of SWNP, SWAP-1 and SWAP-2 and Congo red reagent at different NaOH concentrations is shown in FIG. 4, and when the NaOH concentration is from 0 to 0.1 mol/L, the maximum absorption wavelength of the mixed solution of SWNP, SWAP-1 and SWAP-2 and Congo red reagent is red shifted, which indicates that SWNP, SWAP-1 and SWAP-2 have triple helix structures and can be subjected to complexation reaction with the Congo red reagent; at NaOH concentrations from 0.1 to 0.5 mol/L, the maximum absorption wavelength of the mixed solution of SWNP, SWAP-1, and SWAP-2 with Congo red reagent was reduced, which indicates that at this time the triple helix structure of SWNP, SWAP-1, and SWAP-2 was destroyed and no complexation reaction with Congo red reagent could occur.
Example 2
Structural analysis was performed on the bacteriostatic yeast active polysaccharides SWNP, SWAP-1, and SWAP-2 using infrared spectroscopy and Ion Chromatography (IC).
Test method (one)
1. Infrared spectrum testing method
Grinding the dried analysis sample with KBr, tabletting, and measuring 4000-400cm with FT/IR -1 Scanning in range.
2. Test method for measuring monosaccharide composition by Ion Chromatography (IC)
IC chromatographic conditions: chromatographic column: dionex CarbopacTMPA20 (150 mm. Times.3 mm); mobile phase: AH 2 O；B:15mmol/LNaOH；C:15mmol/LNaOH&100mmol/LNaOAC; flow rate: 0.3mL/min; sample injection amount: 5. Mu.L; column temperature: 30 ℃; a detector: an electrochemical detector.
Sample pretreatment protocol: 10mg of the sample is accurately weighed and placed in an ampoule bottle, 10mL of 3mol/LTFA is added, and hydrolysis is carried out for 3 hours at 120 ℃. Accurately sucking the acid hydrolysis solution, transferring to a pipe, blowing and drying by nitrogen, adding 5mL of deionized water, mixing uniformly by vortex, sucking 100uL of deionized water, adding 900uL of deionized water, and centrifuging at 12000rpm for 5min. The supernatant was analyzed by IC.
Standard curve: 16 standard solutions of 10mg/mL monosaccharides (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, mannuronic acid) were prepared, and gradient concentrations of 0.01, 0.1, 0.5, 1, 5, 10, and 20mg/L were precisely prepared.
And (3) calculating results: according to the absolute quantitative method, the mass of the different monosaccharides is determined, and the molar ratio is calculated according to the molar mass of the monosaccharides.
(II) test results
1. Results of the IR spectrum analysis
The results of IR spectrum analysis for SWNP, SWAP-1, and SWAP-2 are shown in FIGS. 5-7. As can be seen from FIG. 5, SWNP was at 3262cm -1 、2935cm -1 、1636cm -1 、1410cm -1 、1022cm -1 、915cm -1 、810cm -1 And 579cm -1 A stronger absorption peak is arranged at the position; as can be seen from FIG. 6, SWAP-1 was measured at 3290cm -1 、2933cm -1 、1651cm -1 、1386cm -1 、1021cm -1 、911cm -1 、811cm -1 And 588cm -1 A stronger absorption peak is arranged at the position; as can be seen from FIG. 7, SWAP-2 was measured at 3281cm -1 、2933cm -1 、1645cm -1 、1362cm -1 、1019cm -1 、915cm -1 、808cm -1 And 599cm -1 There is a strong absorption peak. 3262cm therein -1 、3290cm -1 And 3281cm -1 The absorption peak is wider, and is the stretching vibration of polysaccharide-OH, at 2935cm -1 And 2933cm -1 The absorption peak of (C-H) is C-H stretching vibration of polysaccharide at 1636cm -1 、1651cm -1 And 1645cm -1 The absorption peak of (C) is the flexural vibration of polysaccharide O-H at 1410cm -1 、1386cm -1 And 1362cm -1 The absorption peak of (C-H) is C-H angular vibration of polysaccharide, at 1126cm -1 、1128cm -1 And 1127cm -1 The absorption peak of (C) is C-O-C stretching vibration on the pyran ring, and the absorption peak is 1022cm -1 、1021cm -1 And 1019cm -1 The absorption peak of (C) is C-O-H stretching vibration on the pyran ring at 915cm -1 And 911cm -1 The absorption peak of (C) indicates the presence of a beta-glycosidic bond at 810cm -1 、811cm -1 And 808cm -1 The absorption peak of (2) indicates the presence of an alpha-glycosidic bond, 579cm -1 、588cm -1 And 599cm -1 The absorption peak of (2) is the symmetrical stretching vibration of the pyranose skeleton. Taken together, SWNP, SWAP-1 and SWAP-2 are polysaccharides having a pyran ring structure, saccharides andthe saccharides are linked by an alpha-glycosidic bond and a beta-glycosidic bond.
