WO2023217056A1 - Composite microbial deodorant, method for preparing same, and use thereof - Google Patents
Composite microbial deodorant, method for preparing same, and use thereof Download PDFInfo
- Publication number
- WO2023217056A1 WO2023217056A1 PCT/CN2023/092640 CN2023092640W WO2023217056A1 WO 2023217056 A1 WO2023217056 A1 WO 2023217056A1 CN 2023092640 W CN2023092640 W CN 2023092640W WO 2023217056 A1 WO2023217056 A1 WO 2023217056A1
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- WIPO (PCT)
- Prior art keywords
- lactobacillus
- composite microbial
- microbial deodorant
- fermentation
- harbinensis
- Prior art date
Links
- 239000002781 deodorant agent Substances 0.000 title claims abstract description 68
- 230000000813 microbial effect Effects 0.000 title claims abstract description 64
- 239000002131 composite material Substances 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 92
- 241000925032 Lactobacillus harbinensis Species 0.000 claims abstract description 80
- 229910021529 ammonia Inorganic materials 0.000 claims abstract description 42
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 claims abstract description 28
- 229910000037 hydrogen sulfide Inorganic materials 0.000 claims abstract description 28
- 238000004332 deodorization Methods 0.000 claims abstract description 14
- 244000144972 livestock Species 0.000 claims abstract description 4
- 244000144977 poultry Species 0.000 claims abstract description 4
- 238000009629 microbiological culture Methods 0.000 claims abstract 2
- 238000000855 fermentation Methods 0.000 claims description 77
- 230000004151 fermentation Effects 0.000 claims description 77
- 244000063299 Bacillus subtilis Species 0.000 claims description 54
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 54
- 244000199866 Lactobacillus casei Species 0.000 claims description 48
- 235000013958 Lactobacillus casei Nutrition 0.000 claims description 48
- 229940017800 lactobacillus casei Drugs 0.000 claims description 48
- 230000001580 bacterial effect Effects 0.000 claims description 39
- 239000007788 liquid Substances 0.000 claims description 35
- 241000186660 Lactobacillus Species 0.000 claims description 22
- 229940039696 lactobacillus Drugs 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 241000894006 Bacteria Species 0.000 claims description 16
- 210000003608 fece Anatomy 0.000 claims description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 11
- 238000009423 ventilation Methods 0.000 claims description 11
- 239000010871 livestock manure Substances 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 9
- 239000002054 inoculum Substances 0.000 claims description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 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 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 239000001301 oxygen Substances 0.000 claims description 7
- 229910052760 oxygen Inorganic materials 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 235000015278 beef Nutrition 0.000 claims description 4
- 235000013379 molasses Nutrition 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 235000012054 meals Nutrition 0.000 claims description 3
- 238000005273 aeration Methods 0.000 claims description 2
- 238000005507 spraying Methods 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 34
- 244000005700 microbiome Species 0.000 abstract description 4
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 238000012545 processing Methods 0.000 abstract description 3
- 241000186605 Lactobacillus paracasei Species 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 238000011109 contamination Methods 0.000 abstract 1
- 230000007774 longterm Effects 0.000 abstract 1
- 230000004913 activation Effects 0.000 description 31
- 150000001875 compounds Chemical class 0.000 description 29
- 239000003795 chemical substances by application Substances 0.000 description 28
- 239000000243 solution Substances 0.000 description 25
- 238000011218 seed culture Methods 0.000 description 23
- 239000001963 growth medium Substances 0.000 description 17
- 239000002609 medium Substances 0.000 description 16
- 239000007789 gas Substances 0.000 description 12
- 239000007921 spray Substances 0.000 description 11
- 241000272525 Anas platyrhynchos Species 0.000 description 10
- 241000282326 Felis catus Species 0.000 description 10
- 230000001877 deodorizing effect Effects 0.000 description 10
- 239000011777 magnesium Substances 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- 238000011081 inoculation Methods 0.000 description 8
- 239000006872 mrs medium Substances 0.000 description 8
- 239000008223 sterile water Substances 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 238000003756 stirring Methods 0.000 description 7
- 238000009630 liquid culture Methods 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 230000005923 long-lasting effect Effects 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 description 3
- -1 ethyl ethyl sulfate morpholine Chemical compound 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 238000003760 magnetic stirring Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000002195 synergetic effect Effects 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 240000006024 Lactobacillus plantarum Species 0.000 description 2
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 229940072205 lactobacillus plantarum Drugs 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 235000005985 organic acids Nutrition 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 239000010865 sewage Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 240000001046 Lactobacillus acidophilus Species 0.000 description 1
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 description 1
- 241000214517 Lactobacillus casei group Species 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 229920000180 alkyd Polymers 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- HIVLDXAAFGCOFU-UHFFFAOYSA-N ammonium hydrosulfide Chemical compound [NH4+].[SH-] HIVLDXAAFGCOFU-UHFFFAOYSA-N 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000443 biocontrol Effects 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000000149 chemical water pollutant Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 210000000750 endocrine system Anatomy 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- 239000010903 husk Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 150000002475 indoles Chemical class 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000001726 jatropha manihot extract Substances 0.000 description 1
- 229940039695 lactobacillus acidophilus Drugs 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 230000006996 mental state Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 231100000344 non-irritating Toxicity 0.000 description 1
- 230000009965 odorless effect Effects 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 239000003440 toxic substance 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
- 239000001393 triammonium citrate Substances 0.000 description 1
- 235000011046 triammonium citrate Nutrition 0.000 description 1
- 229940106668 yucca extract Drugs 0.000 description 1
- 239000010457 zeolite Substances 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
- B01D53/34—Chemical or biological purification of waste gases
- B01D53/46—Removing components of defined structure
- B01D53/54—Nitrogen compounds
- B01D53/58—Ammonia
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
- B01D53/34—Chemical or biological purification of waste gases
- B01D53/74—General processes for purification of waste gases; Apparatus or devices specially adapted therefor
- B01D53/84—Biological processes
Definitions
- the present invention relates to a composite microbial deodorant, in particular to a composite microbial deodorant with Lactobacillus harbinensis and Lactobacillus casei as main active ingredients, and belongs to the technical field of environmental microorganisms.
- Odorous substances include thiols, thioethers, amines, amides, indoles and some inorganic substances. These substances harm people's respiratory system, circulatory system, digestive system, endocrine system and nervous system, seriously affect people's mental state and reduce their quality of life.
- Deodorants are mainly divided into physical deodorants, chemical deodorants, microbial deodorants, plant-based deodorants and compound deodorants.
- Physical deodorants mainly use adsorption and masking to achieve the purpose of deodorization.
- Commonly used materials for this type of deodorants include activated carbon, zeolites, aromatic compounds, etc. The characteristic is that it is difficult to completely change the composition of the odor gas, which is harmful to people, animals, and equipment. There is still a certain degree of damage to the environment and the environment.
- Chemical deodorants use chemical reagents to react with odor molecules to achieve the purpose of deodorization.
- Common chemical deodorant product raw materials include castor oil zinc alkyd, soybean ethyl ethyl sulfate morpholine, methacrylic fatty alcohol, etc. , which is characterized by rapid response but short-lasting effect, and is prone to secondary pollution.
- the active ingredients of plant deodorants come from tea extract, yucca extract, phytoncidin and other plants.
- the chemical substances contained in these plants can chemically react with the odor molecule groups and weaken the chemical bonds in the odor molecules. , and finally produce odorless and non-toxic substances, but the extraction process of active ingredients is complicated, the production cost is high, and the deodorization target is single, and some unpredictable chemical reactions may occur.
- Microbial deodorants use the metabolism of microbial flora or their metabolites to effectively degrade common odor factors such as volatile fatty acids and hydrogen sulfide. They are safe, non-toxic, non-irritating and biodegradable for humans and animals. Degradation, through the continuous reproduction of microorganisms, the biological deodorization effect is long-lasting, without secondary pollution, easy to use and low cost.
- Microbial deodorants have attracted more and more attention due to their environmental protection and reusability advantages.
- the existing production process of microbial deodorants is relatively complex, compound strains with synergistic effects are difficult to obtain, and the effect of decomposing odor is not good, which limits the further application of microbial deodorants.
- the present invention provides a composite microbial deodorant.
- the composite microbial deodorant has a high efficiency in treating ammonia gas generated by pet feces, livestock and poultry feces, and sewers when used for 24 hours. It can reach more than 90%, the hydrogen sulfide treatment efficiency can reach more than 85%, and the deodorization is long-lasting, efficient and has no secondary pollution.
- a composite microbial deodorant including Lactobacillus harbinensis (Lactobacillus harbinensis) and Lactobacillus casei-like, the Lactobacillus harbinensis is preserved in the General Microbiology Center of the China Committee for the Collection of Microorganisms, the address is: Beichen West Road, Chaoyang District, Beijing No. 1, No. 3, Institute of Microbiology, Chinese Academy of Sciences, the deposit number is: CGMCC No. 24253, the deposit date is: January 5, 2022, and its 16S rDNA sequence is shown in SEQ ID No: 1.
- the Lactobacillus harbinensis described in the present invention refers to the Lactobacillus harbinensis MD-1 strain, which is isolated from landfill leachate. Its physiological and biochemical characteristics are: in MRS culture medium When cultured, the colonies are round, milky white, opaque, convex, with a rough surface, and the cells are short rods.
- the Lactobacillus casei described in the present invention is also called Lactobacillus paracasei and belongs to the Lactobacillus casei group in the genus Lactobacillus.
- the ratio of the viable bacterial numbers of Lactobacillus harbinensis and Lactobacillus casei-like bacteria is 1: (1-2), Preferred 1: (1.2-1.6).
- the composite microbial deodorant also contains Bacillus subtilis.
- Bacillus subtilis Preferably, the ratio of the viable bacterial numbers of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis is 1: (0.5-3): (0.5 -2.5), more preferably 1: (0.7-2.1): (0.9-1.5).
- the amount of viable bacteria in the composite microbial deodorant is not less than 10 ⁇ 10 8 CFU/mL, preferably (10-50) ⁇ 10 8 CFU/mL.
- Lactobacillus harbinensis and Lactobacillus casei-like bacteria secrete lactobacilli during the growth process and produce organic acids, alcohol and carbon dioxide, which can significantly inhibit the growth of harmful bacteria and thereby reduce the production of odorous substances;
- Bacillus subtilis can quickly consume free oxygen in the environment, promote the growth of lactic acid bacteria, and produce organic acids such as lactic acid, lowering the pH value of the environment and indirectly inhibiting the growth of other harmful bacteria.
- Bacillus subtilis can synthesize and digest by itself Enzymes, such as protease, amylase, lipase, cellulase, etc., these enzymes have a certain decomposition effect on organic matter and have a certain purification effect on the environment.
- strains are small and the cost is low. It only contains two or three strains. Lactobacillus casei and Bacillus subtilis can be selected from any manufacturer on the market, so the cost can be reasonably controlled.
- the preparation method of the above-mentioned composite microbial deodorant includes the following steps:
- Expanded culture Expand the culture of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis to obtain the culture solution of each strain;
- step (1) The culture solution of Lactobacillus acidophilus, Lactobacillus casei and Bacillus subtilis is inoculated into the fermentation medium at an inoculation amount of 5-10vol%.
- the temperature is controlled at 25-35°C and the rotation speed is 150-300 rpm.
- Lactobacillus harbin and Lactobacillus casei Bacillus ferments under the conditions of a ventilation ratio of 1:(0.5-0.8), and stops fermentation when the pH is ⁇ 4;
- Bacillus subtilis ferments under the conditions of aeration ratio of 1:(1-2), and waits until the dissolved oxygen begins to rise. Stop the fermentation and obtain the fermentation liquid of each strain;
- step (3) Prepare a composite microbial deodorant, mix and fill the fermentation broth of each strain obtained in step (2) according to a volume ratio, to obtain a composite microbial deodorant.