2. Ion Chromatography (IC) assay results
As shown in FIGS. 8 to 10 and Table 4, the monosaccharide composition of SWNP was composed of glucosamine hydrochloride, glucose, mannose and glucuronic acid in the molar ratio of 0.086:0.552:0.344:0.018; as can be seen from FIG. 9 and Table 4, SWAP-1 has a monosaccharide composition consisting of glucosamine hydrochloride, glucose and mannose in a molar ratio of 0.109:0.422:0.469; as can be seen from FIG. 10 and Table 4, SWAP-2 has a monosaccharide composition consisting of glucosamine hydrochloride, glucose and mannose in a molar ratio of 0.090:0.551:0.349.
TABLE 4 monosaccharide composition and molar ratio of bacteriostatic Yeast active polysaccharide
/>
Experimental example 1
MIC (minimum inhibitory concentration) experiments were performed on the yeast cell walls, SWNP, SWAP-1, and SWAP-2 obtained in step (one) of example 1. The specific experimental process and experimental results are as follows:
1. test method
The MIC of the sample for the experimental strain was determined according to the macrobroth dilution method (test tube method) in CLSI M07-A9, and the specific method is as follows: taking 9 sterilized test tubes, adding 1mL of sterilized broth into each tube, adding 1mL of tested liquid medicine into the 1 st tube, uniformly mixing, taking out 1mL to the 2 nd tube, and removing 1mL from the 7 th tube by analogy. Tube 8 was not dosed and tube 9 was blank broth without bacteria as a control. For example, the sample gradient is: 200. 100, 50, 25, 12.5, 6.25 and 3.13mg/mL. And adding 1mL of diluted bacterial suspension into the 1 st to 8 th tubes, uniformly mixing, placing the mixture in a constant temperature incubator at 37 ℃ for incubation for 16 to 20 hours, and observing the result, wherein the minimum concentration capable of completely inhibiting bacterial growth is MIC.
2. Test results
The MIC (minimum inhibitory concentration) test results are shown in Table 5, and compared with the yeast cell wall before separation and purification, the antibacterial effect of SWNP, SWAP-1 and SWAP-2 prepared by the invention on Escherichia coli is improved by 4 times compared with the yeast cell wall before separation and purification, the antibacterial effect on Staphylococcus aureus is improved by 8 times compared with the yeast cell wall before separation and purification, and the antibacterial effect of SWNP, SWAP-1 and SWAP-2 are improved by 4 times compared with the yeast cell wall before separation and purification.
TABLE 5 MIC (minimum inhibitory concentration) test results
In conclusion, the bacteriostatic yeast active polysaccharide with the total sugar content of 65-85% and the weight average molecular weight of 90000-110000Da is obtained by extracting, separating and purifying the yeast cell wall, the bacteriostatic activity of the bacteriostatic yeast active polysaccharide SWNP, SWAP-1 and SWAP-2 is further evaluated by structural identification, and the result shows that the bacteriostatic activity of the bacteriostatic yeast active polysaccharide is improved by more than 4 times compared with that of the yeast cell wall, the stability of the bacteriostatic effect can be greatly improved when the bacteriostatic yeast active polysaccharide is used in the field of feed addition, and the purpose of replacing antibiotics is realized.
The above description is not intended to limit the invention in any way, but is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (71)
1. The bacteriostatic yeast active polysaccharide is characterized by comprising 65-85% of total sugar content and 2-5% of protein content, wherein the total weight of the bacteriostatic yeast active polysaccharide is 100%, and the weight average molecular weight is 90000-110000Da; the monosaccharide composition of the bacteriostatic yeast active polysaccharide comprises glucosamine hydrochloride, glucose and mannose; or the monosaccharide composition of the bacteriostatic yeast active polysaccharide is glucosamine hydrochloride, glucose, mannose and glucuronic acid;
wherein the bacteriostatic yeast active polysaccharide is SWAP-1, and the monosaccharide composition molar ratio is glucosamine hydrochloride to glucose to mannose=0.109:0.422:0.469; or alternatively
The bacteriostatic yeast active polysaccharide is SWAP-2, and the monosaccharide composition molar ratio is glucosamine hydrochloride, glucose, mannose=0.090:0.551:0.349; or alternatively
The bacteriostatic yeast active polysaccharide is SWNP, and the monosaccharide composition molar ratio of the bacteriostatic yeast active polysaccharide is glucosamine hydrochloride, glucose, mannose, glucuronic acid=0.086:0.552:0.344:0.018.
2. The bacteriostatic yeast active polysaccharide according to claim 1, wherein the bacteriostatic yeast active polysaccharide is a polysaccharide having a pyran ring structure.
3. The bacteriostatic yeast active polysaccharide according to claim 1, wherein the bacteriostatic yeast active polysaccharide has a three-dimensional helical structure.
4. The bacteriostatic yeast active polysaccharide according to claim 2, wherein the bacteriostatic yeast active polysaccharide has a three-dimensional helical structure.
5. The bacteriostatic yeast active polysaccharide according to any one of claims 1 to 4, wherein the bacteriostatic yeast active polysaccharide comprises two or more of the following 3 polysaccharides: (1) SWAP-1: the monosaccharide composition molar ratio is glucosamine hydrochloride to glucose to mannose=0.109:0.422:0.469; (2) SWAP-2: the monosaccharide composition molar ratio is glucosamine hydrochloride to glucose to mannose=0.090 to 0.551 to 0.349; (3) SWNP: the monosaccharide composition molar ratio is glucosamine hydrochloride to glucose to mannose to glucuronic acid=0.086 to 0.552 to 0.344 to 0.018.
6. The method for preparing the bacteriostatic yeast active polysaccharide according to any one of claims 1 to 5, which is characterized by comprising the following steps:
(1) And (3) heat extraction: extracting yeast cell walls with hot water, and centrifuging to obtain supernatant;
(2) And (3) evaporation: evaporating the supernatant obtained in the step (1) to obtain a concentrated solution;
(3) Ethanol precipitation: adding ethanol into the concentrated solution obtained in the step (2), and performing centrifugal separation to obtain solid crude zymosan;
(4) Deproteinization: preparing the crude zymosan obtained in the step (3) into a solution, adding a sevag reagent for repeated deproteinization for 1-10 times, centrifugally separating and collecting filtrate, and repeating the step (3) on the filtrate to obtain deproteinized zymosan;
(5) And (3) separating and purifying: and (3) sequentially carrying out ion exchange chromatography and gel column chromatography on the deproteinized polysaccharide obtained in the step (4) to obtain the bacteriostatic yeast active polysaccharide.
7. The process according to claim 6, wherein the heat extraction temperature in the step (1) is 30 to 50 DEG C
8. The method according to claim 6, wherein the feed liquid weight ratio in the step (1) is 1:5-15.
9. The method according to claim 7, wherein the feed liquid weight ratio in the step (1) is 1:5-15.
10. The method according to claim 6, wherein the volume ratio of the ethanol to the concentrated solution in the ethanol precipitation in the step (3) is 1:1-10.
11. The method according to claim 7, wherein the volume ratio of the ethanol to the concentrated solution in the ethanol precipitation in the step (3) is 1:1-10.
12. The method according to claim 8, wherein the volume ratio of the ethanol to the concentrated solution in the ethanol precipitation in the step (3) is 1:1-10.
13. The method according to claim 9, wherein the volume ratio of the ethanol to the concentrated solution in the ethanol precipitation in the step (3) is 1:1-10.