- the preparation method of the composite microbial deodorant includes the following steps:
- (1) First-level seed culture Under sterile conditions, inoculate Lactobacillus harbinensis and Lactobacillus casei into MRS culture medium, and inoculate Bacillus subtilis into LB culture medium, and inoculate them at 25-35°C and 150-300 rpm. Cultivate for 12-48 hours under the conditions to obtain the first-level seed culture liquid of each strain;
- Secondary seed culture Under aseptic conditions, inoculate the primary seed culture liquid of Lactobacillus harbinensis and Lactobacillus casei into the MRS medium at an inoculum volume of 1-5 vol%, and the primary seed culture of Bacillus subtilis.
- the seed culture liquid is inoculated into LB medium at an inoculation amount of 1-5 vol%, and cultured for 12-48 hours at 25-35°C and 150-300 rpm to obtain the secondary seed culture liquid of each strain;
- the ventilation ratio mentioned in the present invention refers to the ratio of the volume of air flowing into the fermentation tank per minute to the total volume of the fermentation liquid.
- the composition of the MRS medium is: 10g peptone, 10g beef meal, 5g yeast powder, 20g glucose, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.1g magnesium sulfate, 0.05 g manganese sulfate, 1000mL distilled water; the composition of LB culture medium is: 5g yeast powder, 10g peptone, 10g sodium chloride, 1000mL distilled water.
- the source of K + is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium sulfate, potassium chloride, and potassium nitrate, and the source of Mg 2+ is magnesium sulfate or magnesium chloride. one or a combination of both.
- the carbon source is selected from one or more combinations of glucose, brown sugar, and molasses.
- the nitrogen source is selected from one or more combinations of yeast powder, peptone, ammonium sulfate or beef powder.
- the present invention also claims a method for using a composite microbial deodorant to remove odor in the environment, which includes the step of spraying the composite microbial deodorant into the environment.
- the present invention also claims the application of the above-mentioned composite microbial deodorant in the field of deodorization.
- the composite microbial deodorant is used to remove ammonia and hydrogen sulfide, and more preferably, it is used to remove pet feces, livestock and poultry Ammonia and hydrogen sulfide from feces and sewers.
- Lactobacillus casei used in the examples is the JLPF-176 product purchased from Shandong Zhongke Jiayi Bioengineering Co., Ltd.
- Bacillus subtilis is the biocontrol 120 product purchased from Shandong Weilan Biotechnology Co., Ltd.
- Example 1 Screening, isolation and identification of Lactobacillus harbinensis
- a total of 5 strains were obtained according to the above isolation method, which were numbered: MD-1, MD-2, MD-3, MD-4 and MD-5.
- the MD-1 strain shows great advantages in processing ammonia gas.
- the ammonia gas processing efficiency can reach 82% in 1 hour, and the ammonia removal ability is strong.
- the strain slant of the MD-1 strain has been identified through 16S rDNA gene sequence detection. Its 16S rDNA sequence is shown as SEQ ID No: 1. The sequencing results are compared in NCBI, and the sequence with the greatest similarity is selected as the species identification result. The result is lactobacillus harbinensis.
- Lactobacillus harbinensis Under sterile conditions, Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis were respectively inoculated into 100 mL of MRS, MRS and LB culture media, and cultured at 30°C and 180 rpm for 24 hours to obtain the activation solution of each strain.
- the viable bacterial counts of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis activation solution are 50 ⁇ 10 8 CFU/mL, 80 ⁇ 10 8 CFU/mL and 120 ⁇ 10 8 CFU/mL respectively.
- Control group add sterile water
- Experimental group 1 Lactobacillus harbinensis
- Experimental group 4 Lactobacillus harbinensis and Lactobacillus casei;
- Experimental group 5 Lactobacillus casei, Bacillus subtilis;
- Experimental group 6 Lactobacillus harbinensis, Lactobacillus casei, and Bacillus subtilis;
- Each formula in experimental groups 4-6 is prepared with equal volumes of strain activation solution.
- the number of viable bacteria in the final compounded bacterial agent is 10 ⁇ 10 8 -50 ⁇ 10 8 CFU/mL.
- the results are shown in Table 2 .
- ammonia treatment effects of experimental groups 1 and 4 are also excellent, and the ammonia treatment efficiency in 1 hour is respectively 80% and 85%, it can be seen that the efficiency of the activation solution of individual strains of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis in treating ammonia gas is significantly lower than that of the composite inoculant, and the strains have a synergistic effect. .
- Example 3 Effect evaluation of different proportions of each bacterial species in the compound bacterial agent formula
- Lactobacillus harbinensis Under aseptic conditions, Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis were respectively inoculated into 100 mL of MRS, MRS and LB culture media, and cultured at 30°C and 180 rpm for 24 hours to obtain the activation solution of each strain.
- the viable bacterial counts of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis activation solution were 55 ⁇ 10 8 CFU/mL, 76 ⁇ 10 8 CFU/mL and 100 ⁇ 10 8 CFU/mL respectively.
- Control group add sterile water
- Experimental group 1 Lactobacillus harbinensis 50%, Lactobacillus casei 25%, Bacillus subtilis 25%;
- Experimental group 2 Lactobacillus harbinensis 43%, Lactobacillus casei 22%, Bacillus subtilis 35%;
- Experimental group 3 Lactobacillus harbinensis 40%, Lactobacillus casei 40%, Bacillus subtilis 20%;
- Experimental group 4 Lactobacillus harbinensis 36%, Lactobacillus casei 36%, Bacillus subtilis 28%;
- Experimental group 5 Lactobacillus harbinensis 33%, Lactobacillus casei 50%, Bacillus subtilis 17%;
- Each compound bacterial agent in experimental groups 1-5 is prepared from the activation solution of three bacterial strains according to volume ratio.
- the number of viable bacteria in the final compounded compound agent is 10 ⁇ 10 8 -50 ⁇ 10 8 CFU/mL.
- the results are shown in Table 3.
- Example 4 Composite microbial deodorizer is used to deodorize duck manure
- Second-level seed liquid culture Lactobacillus harbinensis and Lactobacillus casei were inoculated into MRS medium under aseptic conditions, and Bacillus subtilis was inoculated into LB medium, and cultured for 24 hours at 30°C and 150 rpm. Obtain the primary seed culture liquid of each strain;
- Secondary seed liquid culture Under aseptic conditions, inoculate the primary seed culture liquid of Lactobacillus harbinensis and Lactobacillus casei into the MRS medium at an inoculum volume of 1 vol%, and the primary seed culture liquid of Bacillus subtilis. Inoculate the LB medium at an inoculation amount of 1 vol%, and cultivate it for 24 hours at 30°C and 150 rpm to obtain the secondary seed culture liquid of each strain;
- the fermentation conditions of Lactobacillus harbin and Lactobacillus casei are: control temperature is 25-35°C, ventilation ratio is 1: (0.5-0.8), rotation speed is 150-300rpm, fermentation is 24-48h, and the fermentation is stopped when the pH is ⁇ 4; subtilis
- the fermentation conditions of Bacillus are: control the temperature to 25-35°C, the ventilation ratio to 1:(1-2), and the fermentation speed to 150-300 rpm. Ferment for 24-36 hours. Stop the fermentation when the dissolved oxygen begins to rise.
- the above-mentioned fermentation broth is mixed according to an appropriate volume ratio to obtain a liquid composite microbial deodorant.
- the viable bacterial counts of the fermentation broth of Lactobacillus harbinensis, Lactobacillus casei, and Bacillus subtilis are 55 ⁇ 10 8 CFU/mL and 90 ⁇ 10 8 respectively. CFU/mL and 120 ⁇ 10 8 CFU/mL.
- Deodorization of duck manure At room temperature, add 1 to 2 g of duck manure (taken from a duck farm in Hebei, duck manure has been mixed with rice husk) into a 1L experimental bottle, seal it and let it stand to allow the gas Mix evenly, and after 1 hour, use an ammonia and hydrogen sulfide detector to measure the ammonia and hydrogen sulfide concentrations;
- Set up a control group use sterile water instead of activation solution as the control group, set up 3 experimental groups, including 2 compound bacteria and a Lactobacillus harbinensis activation solution, and set up 3 parallels in each experimental group.
- the bacterial agents of the two compound bacterial agent experimental groups are compounded from the fermentation broth of each strain according to the following volume ratio:
- Compound bacterial agent 1 Lactobacillus harbinensis 50%, Lactobacillus casei-like 50%;
- Compound bacterial agent 2 Lactobacillus harbinensis 40%, Lactobacillus casei 40%, Bacillus subtilis 20%;
- the preparation method of the Lactobacillus Harbinensis activation liquid is the same as in Example 3. After diluting the composite bacterial agents 1 and 2 and the Lactobacillus Harbini activation liquid 30 times with tap water, spray about 1 mL of the diluent into the experimental bottle with a spray bottle, and seal it. Regularly measure the ammonia and hydrogen sulfide concentrations.
- both the composite microbial deodorant and the single Lactobacillus harbinensis activation solution provided by the present invention have a good deodorizing effect on duck manure, but the composite microbial deodorant has a better deodorizing effect than the single Lactobacillus harbinensis activation liquid.
- the efficiency of Lactobacillus Harbinensis in removing ammonia and hydrogen sulfide in 24 hours is 87% and 71% respectively.
- the compound microbial agent 2 has the best effect.
- the efficiency of removing ammonia and hydrogen sulfide in 24 hours is 94% and 93% respectively, so the compound Microbial inoculants are quick-acting and long-lasting.
- Example 5 Composite microbial deodorizer is used to deodorize cat litter
- Second-level seed liquid culture Lactobacillus harbinensis and Lactobacillus casei were inoculated into MRS medium under aseptic conditions, and Bacillus subtilis was inoculated into LB medium, and cultured at 35°C and 300rpm for 12 hours. Obtain the primary seed culture liquid of each strain;
- Lactobacillus harbinensis and Lactobacillus casei were cultured under sterile conditions.
- the first-level seed culture liquid was inoculated into the MRS medium at an inoculation amount of 2.5 vol%.
- the primary seed culture liquid of Bacillus subtilis was inoculated into the LB medium at an inoculum amount of 2.5 vol%. Under the conditions of 35°C and 300 rpm Cultivate for 12 hours to obtain the secondary seed culture liquid of each strain;
- the fermentation conditions of Lactobacillus harbin and Lactobacillus plantarum are: control temperature is 25-35°C, ventilation ratio is 1: (0.5-0.8), rotation speed is 150-300rpm, fermentation is 24-48h, fermentation is stopped when pH is ⁇ 4; Bacillus subtilis
- the fermentation conditions of the bacilli are: control the temperature to 25-35°C, the ventilation ratio to 1:(1-2), and the fermentation speed to 150-300 rpm. Ferment for 24-36 hours. Stop the fermentation when the dissolved oxygen begins to rise. Add the above The fermentation broth is mixed according to an appropriate volume ratio to obtain a liquid composite bacterial agent.
- the viable bacterial counts of the fermentation broth of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis are 85 ⁇ 10 8 CFU/mL and 100 ⁇ 10 8 CFU/mL respectively. and 150 ⁇ 10 8 CFU/mL.
- Set up a control group use sterile water instead of activation solution as the control group, set up 3 experimental groups, including 2 compound bacteria and a Lactobacillus harbinensis activation solution, and set up 3 parallels in each experimental group.
- the bacterial agents of the two compound bacterial agent experimental groups are compounded from the fermentation broth of each strain according to the following volume ratio:
- Compound bacterial agent 1 Lactobacillus harbinensis 50%, Lactobacillus casei-like 50%;
- Compound bacterial agent 2 Lactobacillus harbinensis 40%, Lactobacillus casei 40%, Bacillus subtilis 20%;
- the preparation method of the Lactobacillus Harbinensis activation liquid is the same as in Example 3. After diluting the composite bacterial agents 1 and 2 and the Lactobacillus Harbini activation liquid 30 times with tap water, spray about 1 mL of the diluent into the experimental bottle with a spray bottle, and seal it. Regularly measure the ammonia and hydrogen sulfide concentrations.