14. The method according to claim 6, wherein the step (3) further comprises refrigerating the mixture of ethanol and the concentrate before centrifuging.
15. The method according to claim 7, wherein the step (3) further comprises refrigerating the mixture of ethanol and the concentrate before centrifuging.
16. The method according to claim 8, wherein the step (3) further comprises refrigerating the mixture of ethanol and the concentrate before centrifuging.
17. The method according to claim 9, wherein the step (3) further comprises refrigerating the mixture of ethanol and the concentrate before centrifuging.
18. The method according to claim 10, wherein the step (3) further comprises refrigerating the mixture of ethanol and the concentrate before centrifuging.
19. The method according to claim 11, wherein the step (3) further comprises refrigerating the mixture of ethanol and the concentrate before centrifuging.
20. The method of claim 12, wherein the step (3) further comprises refrigerating the mixture of ethanol and concentrate prior to centrifugation.
21. The method of claim 13, wherein the step (3) further comprises refrigerating the mixture of ethanol and concentrate prior to centrifugation.
22. The method of claim 14, wherein the refrigeration temperature is 2-6 ℃.
23. The method of claim 15, wherein the refrigeration temperature is 2-6 ℃.
24. The method of claim 16, wherein the refrigeration temperature is 2-6 ℃.
25. The method of claim 17, wherein the refrigeration temperature is 2-6 ℃.
26. The method of claim 18, wherein the refrigeration temperature is 2-6 ℃.
27. The method of claim 19, wherein the refrigeration temperature is 2-6 ℃.
28. The method of claim 20, wherein the refrigeration temperature is 2-6 ℃.
29. The method of claim 21, wherein the refrigeration temperature is 2-6 ℃.
30. The method of claim 14, wherein the refrigeration time is 12-24 hours.
31. The method of claim 15, wherein the refrigeration time is 12-24 hours.
32. The method of claim 16, wherein the refrigeration time is 12-24 hours.
33. The method of claim 17, wherein the refrigeration time is 12-24 hours.
34. The method of claim 18, wherein the refrigeration time is 12-24 hours.
35. The method of claim 19, wherein the refrigeration time is 12-24 hours.
36. The method of claim 20, wherein the refrigeration time is 12-24 hours.
37. The method of claim 21, wherein the refrigeration time is 12-24 hours.
38. The method of claim 22, wherein the refrigeration time is 12-24 hours.
39. The method of claim 23, wherein the refrigeration time is 12-24 hours.
40. The method of claim 24, wherein the refrigeration time is 12-24 hours.
41. The method of claim 25, wherein the refrigeration time is 12-24 hours.
42. The method of claim 26, wherein the refrigeration time is 12-24 hours.
43. The method of claim 27, wherein the refrigeration time is 12-24 hours.
44. The method of claim 28, wherein the refrigeration time is 12-24 hours.
45. The method of claim 29, wherein the refrigeration time is 12-24 hours.
46. The method according to claim 6, wherein the step (3) further comprises freeze-drying the solid obtained by centrifugal separation.
47. The method according to claim 7, wherein the step (3) further comprises freeze-drying the solid obtained by centrifugal separation.
48. The method according to claim 8, wherein the step (3) further comprises freeze-drying the solid obtained by centrifugal separation.
49. The method according to claim 10, wherein the step (3) further comprises freeze-drying the solid obtained by centrifugal separation.
50. The method of claim 14, wherein step (3) further comprises freeze-drying the solids obtained by centrifugation.
51. The method of claim 22, wherein step (3) further comprises freeze-drying the solids obtained by centrifugation.
52. The method of claim 30, wherein step (3) further comprises freeze-drying the solids obtained by centrifugation.
53. The method of claim 6, wherein the volume ratio of the crude zymosan solution to sevag reagent in step (4) is 3-5:1.
54. The method of claim 7, wherein the volume ratio of the crude zymosan solution to sevag reagent in step (4) is 3-5:1.
55. The method of claim 8, wherein the volume ratio of the crude zymosan solution to sevag reagent in step (4) is 3-5:1.
56. The method of claim 10, wherein the volume ratio of the crude zymosan solution to sevag reagent in step (4) is 3-5:1.
57. The method of claim 14, wherein the volume ratio of the crude zymosan solution to sevag reagent in step (4) is 3-5:1.
58. The method of claim 22, wherein the volume ratio of the crude zymosan solution to sevag reagent in step (4) is 3-5:1.
59. The method of claim 30, wherein the volume ratio of the crude zymosan solution to sevag reagent in step (4) is 3-5:1.
60. The method of claim 46, wherein the volume ratio of the crude zymosan solution to sevag reagent in step (4) is 3-5:1.
61. The process according to claim 6, wherein step (1)
The yeast raw material of the yeast cell wall is derived from Saccharomyces cerevisiae and/or candida.
62. The process according to claim 7, wherein the yeast material of the yeast cell wall of step (1) is derived from Saccharomyces cerevisiae and/or Candida.
63. The method according to claim 8, wherein the yeast material of the yeast cell wall in step (1) is derived from Saccharomyces cerevisiae and/or Candida.
64. The method according to claim 10, wherein the yeast material of the yeast cell wall of step (1) is derived from Saccharomyces cerevisiae and/or Candida.
65. The method according to claim 14, wherein the yeast material of the yeast cell wall of step (1) is derived from Saccharomyces cerevisiae and/or Candida.
66. The method of claim 22, wherein the yeast material of the yeast cell wall of step (1) is derived from Saccharomyces cerevisiae and/or Candida.
67. The method of claim 30, wherein the yeast material of the yeast cell wall of step (1) is derived from Saccharomyces cerevisiae and/or Candida.
68. The process of claim 46, wherein the yeast material of the yeast cell wall of step (1) is derived from Saccharomyces cerevisiae and/or Candida.
69. The process of claim 53, wherein the yeast material of the yeast cell wall of step (1) is derived from Saccharomyces cerevisiae and/or Candida.
70. The identification method of the bacteriostatic yeast active polysaccharide according to any one of claims 1 to 5 or the bacteriostatic yeast active polysaccharide prepared by the preparation method according to any one of claims 6 to 69, characterized by comprising the steps of: taking a bacteriostatic yeast active polysaccharide sample, and respectively carrying out ion chromatography detection and infrared spectrum detection.
71. Use of the bacteriostatic yeast active polysaccharide according to any one of claims 1-5 or the bacteriostatic yeast active polysaccharide prepared by the preparation method according to any one of claims 6-60 in livestock and poultry cultivation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110598906.8A CN115477708B (en) | 2021-05-31 | 2021-05-31 | Antibacterial yeast active polysaccharide, preparation method, identification method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110598906.8A CN115477708B (en) | 2021-05-31 | 2021-05-31 | Antibacterial yeast active polysaccharide, preparation method, identification method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115477708A CN115477708A (en) | 2022-12-16 |
| CN115477708B true CN115477708B (en) | 2023-11-07 |
Family
ID=84419307
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110598906.8A Active CN115477708B (en) | 2021-05-31 | 2021-05-31 | Antibacterial yeast active polysaccharide, preparation method, identification method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115477708B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003012701A (en) * | 2001-07-03 | 2003-01-15 | Mitsui Chemicals Inc | New polysaccharide originated from yeast |
| CN101020915A (en) * | 2007-03-07 | 2007-08-22 | 中国农业科学院农产品加工研究所 | Process of preparing yeast beta-glucosan |
| CN103570840A (en) * | 2012-07-26 | 2014-02-12 | 宜兴市江山生物科技有限公司 | Yeast polysaccharide separation and purification method |
| CN103613682A (en) * | 2013-11-19 | 2014-03-05 | 济南大学 | Method for preparing yeast glucan and coproducing mannan and trehalose |
| CN104610460A (en) * | 2014-12-17 | 2015-05-13 | 江苏大学 | Method for extraction of polysaccharide from yeast cell wall |
-
2021
- 2021-05-31 CN CN202110598906.8A patent/CN115477708B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003012701A (en) * | 2001-07-03 | 2003-01-15 | Mitsui Chemicals Inc | New polysaccharide originated from yeast |