- both the composite microbial deodorant and the single Lactobacillus harbinensis provided by the present invention have better removal effects on the odor produced by cat litter, but the composite microbial deodorant has a better deodorizing effect than the single Lactobacillus harbinensis.
- the 24-hour ammonia and hydrogen sulfide removal efficiencies of a single Lactobacillus harbinensis agent are 85% and 86% respectively.
- the compound microbial deodorant 2 has the best effect.
- the 24-hour ammonia and hydrogen sulfide removal efficiencies are 94% and 86% respectively. %, and the odor is obviously not felt, indicating that the compound microbial deodorant has good deodorizing effect and good durability.
- Example 6 Composite microbial deodorant used for sewer deodorization
- the fermentation conditions of Lactobacillus harbin and Lactobacillus plantarum are: control temperature is 25-35°C, ventilation ratio is 1: (0.5-0.8), rotation speed is 150-300rpm, fermentation is 24-48h, fermentation is stopped when pH is ⁇ 4; Bacillus subtilis
- the fermentation conditions of the bacilli are: control the temperature to 25-35°C, the ventilation ratio to 1:(1-2), and the fermentation speed to 150-300 rpm. Ferment for 24-36 hours. Stop the fermentation when the dissolved oxygen begins to rise. Add the above The fermentation broth is mixed according to an appropriate volume ratio to obtain a composite microbial deodorant.
- the viable bacterial counts of the fermentation broth of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis are 45 ⁇ 10 8 CFU/mL and 60 ⁇ 10 8 CFU/mL respectively. mL and 90 ⁇ 10 8 CFU/mL.
- the fermentation culture medium of Lactobacillus harbinensis and Lactobacillus casei-like is composed of: molasses 50g/L, yeast powder 20g/L, K + 0.5-1.2g/L, Mg 2+ 0.1-0.2g/L, and the rest is water ;
- the fermentation medium of Bacillus subtilis is: glucose 20g/L, beef meal 10g/L, peptone 20g/L, K + 0.5-1.2g/L, Mg 2+ 0.1-0.2g/L, and the rest is water.
- Deodorization of sewers At room temperature, add 10-20mL of domestic sewage water into a 1L experimental bottle, seal it and let it stand to make the gases mix evenly. After 1 hour, use an ammonia hydrogen sulfide detector to measure the ammonia and sulfide gas. Hydrogen concentration; then add about 1 mL of single Lactobacillus Harbinensis activation solution and compound inoculant respectively, spray it into the reagent bottle with a spray bottle, seal it, and measure its ammonia and hydrogen sulfide concentration regularly.
- Set up a control group use sterile water instead of activation solution as the control group, set up 3 experimental groups, including 2 compound bacteria and a Lactobacillus harbinensis activation solution, and set up 3 parallels in each experimental group.
- the bacterial agents of the two compound bacterial agent experimental groups are compounded from the activation solution of each strain according to the following volume ratio:
- Compound bacterial agent 1 Lactobacillus harbinensis 50%, Lactobacillus casei-like 50%;
- Compound bacterial agent 2 Lactobacillus harbinensis 40%, Lactobacillus casei 40%, Bacillus subtilis 20%;
- Lactobacillus Harbinensis activation liquid is the same as in Example 3.
- the composite microbial deodorant provided by the present invention and the single Lactobacillus harbinensis activation liquid have good deodorizing effects on sewer odor, but the composite microbial deodorant Compared with the single Lactobacillus Harbinensis activation liquid, the deodorization effect is better.
- the efficiency of Lactobacillus Harbini activation liquid in removing ammonia gas and hydrogen sulfide in 24 hours is 83% and 73% respectively, while the compound bacterial agent 2 has the best effect, removing ammonia gas in 24h.
- the efficiency of hydrogen sulfide and hydrogen sulfide are 90% and 85% respectively, so the compound microbial deodorant is quick and long-lasting.
- due to the use of compound microbial deodorant not only the treatment efficiency of ammonia and hydrogen sulfide is high, but also through sensory evaluation, You can also smell something like brewing.
- Lactobacillus harbinensis provided by the present invention is compounded with Lactobacillus casei-like or with Lactobacillus casei-like and Bacillus subtilis, it has the ability to treat ammonia gas and hydrogen sulfide gas that produce odor. Very good handling.
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Abstract
The present invention belongs to the technical field of environmental microorganisms, and in particular, provides a composite microbial deodorant, a method for preparing same, and use thereof. The composite microbial deodorant comprises Lactobacillus harbinensis and Lactobacillus paracasei. The L. harbinensis strain is preserved at China General Microbiological Culture Collection Center, with Accession Number CGMCC No. 24253. The composite microbial deodorant has a 24-hour process efficiency of 90% or greater for ammonia and 85% or greater for hydrogen sulfide when used for processing pet excrement, livestock and poultry excrement, and sewerage, and features a long-term deodorization effect, high efficiency, and no secondary contamination.
Description
本发明涉及一种复合微生物除臭剂，尤其涉及一种以哈尔滨乳杆菌和类干酪乳杆菌作为主要活性成分的复合微生物除臭剂，属于环境微生物技术领域。The present invention relates to a composite microbial deodorant, in particular to a composite microbial deodorant with Lactobacillus harbinensis and Lactobacillus casei as main active ingredients, and belongs to the technical field of environmental microorganisms.
恶臭物质包括硫醇类、硫醚类、胺类、酰胺类、吲哚类及一些无机物质等。这些物质危害人的呼吸***、循环***、消化***、内分泌***和神经***，严重影响了人们的精神状态，降低了生活质量。Odorous substances include thiols, thioethers, amines, amides, indoles and some inorganic substances. These substances harm people's respiratory system, circulatory system, digestive system, endocrine system and nervous system, seriously affect people's mental state and reduce their quality of life.
除臭剂主要分为物理除臭剂、化学除臭剂、微生物除臭剂、植物型除臭剂和复合型除臭剂等。物理除臭剂主要是利用吸附和掩盖作用达到除臭的目的，这类除臭剂常用的材料有活性炭、沸石、芳香化合物等，特点是很难完全改变臭气气体的成分，对人畜、设备和环境等仍有一定程度的损害。化学除臭剂是利用化学试剂与臭气分子发生化学反应达到除臭的目的，常见的化学除臭产品原料有蓖麻油醇酸锌、大豆乙基硫酸乙酯吗啉、甲基丙烯酸脂肪醇等，其特点是反应迅速但是效果不持久，且易产生二次污染。植物除臭剂的有效成分来自于茶叶提取物、丝兰提取物、芬多精等植物，这些植物中含有的化学物质，能与臭味分子基团发生化学反应，削弱异味分子中的化合键，最后生成无味、无毒的物质，但有效成分的提取工艺复杂，生产成本较高，且除臭对象单一，还可能产生一些难以预料的化学反应。Deodorants are mainly divided into physical deodorants, chemical deodorants, microbial deodorants, plant-based deodorants and compound deodorants. Physical deodorants mainly use adsorption and masking to achieve the purpose of deodorization. Commonly used materials for this type of deodorants include activated carbon, zeolites, aromatic compounds, etc. The characteristic is that it is difficult to completely change the composition of the odor gas, which is harmful to people, animals, and equipment. There is still a certain degree of damage to the environment and the environment. Chemical deodorants use chemical reagents to react with odor molecules to achieve the purpose of deodorization. Common chemical deodorant product raw materials include castor oil zinc alkyd, soybean ethyl ethyl sulfate morpholine, methacrylic fatty alcohol, etc. , which is characterized by rapid response but short-lasting effect, and is prone to secondary pollution. The active ingredients of plant deodorants come from tea extract, yucca extract, phytoncidin and other plants. The chemical substances contained in these plants can chemically react with the odor molecule groups and weaken the chemical bonds in the odor molecules. , and finally produce odorless and non-toxic substances, but the extraction process of active ingredients is complicated, the production cost is high, and the deodorization target is single, and some unpredictable chemical reactions may occur.
而微生物除臭剂是利用微生物菌群的代谢或其代谢产物，有效降解挥发性脂肪酸和硫化氢等常见的臭味因子，对人畜安全无毒，无刺激性，可生物
降解，通过微生物的不断繁殖，生物除臭作用较持久，无二次污染，且使用方便，成本低廉。Microbial deodorants use the metabolism of microbial flora or their metabolites to effectively degrade common odor factors such as volatile fatty acids and hydrogen sulfide. They are safe, non-toxic, non-irritating and biodegradable for humans and animals. Degradation, through the continuous reproduction of microorganisms, the biological deodorization effect is long-lasting, without secondary pollution, easy to use and low cost.
微生物型除臭剂由于其具有环保、可重复利用的优势，越来越受到人们的重视。但现有的微生物除臭剂生产工艺较为复杂，具有协同作用的复合菌株不易获得，且分解恶臭的效果不佳，这些都限制了微生物除臭剂的进一步应用。Microbial deodorants have attracted more and more attention due to their environmental protection and reusability advantages. However, the existing production process of microbial deodorants is relatively complex, compound strains with synergistic effects are difficult to obtain, and the effect of decomposing odor is not good, which limits the further application of microbial deodorants.
发明内容Contents of the invention
本发明针对目前市场上的微生物除臭产品所存在的不足，提供一种复合微生物除臭剂，该复合微生物除臭剂在应用24h时对宠物粪便、畜禽粪便、下水道产生的氨气处理效率可达90％以上，硫化氢处理效率可达85％以上，且除臭持久、高效并无二次污染。In view of the shortcomings of microbial deodorization products currently on the market, the present invention provides a composite microbial deodorant. The composite microbial deodorant has a high efficiency in treating ammonia gas generated by pet feces, livestock and poultry feces, and sewers when used for 24 hours. It can reach more than 90%, the hydrogen sulfide treatment efficiency can reach more than 85%, and the deodorization is long-lasting, efficient and has no secondary pollution.
本发明解决上述技术问题的技术方案如下：The technical solutions of the present invention to solve the above technical problems are as follows:
一种复合微生物除臭剂，包含哈尔滨乳杆菌(Lactobacillus harbinensis)和类干酪乳杆菌，所述哈尔滨乳杆菌保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址为：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为：CGMCC No.24253，保藏日期为：2022年01月05日，其16S rDNA序列如SEQ ID No:1所示。A composite microbial deodorant, including Lactobacillus harbinensis (Lactobacillus harbinensis) and Lactobacillus casei-like, the Lactobacillus harbinensis is preserved in the General Microbiology Center of the China Committee for the Collection of Microorganisms, the address is: Beichen West Road, Chaoyang District, Beijing No. 1, No. 3, Institute of Microbiology, Chinese Academy of Sciences, the deposit number is: CGMCC No. 24253, the deposit date is: January 5, 2022, and its 16S rDNA sequence is shown in SEQ ID No: 1.
在没有特别说明的情况下，本发明中所述的哈尔滨乳杆菌均是指哈尔滨乳杆菌MD-1菌株，该菌株是从垃圾渗滤液中分离得到的，其生理生化特征为：在MRS培养基上培养时，其菌落为圆形，乳白色，不透明，凸起，表面粗糙，细胞为短杆。Unless otherwise specified, the Lactobacillus harbinensis described in the present invention refers to the Lactobacillus harbinensis MD-1 strain, which is isolated from landfill leachate. Its physiological and biochemical characteristics are: in MRS culture medium When cultured, the colonies are round, milky white, opaque, convex, with a rough surface, and the cells are short rods.
本发明中所述的类干酪乳杆菌又称为副干酪乳杆菌，属于乳酸菌属中的干酪乳杆菌群。The Lactobacillus casei described in the present invention is also called Lactobacillus paracasei and belongs to the Lactobacillus casei group in the genus Lactobacillus.
进一步，所述哈尔滨乳杆菌与类干酪乳杆菌的活菌数之比为1：(1-2)，
优选1：(1.2-1.6)。Further, the ratio of the viable bacterial numbers of Lactobacillus harbinensis and Lactobacillus casei-like bacteria is 1: (1-2), Preferred 1: (1.2-1.6).
进一步，所述复合微生物除臭剂中还包含枯草芽孢杆菌，优选的，所述哈尔滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌的活菌数之比为1：(0.5-3)：(0.5-2.5)，更优选1：(0.7-2.1)：(0.9-1.5)。Further, the composite microbial deodorant also contains Bacillus subtilis. Preferably, the ratio of the viable bacterial numbers of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis is 1: (0.5-3): (0.5 -2.5), more preferably 1: (0.7-2.1): (0.9-1.5).
进一步，所述复合微生物除臭剂中的活菌量不低于10×108CFU/mL，优选(10-50)×108CFU/mL。Furthermore, the amount of viable bacteria in the composite microbial deodorant is not less than 10×10 8 CFU/mL, preferably (10-50)×10 8 CFU/mL.
本发明提供的复合微生物除臭剂中所选用的菌株特性及功能如下：The characteristics and functions of the bacterial strains selected in the composite microbial deodorant provided by the invention are as follows:
(1)哈尔滨乳杆菌和类干酪乳杆菌的生长过程中会分泌乳酸菌素，产生有机酸、酒精和二氧化碳等，可以显著抑制有害菌的生长，从而减少恶臭物质的产生；(1) Lactobacillus harbinensis and Lactobacillus casei-like bacteria secrete lactobacilli during the growth process and produce organic acids, alcohol and carbon dioxide, which can significantly inhibit the growth of harmful bacteria and thereby reduce the production of odorous substances;
(2)枯草芽孢杆菌能迅速消耗环境中的游离氧，促进乳酸菌的生长，并产生乳酸等有机酸类，降低环境的pH值，间接抑制其他有害菌的生长，芽孢杆菌菌体能自身合成消化酶类，如蛋白酶、淀粉酶、脂肪酶、纤维素酶等，这些酶对有机物有一定的分解作用，对环境有一定的净化作用。(2) Bacillus subtilis can quickly consume free oxygen in the environment, promote the growth of lactic acid bacteria, and produce organic acids such as lactic acid, lowering the pH value of the environment and indirectly inhibiting the growth of other harmful bacteria. Bacillus subtilis can synthesize and digest by itself Enzymes, such as protease, amylase, lipase, cellulase, etc., these enzymes have a certain decomposition effect on organic matter and have a certain purification effect on the environment.
本发明提供的复合微生物除臭剂的有益效果为：The beneficial effects of the composite microbial deodorant provided by the invention are:
(1)将特定比例的复合微生物除臭剂稀释30倍后喷施于鸭粪和猫砂上方，应用24h时氨气去除率可达90％以上，硫化氢去除率可达85％以上；(1) Dilute a specific proportion of compound microbial deodorant 30 times and spray it on top of duck manure and cat litter. After 24 hours of application, the ammonia removal rate can reach more than 90%, and the hydrogen sulfide removal rate can reach more than 85%;
(2)两种或三种菌株之间协同配合发挥作用，拓宽了可处理污染物的种类，且除臭效果明显优于单一菌株，效果更稳定，除臭效果更持久。(2) The synergistic cooperation between two or three bacterial strains broadens the types of pollutants that can be treated, and the deodorizing effect is significantly better than that of a single strain, the effect is more stable, and the deodorizing effect is more lasting.
(3)菌种数量少，成本低，仅包含两至三种菌株，且类干酪乳杆菌和枯草芽孢杆菌可选自市场上任一厂家的产品，可合理控制成本。(3) The number of strains is small and the cost is low. It only contains two or three strains. Lactobacillus casei and Bacillus subtilis can be selected from any manufacturer on the market, so the cost can be reasonably controlled.
上述复合微生物除臭剂的制备方法，包括如下步骤：The preparation method of the above-mentioned composite microbial deodorant includes the following steps:
(1)扩大培养：将哈尔滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌进行扩大培养，得到各菌株的培养液；(1) Expanded culture: Expand the culture of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis to obtain the culture solution of each strain;
(2)发酵：待发酵罐内的发酵培养基消毒完毕后，分别将步骤(1)所得的哈尔
滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌的培养液按照5-10vol％的接种量接种于发酵培养基中，控制温度为25-35℃、转速150-300rpm，哈尔滨乳杆菌和类干酪乳杆菌在通气比为1:(0.5-0.8)的条件下发酵，待pH＜4时停止发酵；枯草芽孢杆菌在通气比为1:(1-2)的条件下发酵，待溶氧开始上升时停止发酵，得各菌株的发酵液；(2) Fermentation: After the fermentation medium in the fermentation tank has been sterilized, the Hal obtained in step (1) The culture solution of Lactobacillus acidophilus, Lactobacillus casei and Bacillus subtilis is inoculated into the fermentation medium at an inoculation amount of 5-10vol%. The temperature is controlled at 25-35°C and the rotation speed is 150-300 rpm. Lactobacillus harbin and Lactobacillus casei Bacillus ferments under the conditions of a ventilation ratio of 1:(0.5-0.8), and stops fermentation when the pH is <4; Bacillus subtilis ferments under the conditions of aeration ratio of 1:(1-2), and waits until the dissolved oxygen begins to rise. Stop the fermentation and obtain the fermentation liquid of each strain;
(3)制备复合微生物除臭剂，将步骤(2)所得的各菌株的发酵液按体积比混合灌装，即得复合微生物除臭剂。(3) Prepare a composite microbial deodorant, mix and fill the fermentation broth of each strain obtained in step (2) according to a volume ratio, to obtain a composite microbial deodorant.
优选的，所述复合微生物除臭剂的制备方法包括如下步骤：Preferably, the preparation method of the composite microbial deodorant includes the following steps:
(1)一级种子培养：无菌条件下，分别取哈尔滨乳杆菌和类干酪乳杆菌接种于MRS培养基中，枯草芽孢杆菌接种于LB培养基中，于25-35℃、150-300rpm的条件下培养12-48h，得到各菌株的一级种子培养液；(1) First-level seed culture: Under sterile conditions, inoculate Lactobacillus harbinensis and Lactobacillus casei into MRS culture medium, and inoculate Bacillus subtilis into LB culture medium, and inoculate them at 25-35°C and 150-300 rpm. Cultivate for 12-48 hours under the conditions to obtain the first-level seed culture liquid of each strain;
(2)二级种子培养：无菌条件下，分别将哈尔滨乳杆菌和类干酪乳杆菌的一级种子培养液按照1-5vol％的接种量接种于MRS培养基中，枯草芽孢杆菌的一级种子培养液按照1-5vol％的接种量接种于LB培养基中，于25-35℃、150-300rpm的条件下培养12-48h，得到各菌株的二级种子培养液；(2) Secondary seed culture: Under aseptic conditions, inoculate the primary seed culture liquid of Lactobacillus harbinensis and Lactobacillus casei into the MRS medium at an inoculum volume of 1-5 vol%, and the primary seed culture of Bacillus subtilis. The seed culture liquid is inoculated into LB medium at an inoculation amount of 1-5 vol%, and cultured for 12-48 hours at 25-35°C and 150-300 rpm to obtain the secondary seed culture liquid of each strain;
(3)发酵：待发酵罐内的发酵培养基消毒完毕后，分别将步骤(2)所得的哈尔滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌的二级种子培养液按照5-10vol％的接种量接种于发酵培养基中，控制温度为25-35℃、转速150-300rpm，哈尔滨乳杆菌和类干酪乳杆菌在通气比为1:(0.5-0.8)的条件下发酵，待pH＜4时停止发酵；枯草芽孢杆菌在通气比为1:(1-2)的条件下发酵，待溶氧开始上升时停止发酵，得各菌株的发酵液；(3) Fermentation: After the fermentation medium in the fermentation tank is sterilized, inoculate the secondary seed culture liquid of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis obtained in step (2) at 5-10vol%. Inoculate a certain amount into the fermentation medium, control the temperature to 25-35°C, and the rotation speed to 150-300 rpm. Lactobacillus harbinensis and Lactobacillus casei are fermented under the conditions of a ventilation ratio of 1: (0.5-0.8), until the pH is < 4 Stop the fermentation; Bacillus subtilis ferments under the condition of a ventilation ratio of 1: (1-2), stop the fermentation when the dissolved oxygen begins to rise, and obtain the fermentation liquid of each strain;
(4)制备复合微生物除臭剂：将步骤(3)所得的各菌株的发酵液按体积比混合灌装，即得复合微生物除臭剂。(4) Preparing the composite microbial deodorant: Mix and fill the fermentation broth of each strain obtained in step (3) according to the volume ratio to obtain the composite microbial deodorant.
本发明中所述的通气比是指每分钟内通入发酵罐的空气体积与发酵液总体积之比。
The ventilation ratio mentioned in the present invention refers to the ratio of the volume of air flowing into the fermentation tank per minute to the total volume of the fermentation liquid.
所述MRS培养基的组成为：10g蛋白胨、10g牛肉粉、5g酵母粉、20g葡萄糖、1mL吐温80、2g磷酸氢二钾、5g乙酸钠、2g柠檬酸三铵、0.1g硫酸镁、0.05g硫酸锰、1000mL蒸馏水；LB培养基的组成为：5g酵母粉、10g蛋白胨、10g氯化钠、1000mL蒸馏水。The composition of the MRS medium is: 10g peptone, 10g beef meal, 5g yeast powder, 20g glucose, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.1g magnesium sulfate, 0.05 g manganese sulfate, 1000mL distilled water; the composition of LB culture medium is: 5g yeast powder, 10g peptone, 10g sodium chloride, 1000mL distilled water.
进一步，所述哈尔滨乳杆菌和类干酪乳杆菌的发酵培养基组成为：碳源40-60g/L、氮源15-30g/L、K+0.5-1.2g/L、Mg2+0.1-0.2g/L，溶剂为水，且pH＝6～7；所述枯草芽孢杆菌的发酵培养基组成为：碳源20-50g/L、氮源10-30g/L、K+0.3-0.8g/L、Mg2+0.5-1.5g/L，溶剂为水，且pH＝6.5-8。Further, the fermentation medium composition of Lactobacillus harbinensis and Lactobacillus casei-like is as follows: carbon source 40-60g/L, nitrogen source 15-30g/L, K + 0.5-1.2g/L, Mg 2+ 0.1-0.2 g/L, the solvent is water, and the pH=6-7; the fermentation medium of Bacillus subtilis is composed of: carbon source 20-50g/L, nitrogen source 10-30g/L, K + 0.3-0.8g/ L, Mg 2+ 0.5-1.5g/L, solvent is water, and pH=6.5-8.
优选的，所述K+的来源为磷酸氢二钾、磷酸二氢钾、硫酸钾、氯化钾、硝酸钾中的一种或多种，所述Mg2+的来源为硫酸镁、氯化镁中的一种或二者的复配。Preferably, the source of K + is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium sulfate, potassium chloride, and potassium nitrate, and the source of Mg 2+ is magnesium sulfate or magnesium chloride. one or a combination of both.
进一步，所述碳源选自葡萄糖、红糖、糖蜜中的一种或多种的复配。Further, the carbon source is selected from one or more combinations of glucose, brown sugar, and molasses.
进一步，所述氮源选自酵母粉、蛋白胨、硫酸铵或牛肉粉中的一种或多种的复配。Further, the nitrogen source is selected from one or more combinations of yeast powder, peptone, ammonium sulfate or beef powder.
本发明还要求保护使用复合微生物除臭剂去除环境中臭味的方法，包括向环境中喷洒所述复合微生物除臭剂的步骤。The present invention also claims a method for using a composite microbial deodorant to remove odor in the environment, which includes the step of spraying the composite microbial deodorant into the environment.
本发明还要求保护上述的复合微生物除臭剂在除臭领域的应用，优选的，所述复合微生物除臭剂用于去除氨气和硫化氢，更优选的，用于去除宠物粪便、畜禽粪便和下水道所产生的氨气和硫化氢。The present invention also claims the application of the above-mentioned composite microbial deodorant in the field of deodorization. Preferably, the composite microbial deodorant is used to remove ammonia and hydrogen sulfide, and more preferably, it is used to remove pet feces, livestock and poultry Ammonia and hydrogen sulfide from feces and sewers.
以下结合实例对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。The principles and features of the present invention are described below with examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.
实施例中使用的类干酪乳杆菌为购自山东中科嘉亿生物工程有限公司JLPF-176型号的产品，枯草芽孢杆菌为购自山东蔚蓝生物科技有限公司生防120型号的产品。
The Lactobacillus casei used in the examples is the JLPF-176 product purchased from Shandong Zhongke Jiayi Bioengineering Co., Ltd., and the Bacillus subtilis is the biocontrol 120 product purchased from Shandong Weilan Biotechnology Co., Ltd.
实施例1：哈尔滨乳杆菌的筛选分离与鉴定Example 1: Screening, isolation and identification of Lactobacillus harbinensis
1、菌株的筛选分离1. Screening and isolation of bacterial strains
采集河南省商丘市某垃圾填埋场的垃圾渗滤液，采用梯度稀释法将该污水稀释至10-2、10-3和10-4，分别吸取各稀释液100μl至MRS培养基，涂布均匀后倒置于30℃条件下培养，约48h长出单菌落。挑选形态相异的单菌落转接至试管斜面分离培养基上，30℃条件下培养约48h，然后转移至4℃冰箱中保藏备用。Collect leachate from a landfill in Shangqiu City, Henan Province. Use gradient dilution method to dilute the sewage to 10 -2 , 10 -3 and 10 -4 . Pipette 100μl of each dilution into the MRS culture medium and spread evenly. Then invert and culture at 30°C, and a single colony will grow in about 48 hours. Select single colonies with different morphologies and transfer them to the test tube slant separation medium, culture them at 30°C for about 48 hours, and then transfer them to a 4°C refrigerator for storage until later use.
按上述分离方法共得到5株菌株，分别编号为：MD-1、MD-2、MD-3、MD-4和MD-5。A total of 5 strains were obtained according to the above isolation method, which were numbered: MD-1, MD-2, MD-3, MD-4 and MD-5.
2、初筛效果评价2. Evaluation of preliminary screening effects
无菌环境中，将初筛得到的5株菌株分别挑取1环接种于盛有100mL MRS培养基的250mL三角瓶中，30℃、220rpm条件下培养48h，进行活化。In a sterile environment, pick 1 loop of each of the 5 strains obtained from the preliminary screening and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL MRS culture medium, and culture it at 30°C and 220 rpm for 48 hours for activation.
室温条件下，将50-100μl的氨水加入到1L实验瓶中，密封后进行磁子搅拌，使气体混合均匀，搅拌1h后，用氨气检测仪测其氨气浓度；然后分别加入筛选出的5株菌的活化液约1mL，用喷壶喷到试剂瓶中，密封，进行磁子搅拌，使气体混合均匀，搅拌1h后测其氨气浓度。设置1组对照组，用无菌水代替活化液作为对照组，各实验组设置3个平行，结果如表1所示。At room temperature, add 50-100 μl of ammonia water into a 1L experimental bottle, seal it and perform magnetic stirring to mix the gas evenly. After stirring for 1 hour, use an ammonia detector to measure the ammonia concentration; then add the screened out About 1 mL of the activation solution of the 5 strains of bacteria was sprayed into the reagent bottle with a spray bottle, sealed, and stirred with a magnet to mix the gas evenly. After stirring for 1 hour, measure the ammonia concentration. Set up a control group and use sterile water instead of the activation solution as the control group. Each experimental group is set up in three parallel groups. The results are shown in Table 1.
表1.各菌株去除氨气的效果
Table 1. Ammonia removal effect of each strain
Table 1. Ammonia removal effect of each strain
根据表1的检测结果可知，与其他菌株相比，MD-1菌株在处理氨气方面表现出巨大的优势，1h处理氨气效率可达82％，除氨能力强。According to the test results in Table 1, compared with other strains, the MD-1 strain shows great advantages in processing ammonia gas. The ammonia gas processing efficiency can reach 82% in 1 hour, and the ammonia removal ability is strong.
3、菌株的检测鉴定3. Detection and identification of bacterial strains
MD-1菌株的菌种斜面经过16S rDNA基因序列检测鉴定，其16S rDNA序列如SEQ ID No:1所示，将测序结果在NCBI中比对，选择相似度最大的序列作为物种鉴定结果，鉴定结果为lactobacillus harbinensis哈尔滨乳杆菌。The strain slant of the MD-1 strain has been identified through 16S rDNA gene sequence detection. Its 16S rDNA sequence is shown as SEQ ID No: 1. The sequencing results are compared in NCBI, and the sequence with the greatest similarity is selected as the species identification result. The result is lactobacillus harbinensis.
实施例2：不同菌剂配方的效果评价Example 2: Effect evaluation of different bacterial agent formulas
无菌条件下分别取哈尔滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌接种于100mL的MRS、MRS和LB培养基中，于30℃、180rpm的条件下培养24h，得到各菌株的活化液，所得哈尔滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌活化液的活菌数分别为50×108CFU/mL、80×108CFU/mL、120×108CFU/mL。Under sterile conditions, Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis were respectively inoculated into 100 mL of MRS, MRS and LB culture media, and cultured at 30°C and 180 rpm for 24 hours to obtain the activation solution of each strain. The viable bacterial counts of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis activation solution are 50×10 8 CFU/mL, 80×10 8 CFU/mL and 120×10 8 CFU/mL respectively.
室温条件下，将50-100μl的氨水加入到1L实验瓶中，密封后进行磁子搅拌，使气体混合均匀，搅拌1h后，用氨气检测仪测其氨气浓度；然后分别加入不同配方的菌株活化液约1mL，用喷壶喷到试剂瓶中，密封，进行磁子搅拌，使气体混合均匀，搅拌1h后采用氨气检测仪测其氨气浓度。设置1组对照组，用无菌水代替活化液作为对照组，各实验组设置3个平行。具体实验安排如下：At room temperature, add 50-100 μl of ammonia water into a 1L experimental bottle, seal it and stir with a magnet to mix the gas evenly. After stirring for 1 hour, use an ammonia detector to measure the ammonia concentration; then add different formulas. About 1 mL of strain activation solution was sprayed into the reagent bottle with a spray bottle, sealed, and magnetically stirred to mix the gas evenly. After stirring for 1 hour, use an ammonia detector to measure the ammonia concentration. Set up a control group, use sterile water instead of activation solution as the control group, and set up three parallel groups in each experimental group. The specific experimental arrangement is as follows:
对照组：加无菌水；Control group: add sterile water;
实验组1：哈尔滨乳杆菌；Experimental group 1: Lactobacillus harbinensis;
实验组2：类干酪乳杆菌；Experimental group 2: Lactobacillus casei;
实验组3：枯草芽孢杆菌；Experimental group 3: Bacillus subtilis;
实验组4：哈尔滨乳杆菌、类干酪乳杆菌；Experimental group 4: Lactobacillus harbinensis and Lactobacillus casei;
实验组5：类干酪乳杆菌、枯草芽孢杆菌；
Experimental group 5: Lactobacillus casei, Bacillus subtilis;
实验组6：哈尔滨乳杆菌、类干酪乳杆菌、枯草芽孢杆菌；Experimental group 6: Lactobacillus harbinensis, Lactobacillus casei, and Bacillus subtilis;
实验组4-6中的各配方均为菌株活化液的等体积配制，最终复配得到菌剂的活菌数均为10×108-50×108CFU/mL，结果如表2所示。Each formula in experimental groups 4-6 is prepared with equal volumes of strain activation solution. The number of viable bacteria in the final compounded bacterial agent is 10×10 8 -50×10 8 CFU/mL. The results are shown in Table 2 .
表2.各菌剂去除氨气的效果
Table 2. Ammonia removal effect of each bacterial agent
Table 2. Ammonia removal effect of each bacterial agent
由表2中的结果可知，各实验组菌剂均有一定的除氨效果，其中不含哈尔滨乳杆菌的实验组2、3和5的效果较差，可见哈尔滨乳杆菌在复合菌剂作用中的重要性，含三个菌种的实验组6的效果最佳，1h处理氨气效率可达96％，实验组1和4的氨气处理效果也很优异，1h的氨气处理效率分别为80％和85％，由此可见，哈尔滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌的单个菌种的活化液处理氨气的效率明显低于复合菌剂，各菌株之间发挥了协同配合作用。From the results in Table 2, it can be seen that the bacterial agents in each experimental group have a certain ammonia removal effect. Among them, the experimental groups 2, 3 and 5 that do not contain Lactobacillus harbinensis have poorer effects. It can be seen that Lactobacillus harbinensis plays an important role in the effect of the compound bacterial agent. importance, experimental group 6 containing three strains of bacteria has the best effect, and the ammonia treatment efficiency can reach 96% in 1 hour. The ammonia treatment effects of experimental groups 1 and 4 are also excellent, and the ammonia treatment efficiency in 1 hour is respectively 80% and 85%, it can be seen that the efficiency of the activation solution of individual strains of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis in treating ammonia gas is significantly lower than that of the composite inoculant, and the strains have a synergistic effect. .
实施例3：复合菌剂配方中各菌种不同比例的效果评价Example 3: Effect evaluation of different proportions of each bacterial species in the compound bacterial agent formula
无菌条件下分别取哈尔滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌接种于100mL的MRS、MRS和LB培养基中，于30℃、180rpm的条件下培养24h，得到各菌株的活化液，所得哈尔滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌活化液的活菌数分别为55×108CFU/mL、76×108CFU/mL、100×108CFU/mL。
Under aseptic conditions, Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis were respectively inoculated into 100 mL of MRS, MRS and LB culture media, and cultured at 30°C and 180 rpm for 24 hours to obtain the activation solution of each strain. The viable bacterial counts of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis activation solution were 55×10 8 CFU/mL, 76×10 8 CFU/mL and 100×10 8 CFU/mL respectively.
室温条件下，将50-100μl的氨水加入到1L实验瓶中，密封后进行磁子搅拌，使气体混合均匀，搅拌1h后，用氨气检测仪测其氨气浓度；然后分别加入三种菌株活化液以不同比例复配所得的复合菌剂约1mL，用喷壶喷到试剂瓶中，密封，进行磁子搅拌，使气体混合均匀，搅拌1h后测其氨气浓度。设置1组对照组，用无菌水代替活化液作为对照组，各实验组分别设置3个平行。At room temperature, add 50-100 μl of ammonia water into a 1L experimental bottle, seal it and perform magnetic stirring to mix the gas evenly. After stirring for 1 hour, use an ammonia detector to measure the ammonia concentration; then add the three strains respectively. The activated solution is mixed with different proportions to obtain about 1 mL of composite inoculant. Spray it into the reagent bottle with a spray bottle, seal it, and perform magnetic stirring to mix the gas evenly. After stirring for 1 hour, measure the ammonia concentration. Set up 1 control group, use sterile water instead of activation solution as the control group, and set up 3 parallels in each experimental group.
具体实验安排如下：The specific experimental arrangement is as follows:
对照组：加无菌水；Control group: add sterile water;
实验组1：哈尔滨乳杆菌50％、类干酪乳杆菌25％、枯草芽孢杆菌25％；Experimental group 1: Lactobacillus harbinensis 50%, Lactobacillus casei 25%, Bacillus subtilis 25%;
实验组2：哈尔滨乳杆菌43％、类干酪乳杆菌22％、枯草芽孢杆菌35％；Experimental group 2: Lactobacillus harbinensis 43%, Lactobacillus casei 22%, Bacillus subtilis 35%;
实验组3：哈尔滨乳杆菌40％、类干酪乳杆菌40％、枯草芽孢杆菌20％；Experimental group 3: Lactobacillus harbinensis 40%, Lactobacillus casei 40%, Bacillus subtilis 20%;
实验组4：哈尔滨乳杆菌36％、类干酪乳杆菌36％、枯草芽孢杆菌28％；Experimental group 4: Lactobacillus harbinensis 36%, Lactobacillus casei 36%, Bacillus subtilis 28%;
实验组5：哈尔滨乳杆菌33％、类干酪乳杆菌50％、枯草芽孢杆菌17％；Experimental group 5: Lactobacillus harbinensis 33%, Lactobacillus casei 50%, Bacillus subtilis 17%;
实验组1-5中的各复合菌剂均为三种菌株的活化液按体积比配制，最终复配得到复合菌剂的活菌数均为10×108-50×108CFU/mL，结果如表3所示。Each compound bacterial agent in experimental groups 1-5 is prepared from the activation solution of three bacterial strains according to volume ratio. The number of viable bacteria in the final compounded compound agent is 10×10 8 -50×10 8 CFU/mL. The results are shown in Table 3.
表3.各复合菌剂去除氨气的效果
Table 3. Ammonia removal effect of each compound bacterial agent
Table 3. Ammonia removal effect of each compound bacterial agent
由表3中的结果可知，各实验组复合菌剂均有一定的除氨效果，1h氨气的处理效率为87-96％，其中实验组3的配方效果最佳。It can be seen from the results in Table 3 that the compound bacterial agents of each experimental group have a certain ammonia removal effect. The ammonia treatment efficiency in 1 hour is 87-96%, among which the formula of experimental group 3 has the best effect.
实施例4：复合微生物除臭剂用于鸭粪除臭Example 4: Composite microbial deodorizer is used to deodorize duck manure
1、复合微生物除臭剂的制备1. Preparation of composite microbial deodorant
1)一级种子液培养：无菌条件下分别取哈尔滨乳杆菌和类干酪乳杆菌接种于MRS培养基中，枯草芽孢杆菌接种于LB培养基中，于30℃、150rpm的条件下培养24h，得到各菌株的一级种子培养液；1) First-level seed liquid culture: Lactobacillus harbinensis and Lactobacillus casei were inoculated into MRS medium under aseptic conditions, and Bacillus subtilis was inoculated into LB medium, and cultured for 24 hours at 30°C and 150 rpm. Obtain the primary seed culture liquid of each strain;
2)二级种子液培养:无菌条件下分别将哈尔滨乳杆菌和类干酪乳杆菌的一级种子培养液按照1vol％的接种量接种于MRS培养基中，枯草芽孢杆菌的一级种子培养液按照1vol％的接种量接种于LB培养基中，于30℃、150rpm的条件下培养24h，得到各菌株的二级种子培养液；2) Secondary seed liquid culture: Under aseptic conditions, inoculate the primary seed culture liquid of Lactobacillus harbinensis and Lactobacillus casei into the MRS medium at an inoculum volume of 1 vol%, and the primary seed culture liquid of Bacillus subtilis. Inoculate the LB medium at an inoculation amount of 1 vol%, and cultivate it for 24 hours at 30°C and 150 rpm to obtain the secondary seed culture liquid of each strain;
3)发酵：在3个发酵罐中分别装入总体积60％的发酵培养基，待发酵培养基消毒完毕后，分别将步骤(2)所得的哈尔滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌的二级种子培养液按照5vol％的接种量接种于3个发酵培养基中。哈尔滨乳杆菌、类干酪乳杆菌的发酵条件是：控制温度为25-35℃，通气比为1:(0.5-0.8)，转速150-300rpm，发酵24-48h，待pH＜4停止发酵；枯草芽孢杆菌的发酵条件是：控制温度为25-35℃，通气比为1:(1-2)，转速150-300rpm的条件下发酵，发酵24-36h，待溶氧开始上升时停止发酵，将上述发酵液按合适体积比进行混合后得液体复合微生物除臭剂，哈尔滨乳杆菌、类干酪乳杆菌、枯草芽孢杆菌的发酵液活菌数分别为55×108CFU/mL、90×108CFU/mL和120×108CFU/mL。3) Fermentation: 60% of the total volume of the fermentation culture medium is loaded into three fermentation tanks respectively. After the fermentation culture medium is sterilized, the Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis obtained in step (2) are added respectively. The secondary seed culture liquid was inoculated into three fermentation media at an inoculation amount of 5vol%. The fermentation conditions of Lactobacillus harbin and Lactobacillus casei are: control temperature is 25-35°C, ventilation ratio is 1: (0.5-0.8), rotation speed is 150-300rpm, fermentation is 24-48h, and the fermentation is stopped when the pH is <4; subtilis The fermentation conditions of Bacillus are: control the temperature to 25-35°C, the ventilation ratio to 1:(1-2), and the fermentation speed to 150-300 rpm. Ferment for 24-36 hours. Stop the fermentation when the dissolved oxygen begins to rise. The above-mentioned fermentation broth is mixed according to an appropriate volume ratio to obtain a liquid composite microbial deodorant. The viable bacterial counts of the fermentation broth of Lactobacillus harbinensis, Lactobacillus casei, and Bacillus subtilis are 55×10 8 CFU/mL and 90×10 8 respectively. CFU/mL and 120×10 8 CFU/mL.
所述哈尔滨乳杆菌、类干酪乳杆菌的发酵培养基组成为：糖蜜40g/L、酵母粉15g/L、K+0.5-1.2g/L、Mg2+0.1-0.2g/L，pH＝6～7，其余为水；枯草芽孢杆菌的发酵培养基为：酵母粉10g/L、硫酸铵10g/L、蛋白胨10g/L，葡萄糖35g/L，红糖15g/L，K+0.5-1.2g/L、Mg2+0.1-0.2g/L，pH＝6.5～7，其余
为水。The fermentation culture medium of Lactobacillus harbinensis and Lactobacillus casei-like is composed of: molasses 40g/L, yeast powder 15g/L, K + 0.5-1.2g/L, Mg 2+ 0.1-0.2g/L, pH=6 ~7, the rest is water; the fermentation medium of Bacillus subtilis is: yeast powder 10g/L, ammonium sulfate 10g/L, peptone 10g/L, glucose 35g/L, brown sugar 15g/L, K + 0.5-1.2g/ L, Mg 2+ 0.1-0.2g/L, pH=6.5~7, the rest for water.
2、实验安排与设计2. Experimental arrangement and design
鸭粪的除臭：室温条件下，将1～2g的鸭粪(取自河北某养鸭场，鸭粪已同稻壳混合在一起)加入到1L实验瓶中，密封后静置，使气体混合均匀，1h后用氨气硫化氢检测仪测其氨气、硫化氢浓度；Deodorization of duck manure: At room temperature, add 1 to 2 g of duck manure (taken from a duck farm in Hebei, duck manure has been mixed with rice husk) into a 1L experimental bottle, seal it and let it stand to allow the gas Mix evenly, and after 1 hour, use an ammonia and hydrogen sulfide detector to measure the ammonia and hydrogen sulfide concentrations;
设置1组对照组，用无菌水代替活化液作为对照组，设置3个实验组，其中包含2个复合菌剂和一个哈尔滨乳杆菌活化液，各实验组分别设置3个平行。Set up a control group, use sterile water instead of activation solution as the control group, set up 3 experimental groups, including 2 compound bacteria and a Lactobacillus harbinensis activation solution, and set up 3 parallels in each experimental group.
2个复合菌剂实验组的菌剂由各菌株的发酵液按如下体积比复配而成：The bacterial agents of the two compound bacterial agent experimental groups are compounded from the fermentation broth of each strain according to the following volume ratio:
复合菌剂1：哈尔滨乳杆菌50％、类干酪乳杆菌50％；Compound bacterial agent 1: Lactobacillus harbinensis 50%, Lactobacillus casei-like 50%;
复合菌剂2：哈尔滨乳杆菌40％、类干酪乳杆菌40％、枯草芽孢杆菌20％；Compound bacterial agent 2: Lactobacillus harbinensis 40%, Lactobacillus casei 40%, Bacillus subtilis 20%;
哈尔滨乳杆菌活化液的制备方法同实施例3，将复合菌剂1、2及哈尔滨乳杆菌活化液用自来水稀释30倍后，分别将约1mL的稀释液用喷壶喷到实验瓶中，密封，定期测其氨气、硫化氢浓度。The preparation method of the Lactobacillus Harbinensis activation liquid is the same as in Example 3. After diluting the composite bacterial agents 1 and 2 and the Lactobacillus Harbini activation liquid 30 times with tap water, spray about 1 mL of the diluent into the experimental bottle with a spray bottle, and seal it. Regularly measure the ammonia and hydrogen sulfide concentrations.
为测试除臭效果，每次测试从感官上进行判断(判断标准为：恶臭“++++”，较臭“+++”，微臭“++”，无明显臭感“+”)In order to test the deodorizing effect, each test is judged from the senses (judgment criteria are: bad smell "++++", relatively smelly "+++", slight smell "++", no obvious smell "+")
表4.鸭粪中的氨气处理效果
Table 4. Ammonia treatment effect in duck manure
Table 4. Ammonia treatment effect in duck manure
表5.鸭粪中的硫化氢处理效果
Table 5. Hydrogen sulfide treatment effect in duck manure
Table 5. Hydrogen sulfide treatment effect in duck manure
表6.鸭粪中的臭味去除效果
Table 6. Odor removal effect in duck manure
Table 6. Odor removal effect in duck manure
由上述结果可知，本发明提供的复合微生物除臭剂及单一的哈尔滨乳杆菌活化液对于鸭粪都具有较好的除臭效果，但复合微生物除臭剂较单一的哈尔滨乳杆菌除臭效果更好，哈尔滨乳杆菌24h除氨气、硫化氢的效率分别为87％、71％，复合微生物菌剂2效果最优，24h除氨气、硫化氢的效率分别为94％、93％，所以复合微生物菌剂见效快，且持久。It can be seen from the above results that both the composite microbial deodorant and the single Lactobacillus harbinensis activation solution provided by the present invention have a good deodorizing effect on duck manure, but the composite microbial deodorant has a better deodorizing effect than the single Lactobacillus harbinensis activation liquid. Well, the efficiency of Lactobacillus Harbinensis in removing ammonia and hydrogen sulfide in 24 hours is 87% and 71% respectively. The compound microbial agent 2 has the best effect. The efficiency of removing ammonia and hydrogen sulfide in 24 hours is 94% and 93% respectively, so the compound Microbial inoculants are quick-acting and long-lasting.
实施例5：复合微生物除臭剂用于猫砂除臭Example 5: Composite microbial deodorizer is used to deodorize cat litter
1、复合微生物除臭剂的制备1. Preparation of composite microbial deodorant
1)一级种子液培养：无菌条件下分别取哈尔滨乳杆菌和类干酪乳杆菌接种于MRS培养基中，枯草芽孢杆菌接种于LB培养基中，于35℃、300rpm的条件下培养12h，得到各菌株的一级种子培养液；1) First-level seed liquid culture: Lactobacillus harbinensis and Lactobacillus casei were inoculated into MRS medium under aseptic conditions, and Bacillus subtilis was inoculated into LB medium, and cultured at 35°C and 300rpm for 12 hours. Obtain the primary seed culture liquid of each strain;
2)二级种子液培养：无菌条件下分别将哈尔滨乳杆菌和类干酪乳杆菌的
一级种子培养液按照2.5vol％的接种量接种于MRS培养基中，枯草芽孢杆菌的一级种子培养液按照2.5vol％的接种量接种于LB培养基中，于35℃、300rpm的条件下培养12h，得到各菌株的二级种子培养液；2) Secondary seed liquid culture: Lactobacillus harbinensis and Lactobacillus casei were cultured under sterile conditions. The first-level seed culture liquid was inoculated into the MRS medium at an inoculation amount of 2.5 vol%. The primary seed culture liquid of Bacillus subtilis was inoculated into the LB medium at an inoculum amount of 2.5 vol%. Under the conditions of 35°C and 300 rpm Cultivate for 12 hours to obtain the secondary seed culture liquid of each strain;
3)发酵：在3个发酵罐中分别装入总体积60％的发酵培养基，待发酵培养基消毒完毕后，分别将步骤(2)所得的哈尔滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌的二级种子培养液按照10vol％的接种量接种于3个发酵培养基中。哈尔滨乳杆菌、植物乳杆菌的发酵条件是：控制温度为25-35℃，通气比为1:(0.5-0.8)，转速150-300rpm，发酵24-48h，待pH＜4停止发酵；枯草芽孢杆菌的发酵条件是：控制温度为25-35℃，通气比为1:(1-2)，转速150-300rpm的条件下发酵，发酵24-36h，待溶氧开始上升时停止发酵，将上述发酵液按合适体积比进行混合后得液体复合菌剂，哈尔滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌的发酵液活菌数分别为85×108CFU/mL、100×108CFU/mL和150×108CFU/mL。3) Fermentation: 60% of the total volume of the fermentation culture medium is loaded into three fermentation tanks respectively. After the fermentation culture medium is sterilized, the Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis obtained in step (2) are added respectively. The secondary seed culture liquid was inoculated into three fermentation media at an inoculation amount of 10vol%. The fermentation conditions of Lactobacillus harbin and Lactobacillus plantarum are: control temperature is 25-35°C, ventilation ratio is 1: (0.5-0.8), rotation speed is 150-300rpm, fermentation is 24-48h, fermentation is stopped when pH is <4; Bacillus subtilis The fermentation conditions of the bacilli are: control the temperature to 25-35°C, the ventilation ratio to 1:(1-2), and the fermentation speed to 150-300 rpm. Ferment for 24-36 hours. Stop the fermentation when the dissolved oxygen begins to rise. Add the above The fermentation broth is mixed according to an appropriate volume ratio to obtain a liquid composite bacterial agent. The viable bacterial counts of the fermentation broth of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis are 85×10 8 CFU/mL and 100×10 8 CFU/mL respectively. and 150×10 8 CFU/mL.
所述哈尔滨乳杆菌、类干酪乳杆菌的发酵培养基组成为：红糖20g/L、葡萄糖40g/L、酵母粉30g/L、K+0.5-1.2g/L、Mg2+0.1-0.2g/L，pH＝6～7，其余为水；枯草芽孢杆菌的发酵培养基为：葡萄糖30g/L、酵母粉10g/L、K+0.3-0.8g/L、Mg2+0.5-1.5g/L，pH＝7～8，其余为水。The fermentation culture medium of Lactobacillus harbinensis and Lactobacillus casei-like is composed of: brown sugar 20g/L, glucose 40g/L, yeast powder 30g/L, K + 0.5-1.2g/L, Mg 2+ 0.1-0.2g/ L, pH=6~7, the rest is water; the fermentation medium of Bacillus subtilis is: glucose 30g/L, yeast powder 10g/L, K + 0.3-0.8g/L, Mg 2+ 0.5-1.5g/L , pH=7~8, the rest is water.
2、实验安排与设计2. Experimental arrangement and design
猫砂的除臭：室温条件下，将1～2g的猫砂(取自宠物猫用过的猫砂)加入到1L实验瓶中，密封后静置，使气体混合均匀，1h后用氨气硫化氢检测仪测其氨气、硫化氢浓度；Deodorization of cat litter: At room temperature, add 1 to 2 g of cat litter (taken from cat litter used by pet cats) into a 1L experimental bottle, seal it and let it stand to allow the gas to mix evenly. After 1 hour, use ammonia gas The hydrogen sulfide detector measures the ammonia and hydrogen sulfide concentrations;
设置1组对照组，用无菌水代替活化液作为对照组，设置3个实验组，其中包含2个复合菌剂和一个哈尔滨乳杆菌活化液，各实验组分别设置3个平行。Set up a control group, use sterile water instead of activation solution as the control group, set up 3 experimental groups, including 2 compound bacteria and a Lactobacillus harbinensis activation solution, and set up 3 parallels in each experimental group.
2个复合菌剂实验组的菌剂由各菌株的发酵液按如下体积比复配而成：
The bacterial agents of the two compound bacterial agent experimental groups are compounded from the fermentation broth of each strain according to the following volume ratio:
复合菌剂1：哈尔滨乳杆菌50％、类干酪乳杆菌50％；Compound bacterial agent 1: Lactobacillus harbinensis 50%, Lactobacillus casei-like 50%;
复合菌剂2：哈尔滨乳杆菌40％、类干酪乳杆菌40％、枯草芽孢杆菌20％；Compound bacterial agent 2: Lactobacillus harbinensis 40%, Lactobacillus casei 40%, Bacillus subtilis 20%;
哈尔滨乳杆菌活化液的制备方法同实施例3，将复合菌剂1、2及哈尔滨乳杆菌活化液用自来水稀释30倍后，分别将约1mL的稀释液用喷壶喷到实验瓶中，密封，定期测其氨气、硫化氢浓度。The preparation method of the Lactobacillus Harbinensis activation liquid is the same as in Example 3. After diluting the composite bacterial agents 1 and 2 and the Lactobacillus Harbini activation liquid 30 times with tap water, spray about 1 mL of the diluent into the experimental bottle with a spray bottle, and seal it. Regularly measure the ammonia and hydrogen sulfide concentrations.
为测试除臭效果，每次测试从感官上进行判断(判断标准为：恶臭“++++”，较臭“+++”，微臭“++”，无明显臭感“+”)In order to test the deodorizing effect, each test is judged from the senses (judgment criteria are: bad smell "++++", relatively smelly "+++", slight smell "++", no obvious smell "+")
表7.猫砂中的氨气处理效果
Table 7. Ammonia treatment effect in cat litter
Table 7. Ammonia treatment effect in cat litter
表8.猫砂中的硫化氢处理效果
Table 8. Hydrogen sulfide treatment effect in cat litter
Table 8. Hydrogen sulfide treatment effect in cat litter
表9.猫砂中的臭味去除效果
Table 9. Odor removal effect in cat litter
Table 9. Odor removal effect in cat litter
由上述结果可知，本发明提供的复合微生物除臭剂及单一的哈尔滨乳杆菌对于猫砂产生的臭味都具有较好的去除效果，但复合微生物除臭剂较单一的哈尔滨乳杆菌除臭效果更好，单一的哈尔滨乳杆菌24h除氨气、硫化氢的效率分别为85％、86％，复合微生物除臭剂2效果最优，24h除氨气、硫化氢的效率分别为94％、86％，且臭味明显感觉不到，说明该复合微生物除臭剂的除臭效果好，且持久性好。It can be seen from the above results that both the composite microbial deodorant and the single Lactobacillus harbinensis provided by the present invention have better removal effects on the odor produced by cat litter, but the composite microbial deodorant has a better deodorizing effect than the single Lactobacillus harbinensis. Even better, the 24-hour ammonia and hydrogen sulfide removal efficiencies of a single Lactobacillus harbinensis agent are 85% and 86% respectively. The compound microbial deodorant 2 has the best effect. The 24-hour ammonia and hydrogen sulfide removal efficiencies are 94% and 86% respectively. %, and the odor is obviously not felt, indicating that the compound microbial deodorant has good deodorizing effect and good durability.
实施例6：复合微生物除臭剂用于下水道除臭Example 6: Composite microbial deodorant used for sewer deodorization
1、复合微生物除臭剂的制备1. Preparation of composite microbial deodorant
1)一级种子液培养：无菌条件下分别取哈尔滨乳杆菌和类干酪乳杆菌接种于MRS培养基中，枯草芽孢杆菌接种于LB培养基中，于25℃、220rpm的条件下培养24h，得到各菌株的一级种子培养液；1) First-level seed liquid culture: Lactobacillus harbinensis and Lactobacillus casei were inoculated into MRS medium under aseptic conditions, and Bacillus subtilis was inoculated into LB medium, and cultured for 24 hours at 25°C and 220 rpm. Obtain the primary seed culture liquid of each strain;
2)二级种子液培养：无菌条件下分别将哈尔滨乳杆菌和类干酪乳杆菌的一级种子培养液按照5vol％的接种量接种于MRS培养基中，枯草芽孢杆菌的一级种子培养液按照5vol％的接种量接种于LB培养基中，于30℃、180rpm的条件下培养24h，得到各菌株的二级种子培养液；2) Secondary seed liquid culture: Under aseptic conditions, the primary seed culture liquid of Lactobacillus harbinensis and Lactobacillus casei were inoculated into the MRS medium at an inoculum volume of 5 vol%, and the primary seed culture liquid of Bacillus subtilis was Inoculate the LB medium at an inoculation amount of 5 vol%, and cultivate it for 24 hours at 30°C and 180 rpm to obtain the secondary seed culture liquid of each strain;
3)发酵：在3个发酵罐中分别装入总体积60％的发酵培养基，待发酵培养基消毒完毕后，分别将步骤(2)所得的哈尔滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌的二级种子培养液按照5vol％的接种量接种于3个发酵培养基中。
哈尔滨乳杆菌、植物乳杆菌的发酵条件是：控制温度为25-35℃，通气比为1:(0.5-0.8)，转速150-300rpm，发酵24-48h，待pH＜4停止发酵；枯草芽孢杆菌的发酵条件是：控制温度为25-35℃，通气比为1:(1-2)，转速150-300rpm的条件下发酵，发酵24-36h，待溶氧开始上升时停止发酵，将上述发酵液按合适体积比进行混合后得复合微生物除臭剂，哈尔滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌的发酵液活菌数分别为45×108CFU/mL、60×108CFU/mL和90×108CFU/mL。3) Fermentation: 60% of the total volume of the fermentation culture medium is loaded into three fermentation tanks respectively. After the fermentation culture medium is sterilized, the Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis obtained in step (2) are added respectively. The secondary seed culture liquid was inoculated into three fermentation media at an inoculation amount of 5vol%. The fermentation conditions of Lactobacillus harbin and Lactobacillus plantarum are: control temperature is 25-35°C, ventilation ratio is 1: (0.5-0.8), rotation speed is 150-300rpm, fermentation is 24-48h, fermentation is stopped when pH is <4; Bacillus subtilis The fermentation conditions of the bacilli are: control the temperature to 25-35°C, the ventilation ratio to 1:(1-2), and the fermentation speed to 150-300 rpm. Ferment for 24-36 hours. Stop the fermentation when the dissolved oxygen begins to rise. Add the above The fermentation broth is mixed according to an appropriate volume ratio to obtain a composite microbial deodorant. The viable bacterial counts of the fermentation broth of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis are 45×10 8 CFU/mL and 60×10 8 CFU/mL respectively. mL and 90×10 8 CFU/mL.
所述哈尔滨乳杆菌、类干酪乳杆菌的发酵培养基组成为：糖蜜50g/L、酵母粉20g/L、K+0.5-1.2g/L、Mg2+0.1-0.2g/L，其余为水；枯草芽孢杆菌的发酵培养基为：葡萄糖20g/L、牛肉粉10g/L、蛋白胨20g/L，K+0.5-1.2g/L、Mg2+0.1-0.2g/L，其余为水。The fermentation culture medium of Lactobacillus harbinensis and Lactobacillus casei-like is composed of: molasses 50g/L, yeast powder 20g/L, K + 0.5-1.2g/L, Mg 2+ 0.1-0.2g/L, and the rest is water ; The fermentation medium of Bacillus subtilis is: glucose 20g/L, beef meal 10g/L, peptone 20g/L, K + 0.5-1.2g/L, Mg 2+ 0.1-0.2g/L, and the rest is water.
2、实验设计与安排：2. Experimental design and arrangement:
下水道的除臭：室温条件下，将10-20mL的生活污水进水加入到1L实验瓶中，密封后静置，使气体混合均匀，1h后用氨气硫化氢检测仪测其氨气、硫化氢浓度；然后分别加入单一的哈尔滨乳杆菌活化液及复合菌剂约1mL，用喷壶喷到试剂瓶中，密封，定期测其氨气、硫化氢浓度。Deodorization of sewers: At room temperature, add 10-20mL of domestic sewage water into a 1L experimental bottle, seal it and let it stand to make the gases mix evenly. After 1 hour, use an ammonia hydrogen sulfide detector to measure the ammonia and sulfide gas. Hydrogen concentration; then add about 1 mL of single Lactobacillus Harbinensis activation solution and compound inoculant respectively, spray it into the reagent bottle with a spray bottle, seal it, and measure its ammonia and hydrogen sulfide concentration regularly.
设置1组对照组，用无菌水代替活化液作为对照组，设置3个实验组，其中包含2个复合菌剂和一个哈尔滨乳杆菌活化液，各实验组分别设置3个平行。Set up a control group, use sterile water instead of activation solution as the control group, set up 3 experimental groups, including 2 compound bacteria and a Lactobacillus harbinensis activation solution, and set up 3 parallels in each experimental group.
2个复合菌剂实验组的菌剂由各菌株的活化液按如下体积比复配而成：The bacterial agents of the two compound bacterial agent experimental groups are compounded from the activation solution of each strain according to the following volume ratio:
复合菌剂1：哈尔滨乳杆菌50％、类干酪乳杆菌50％；Compound bacterial agent 1: Lactobacillus harbinensis 50%, Lactobacillus casei-like 50%;
复合菌剂2：哈尔滨乳杆菌40％、类干酪乳杆菌40％、枯草芽孢杆菌20％；Compound bacterial agent 2: Lactobacillus harbinensis 40%, Lactobacillus casei 40%, Bacillus subtilis 20%;
哈尔滨乳杆菌活化液的制备方法同实施例3。The preparation method of Lactobacillus Harbinensis activation liquid is the same as in Example 3.
为测试除臭效果，每次测试从感官上进行判断(判断标准为：恶臭
“++++”，较臭“+++”，微臭“++”，无明显臭感“+”)In order to test the deodorizing effect, each test is judged from the senses (judgment standard is: odor "++++", relatively smelly "+++", slightly smelly "++", no obvious smell "+")
3、实验结果3. Experimental results
表10.下水道中氨气的处理效果
Table 10. Treatment effect of ammonia in sewers
Table 10. Treatment effect of ammonia in sewers
表11.下水道中硫化氢的处理效果
Table 11. Treatment effect of hydrogen sulfide in sewers
Table 11. Treatment effect of hydrogen sulfide in sewers
表12.下水道中的臭味去除效果
Table 12. Odor removal effect in sewers
Table 12. Odor removal effect in sewers
由上述实验结果可见，本发明提供的复合微生物除臭剂及单一的哈尔滨乳杆菌活化液对于下水道臭味都具有较好的除臭效果，但复合微生物除臭剂
较单一的哈尔滨乳杆菌活化液除臭效果更好，哈尔滨乳杆菌活化液24h除氨气、硫化氢的效率分别为83％、73％，而复合菌剂2的效果最优，24h除氨气、硫化氢的效率分别为90％、85％，所以复合微生物除臭剂见效快且持久，而且由于使用了复合微生物除臭剂，不仅氨气、硫化氢的处理效率高，并且通过感官评价，还能闻到类似酿酒的味道。It can be seen from the above experimental results that the composite microbial deodorant provided by the present invention and the single Lactobacillus harbinensis activation liquid have good deodorizing effects on sewer odor, but the composite microbial deodorant Compared with the single Lactobacillus Harbinensis activation liquid, the deodorization effect is better. The efficiency of Lactobacillus Harbini activation liquid in removing ammonia gas and hydrogen sulfide in 24 hours is 83% and 73% respectively, while the compound bacterial agent 2 has the best effect, removing ammonia gas in 24h. The efficiency of hydrogen sulfide and hydrogen sulfide are 90% and 85% respectively, so the compound microbial deodorant is quick and long-lasting. Moreover, due to the use of compound microbial deodorant, not only the treatment efficiency of ammonia and hydrogen sulfide is high, but also through sensory evaluation, You can also smell something like brewing.
综上，本发明提供的哈尔滨乳杆菌无论是与类干酪乳杆菌二者复配，还是与类干酪乳杆菌和枯草芽孢杆菌三者复配使用，都对产生恶臭的氨气和硫化氢气体具有非常好的处理作用。To sum up, whether the Lactobacillus harbinensis provided by the present invention is compounded with Lactobacillus casei-like or with Lactobacillus casei-like and Bacillus subtilis, it has the ability to treat ammonia gas and hydrogen sulfide gas that produce odor. Very good handling.
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.
Claims (10)
- 一种复合微生物除臭剂，其特征在于，包含哈尔滨乳杆菌(Lactobacillus harbinensis)和类干酪乳杆菌，所述哈尔滨乳杆菌保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为：CGMCC No.24253。A composite microbial deodorant, characterized in that it contains Lactobacillus harbinensis (Lactobacillus harbinensis) and Lactobacillus casei. The Lactobacillus harbinensis is deposited in the General Microbiology Center of the China Microbial Culture Collection Committee, and the preservation number is: CGMCC No. .24253.
- 根据权利要求1所述的复合微生物除臭剂，其特征在于，所述哈尔滨乳杆菌与类干酪乳杆菌的活菌数之比为1：(1-2)，优选1：(1.2-1.6)。The composite microbial deodorant according to claim 1, characterized in that the ratio of the number of viable bacteria of Lactobacillus harbinensis and Lactobacillus casei-like bacteria is 1: (1-2), preferably 1: (1.2-1.6) .
- 根据权利要求1所述的复合微生物除臭剂，其特征在于，还包含枯草芽孢杆菌。The composite microbial deodorant according to claim 1, further comprising Bacillus subtilis.
- 根据权利要求3所述的复合微生物除臭剂，其特征在于，所述哈尔滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌的活菌数之比为1：(0.5-3)：(0.5-2.5)，优选1：(0.7-2.1)：(0.9-1.5)。The composite microbial deodorant according to claim 3, characterized in that the ratio of the viable bacterial numbers of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis is 1: (0.5-3): (0.5-2.5 ), preferably 1: (0.7-2.1): (0.9-1.5).
- 根据权利要求1-4中任一项所述的复合微生物除臭剂，其特征在于，所述复合微生物除臭剂中的活菌量不低于10×108CFU/mL，优选(10-50)×108CFU/mL。The composite microbial deodorant according to any one of claims 1 to 4, characterized in that the amount of viable bacteria in the composite microbial deodorant is not less than 10×10 8 CFU/mL, preferably (10- 50)×10 8 CFU/mL.
- 权利要求1-5中任一项所述的复合微生物除臭剂的制备方法，其特征在于，包括如下步骤：The preparation method of the composite microbial deodorant according to any one of claims 1-5, characterized in that it includes the following steps:(1)扩大培养：将哈尔滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌进行扩大培养，得到各菌株的培养液；(1) Expanded culture: Expand the culture of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis to obtain the culture solution of each strain;(2)发酵：待发酵罐内的发酵培养基消毒完毕后，分别将步骤(1)所得的哈尔滨乳杆菌、类干酪乳杆菌和枯草芽孢杆菌的培养液按照5-10vol％的接种量接种于发酵培养基中，控制温度为25-35℃、转速150-300rpm，哈尔滨乳杆菌和类干酪乳杆菌在通气比为1:(0.5-0.8)的条件下发酵，待pH＜4时停止发酵；枯草芽孢杆菌在通气比为1:(1-2)的条件下发酵，待溶氧开始上升时停止发酵，得各菌株的发酵液； (2) Fermentation: After the fermentation medium in the fermentation tank has been sterilized, inoculate the culture solution of Lactobacillus harbinensis, Lactobacillus casei and Bacillus subtilis obtained in step (1) at an inoculum volume of 5-10vol%. In the fermentation medium, the temperature is controlled at 25-35°C and the rotation speed is 150-300 rpm. Lactobacillus harbinensis and Lactobacillus casei are fermented under the condition of a ventilation ratio of 1: (0.5-0.8). The fermentation is stopped when the pH is <4; Bacillus subtilis is fermented under the condition of aeration ratio of 1:(1-2). The fermentation is stopped when the dissolved oxygen begins to rise, and the fermentation liquid of each strain is obtained;(3)制备复合微生物除臭剂：将步骤(2)所得的各菌株的发酵液按体积比混合灌装，即得复合微生物除臭剂。(3) Preparing the composite microbial deodorant: Mix and fill the fermentation broth of each strain obtained in step (2) according to the volume ratio to obtain the composite microbial deodorant.
- 根据权利要求6所述的制备方法，其特征在于，所述哈尔滨乳杆菌和类干酪乳杆菌的发酵培养基组成为：碳源40-60g/L、氮源15-30g/L、K+0.5-1.2g/L、Mg2+0.1-0.2g/L，溶剂为水，且pH＝6～7；所述枯草芽孢杆菌的发酵培养基组成为：碳源20-50g/L、氮源10-30g/L、K+0.3-0.8g/L、Mg2+0.5-1.5g/L，溶剂为水，且pH＝6.5-8。The preparation method according to claim 6, characterized in that the fermentation medium of Lactobacillus harbinensis and Lactobacillus casei is composed of: carbon source 40-60g/L, nitrogen source 15-30g/L, K + 0.5 -1.2g/L, Mg 2+ 0.1-0.2g/L, the solvent is water, and pH=6~7; the fermentation medium of Bacillus subtilis consists of: carbon source 20-50g/L, nitrogen source 10 -30g/L, K + 0.3-0.8g/L, Mg 2+ 0.5-1.5g/L, the solvent is water, and pH=6.5-8.
- 根据权利要求7所述的制备方法，其特征在于，所述碳源选自葡萄糖、红糖、糖蜜中的一种或多种的复配；The preparation method according to claim 7, characterized in that the carbon source is selected from one or more combinations of glucose, brown sugar, and molasses;所述氮源选自酵母粉、蛋白胨、硫酸铵或牛肉粉中的一种或多种的复配。The nitrogen source is selected from one or more combinations of yeast powder, peptone, ammonium sulfate or beef meal.
- 一种使用权利要求1-5中任一项所述的复合微生物除臭剂去除环境中臭味的方法，包括向环境中喷洒所述复合微生物除臭剂的步骤。A method of using the composite microbial deodorant according to any one of claims 1 to 5 to remove odor in the environment, including the step of spraying the composite microbial deodorant into the environment.
- 权利要求1-5中任一项所述的复合微生物除臭剂在除臭领域的应用，优选的，所述复合微生物除臭剂用于去除氨气和硫化氢，更优选的，用于去除宠物粪便、畜禽粪便和下水道所产生的氨气和硫化氢。 Application of the composite microbial deodorant according to any one of claims 1 to 5 in the field of deodorization. Preferably, the composite microbial deodorant is used to remove ammonia and hydrogen sulfide. More preferably, it is used to remove ammonia and hydrogen sulfide. Ammonia and hydrogen sulfide produced by pet feces, livestock and poultry manure, and sewers.
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