| CN101020915A (en) * | 2007-03-07 | 2007-08-22 | 中国农业科学院农产品加工研究所 | Process of preparing yeast beta-glucosan |
| CN103570840A (en) * | 2012-07-26 | 2014-02-12 | 宜兴市江山生物科技有限公司 | Yeast polysaccharide separation and purification method |
| CN103613682A (en) * | 2013-11-19 | 2014-03-05 | 济南大学 | Method for preparing yeast glucan and coproducing mannan and trehalose |
| CN104610460A (en) * | 2014-12-17 | 2015-05-13 | 江苏大学 | Method for extraction of polysaccharide from yeast cell wall |
Non-Patent Citations (3)
| Title |
|---|
| Paweena Dikit等.Characterization of an unexpected bioemulsifier from spent yeast obtained from Thai traditional liquor distillation.《International Journal of Biological Macromolecules》.2010,第47卷465-470. * |
| Zhaomin Zheng等.Water-soluble yeast β'glucan fractions with different molecular weights: Extraction and separation by acidolysis assisted-size exclusion chromatography and their association with proliferative activity.《International Journal of Biological Macromolecules》.2018,1-45. * |
| 成建国等.酵母细胞壁多糖分子量分布和 结构的初步分析.《食品工业科技》.2011,第33卷(第12期),143-146. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115477708A (en) | 2022-12-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11111317B2 (en) | Cordyceps militaris medium polysaccharide, method for separating and purifying same, and use thereof | |
| US10835552B2 (en) | Method for preparing linseed polysaccharide having antiviral activity and immunological activity, and use of the linseed polysaccharide | |
| CN104945527A (en) | Galactomannan antigen and preparation method thereof | |
| CN105131153A (en) | Sheep enoxaparin sodium compound preparation method, compound and application of compound | |
| CN115960167A (en) | Corn anti-adhesion peptide and preparation method and application thereof | |
| CN115028750B (en) | Paulownia She Zaoyan algal polysaccharide, and preparation method and application thereof | |
| CN113274490A (en) | Preparation method and application of botryococcus longipediculus polysaccharide disease-resistant inducer | |
| CN111363056B (en) | Rhodopseudomonas palustris exopolysaccharide and preparation method and application thereof | |
| CN109295131B (en) | Receptor positioning solid-phase enzymolysis preparation method of dendrobe active oligosaccharide | |
| CN108503724B (en) | Cordyceps militaris culture medium polysaccharide, and separation and purification method and application thereof | |
| CN115477708B (en) | Antibacterial yeast active polysaccharide, preparation method, identification method and application | |
| CN113831419A (en) | Armillariella tabescens mycelium polysaccharide and its preparation method and bacteriostatic use | |
| US6284885B1 (en) | Process for preparing (1→3)-β-D-glucan from fungi | |
| CN113817078B (en) | Poplar yellow fungus polysaccharide with anti-colorectal cancer effect based on immune regulation and control and application thereof | |
| CN113244258A (en) | Preparation method and application of spirulina polysaccharide proinflammatory enzyme inducer | |
| CN115160450A (en) | Rapid preparation method and application of Pholiota nameko polysaccharide | |
| CN109988249A (en) | The preparation method and applications of flammulina velutipes FVP | |
| EP4368697A1 (en) | High immune yeast cell wall, and preparation method therefor and use thereof | |
| CN113604522A (en) | Penicillium D306 strain capable of producing extracellular polysaccharide and application thereof in preparation of bile acid binder | |
| CN111281859B (en) | Chitosan derivative nanoparticles with effect of enhancing antigen presenting cell to present antigen and preparation method thereof | |
| CN114456246A (en) | Method for improving antibacterial activity and thermal stability of roxburgh rose protein and application | |
| CN108641006B (en) | Method for extracting, separating and purifying polysaccharide from cordyceps militaris culture medium leftovers | |
| CN114989322B (en) | Method for improving bioactivity of pleurotus eryngii polysaccharide | |
| CN116731217B (en) | Ampelopsis grossedentata acidic polysaccharide AGP-2a, preparation method thereof and application thereof in preparing anti-inflammatory cosmetics | |
| CN113801798B (en) | Acidocella adephagia strain A50, extracellular polysaccharide produced by same 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |