CN101199319A - Enzyme composite bactericide with broad-spectrum high-efficient bactericidal action and its preparation method - Google Patents
Enzyme composite bactericide with broad-spectrum high-efficient bactericidal action and its preparation method Download PDFInfo
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- CN101199319A CN101199319A CNA200710135415XA CN200710135415A CN101199319A CN 101199319 A CN101199319 A CN 101199319A CN A200710135415X A CNA200710135415X A CN A200710135415XA CN 200710135415 A CN200710135415 A CN 200710135415A CN 101199319 A CN101199319 A CN 101199319A
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Abstract
Disclosed is an enzyme combination and sterilization preparation having the broad-spectrum high efficiency sterilization function and the preparation method, which relates to the food safety and enzyme preparation technique field. The invention provides the enzyme combination and sterilization preparation which is combined by lysozyme, Beta-1, 3- dextranase, Beta-mannase, enzyme and neutral protein enzyme, and adopts the coating agent formulation of fucose, xanthan gum, sodium carboxymethylcellulose and pullulan to coat the combined enzyme. The invention adopts the multi-enzyme combination and coating technology to optimize the combination of the enzyme, meanwhile protecting the enzyme by the coating technology so as to effectively kill gram positive and negative bacteria with wide control spectrum and high efficiency, thus effectively killing pathogenic bacteria such as salmonella, Escherichia coli and golden yellow staphylococcus, and the invention and is a perfect alternative to antibiotics within feedstuff of pig and poultry for the preservation of animal and plant products. The coating protection promotes the resistance of enzyme towards temperature, oxidant and acid, thereby enhancing the sterilization effects under unfavorable circumstances and expanding the field of applications of enzyme fungicide.
Description
Technical field
A kind of enzyme combination biocide preparation that the broad-spectrum high efficacy bactericidal action is arranged and preparation method thereof the present invention relates to food security and enzyme preparation technical field.Also relate to antibiotic in the alternative feed, the preservation field of animals and plants product is a kind of combination with broad-spectrum high efficacy bactericidal action enzyme specifically.
Background technology
Antibiotic is the most widely used antibacterials of a class, not only is used for clinically, also is used for its feeding and agriculture aspect.In in the past more than 50 year, use the generation that causes a large amount of antibody-resistant bacterium because antibiotic is long-term, and the pathogen resistance to the action of a drug strengthens year by year, cause curative effect to descend, dosage improves.Therefore, the World Health Organization has proposed caution in the monitoring result with regard to bacterial drug resistance in 1994 to the whole world: the drug resistance that bacterial antibiotic produces increases with surprising rapidity, and existing antibiotic medicine is losing original curative effect.Therefore seek wide spectrum, antibacterials of new generation have become the problem of extremely urgent pendulum in face of the mankind efficiently.
Enzyme extensively is present in the organism, participates in different physiological roles such as metabolism, and wherein the enzyme that microorganism wall is had a hydrolysis function can dissolve microorganism wall and make microbial death.Because the specificity of hydrolase is very strong, cells of microorganisms wall construction and chemical composition there are differences again, and therefore a kind of enzyme can only have hydrolysis to a certain quasi-microorganism.Even for a certain specified microorganisms,, just better action effect can be arranged because the complexity of cell membrane chemical composition also needs the combination of dissimilar hydrolases.
The bactericidal action of hydrolase is compared with traditional antibiotic medicine, have all effective advantage of all serotypes of a certain pathogen, after several enzymes are compound, all effective to dissimilar pathogens, overcome a kind of antibiotic and can only prevent deficiency a kind of or wherein a kind of serotype pathogen, more do not had medicament residue and chemical sproof problem.
Lysozyme is brought into use in medicine and food, and then at the early-stage as additive for farm animal feed, only some Primary Study have been done by the former Soviet Union, France, Germany and the U.S., and is present, domesticly also begins to carry out correlative study.And the research of composite bactericidal enzyme medicine is all at the early-stage both at home and abroad.
Summary of the invention
The purpose of this invention is to provide a kind of enzyme combination biocide preparation that the broad-spectrum high efficacy bactericidal action is arranged and preparation method thereof, is that a kind of spectrum is efficient, noresidue, biocide preparation pollution-free, applied widely.
Technical scheme of the present invention: a kind of enzyme combination biocide preparation, in the content alive of enzyme in the preparation, press lysozyme 400～1000U/g, β-1, the proportioning of 3-dextranase 20000～80000U/g, 'beta '-mannase 5000～10000U/g and neutral proteinase 80000～140000U/g is formulated as the combination enzyme, adopt the proportioning of trehalose 30%～50%, xanthans 20%～40%, sodium carboxymethylcellulose 15%～30% and pullulan 5%～10% to be formulated as coating agent, will make up enzyme and be packaged as enzyme combination biocide preparation.
The preparation method of described enzyme combination biocide preparation:
1) preparation of combination enzyme: be converted into quality according to the enzyme of the used enzyme content of living, take by weighing lysozyme, the β-1 of respective quality by the proportioning of combination enzyme, 3-dextranase, 'beta '-mannase and neutral proteinase, mixing then is the combination enzyme;
2) coated for protecting of enzyme: take by weighing trehalose, xanthans, sodium carboxymethylcellulose and pullulan by the coating agent proportioning; mix and be coating agent; steeped 3-5 hour with 30-50 ℃ the water logging that coating agent weight 8-10 doubly measures; add operation 1) gained combination enzyme; grind twice with colloid mill; adopt white carbon absorption to be prepared into powder particle, or adopt spray-drying to be prepared into microcapsules.
The application of enzyme combination biocide preparation is used for feed: the combination enzyme adds directly use in the feed to powder type, addition: enzyme combination biocide preparation/complete feed is the 40-50g/ ton; Or be used for food preservation: will make up enzyme with after the suitable quantity of water dissolving, and filter with quick filter paper, and get filtrate use, it is 0.03%-0.05% that enzyme makes up the biocide preparation solution concentration.
Beneficial effect of the present invention:
1, fungicidal spectrum is wide: can kill gram-positive bacteria and negative bacterium, particularly pathogenic bacteria, as salmonella, staphylococcus aureus, Escherichia coli are had the good sterilization effect, and fungi also be had bactericidal action.
2, sterilizing ability is strong: the combination biocide preparation of several enzymes, and not only have the wide characteristics of fungicidal spectrum, and have the strong advantage of sterilizing ability, different enzyme combinations can strengthen bactericidal effect, increase more than 10 times than single use lysozyme sterilizing ability.
3, have no drug resistance and residual: the sterilization mechanism of enzyme is the cell membrane of cracking bacterium, and bacterium can not produce drug resistance, can not remain in the histoorgan, enzyme is a protein simultaneously, sex change inactivation in the food cooking process helps ensuring food safety, and improves human health.
4, strong stress resistance: bag is become the enzyme combined bactericide of microcapsules can effectively tolerate high temperature, the strong oxidizer in the process and is hanged down acid, keeps high disinfection vitality, can satisfy the needs that use under the different condition.
5, usage range is wide: can substitute the antibiotic in the feed, be used for the treatment of Animal diseases as veterinary drug; Also can be used for food industry, be used for the preservation of animals and plants product, prolong shelf life, improve food quality; Can be used as disinfectant in medical treatment uses.
The specific embodiment
Below in conjunction with embodiment in detail the present invention is described in detail.
The preparation of embodiment 1 enzyme combination biocide preparation
In the content alive of enzyme in the preparation, with lysozyme 400～1000U/g, β-1,3-dextranase 20000～80000U/g, 'beta '-mannase 5000～10000U/g and neutral proteinase 80000～140000U/g combination.Adopt the coating agent of trehalose 30%～50%, xanthans 20%～40%, sodium carboxymethylcellulose 15%～30% and pullulan 5%～10%, will make up enzyme and be prepared into microcapsules or powder enzyme combination biocide preparation.
Its preparation method is:
1) preparation of combination enzyme: according to the enzyme of enzyme content alive, enzyme work is converted into required quality, takes by weighing lysozyme, the β-1 of respective quality, 3-dextranase, 'beta '-mannase and neutral proteinase mix.
2) enzyme activity protection: the coating agent prescription that adopts trehalose 30%～50%, xanthans 20%～40%, sodium carboxymethylcellulose 15%～30% and pullulan 5%～10%; soaked coating agent 3-5 hour with an amount of warm water (8-10 doubly measures, 30-50 ℃) dissolving; to make up enzyme and coating agent mixes by proportioning; grind twice with colloid mill; adopt white carbon absorption to be prepared into powder particle, or adopt spray-drying to be prepared into microcapsules.
As be used for feed and can directly use, as be used for food preservation and said preparation need be dissolved in suitable quantity of water, filter with quick filter paper, get filtrate use, enzyme combination biocide preparation solution concentration is 0.03%-0.05%.
Embodiment 2
After salmonella is cultivated 5h, the lysozyme liquid 0.5mL that adds optium concentration at No. 1 test tube, (the sixth of the twelve Earthly Branches is with the solid bactericide wiring solution-forming for No. 2 enzyme-added combined bactericide liquid of test tube 0.5mL, wherein the enzyme work of each enzyme is in the described scope of claim 1, down together), No. 3 test tube adds phosphate buffer 0.5mL, and every number pipe is done three repetitions.Place the water-bath constant-temperature shaking culture to take out after 10 minutes again.1000 times of No. 1 test tube dilutions, 100 times of No. 2 test tube dilutions, 100000 times of No. 3 test tube dilutions are respectively got 1mL in sterile petri dish, pour the about 15mL of nutrient agar into, and the upset of cooling back places 37 ℃ of constant incubators to cultivate and takes out counting after 48 hours.The results are shown in Table 1.
Table 1 salmonella plate count result
| Project | No. 1 test tube | No. 2 test tubes | No. 3 test tubes |
| Clump count (cfu/mL) | 3.4×10 4 | 3.5×10 3 | 1.98×10 7 |
By table 1 result as can be known, enzyme combined bactericide bactericidal effect is the strongest, and the salmonella number has reduced 10000 times than control group, even compare with the lysozyme group over against the best of photograph, also reduced 10 times, this has confirmed that fully the enzyme combined bactericide has stronger bactericidal effect.
Embodiment 3
After staphylococcus aureus is cultivated 5h, add the lysozyme liquid 0.5mL of optium concentration at No. 1 test tube, No. 2 enzyme-added combined bactericide liquid of test tube 0.5mL, No. 3 test tube adds phosphate buffer 0.5mL, and every number pipe is done three repetitions.Place the water-bath constant-temperature shaking culture to take out after 10 minutes again.1000 times of No. 1 test tube dilutions, 100 times of No. 2 test tube dilutions, 100000 times of No. 3 test tube dilutions are respectively got 1mL in sterile petri dish, pour the about 15mL of nutrient agar into, and the upset of cooling back places 37 ℃ of constant incubators to cultivate and takes out counting after 36 hours.The results are shown in Table 2.
Table 2 staphylococcus aureus plate count result
| Project | No. 1 test tube | No. 2 test tubes | No. 3 test tubes |
| Clump count (cfu/mL) | 2.3×10 5 | 4.5×10 3 | 5.2×10 8 |
By table 2 result as can be known, enzyme combined bactericide bactericidal effect is the strongest, and the staphylococcus aureus number has reduced about 100000 times than control group, even compare with the lysozyme group over against the best of photograph, reduced about 100 times, this has confirmed that fully the enzyme combined bactericide has stronger bactericidal effect.
Embodiment 4
After Escherichia coli (JM109) are cultivated 5h, add the lysozyme liquid 0.5mL of optium concentration at No. 1 test tube, No. 2 enzyme-added combined bactericide liquid of test tube 0.5mL, No. 3 test tube adds phosphate buffer 0.5mL, and every number pipe is done three repetitions.Place the water-bath constant-temperature shaking culture to take out after 10 minutes again.1000 times of No. 1 test tube dilutions, 100 times of No. 2 test tube dilutions, 100000 times of No. 3 test tube dilutions are respectively got 1mL in sterile petri dish, pour the about 15mL of nutrient agar into, and the upset of cooling back places 37 ℃ of constant incubators to cultivate and takes out counting after 36 hours.The results are shown in Table 3.
Table 3 Escherichia coli plate count result
| Project | No. 1 test tube | No. 2 test tubes | No. 3 test tubes |
| Clump count (cfu/ml) | 4.6×10 6 | 3.1×10 4 | 2.7×10 9 |
By table 3 result as can be known, enzyme combined bactericide bactericidal effect is the strongest, and Escherichia coli number comparison has reduced about 100000 times according to group, even compare with the lysozyme group over against the best of photograph, reduced about 100 times, this has confirmed that fully the enzyme combined bactericide has stronger bactericidal effect.
Embodiment 5
Test is carried out at poultry test site, Jiangsu Province, and test finishes since 1 age in days to 49 age in days, and the date is on August 14,25 days to 2006 June in 2006, and ground flat is supported, the sawdust bedding and padding.Control group adds medicine (comprising coccidia medicine and antibiotic); Test group is not added any other medicines except that adding the combination of sterilization enzyme.
Arrangement of table 4 broiler trial and result
| Project | Control group | Test group |
| The fryer number of elements | 320 | 320 |
| Medicine | Add | 0 |
| Sterilization enzyme combination (g/ ton complete feed) | 0 | 40 |
| 49 age in days body weight (kg) | 1.41 | 1.45 |
| 49 age in days material anharmonic ratioes | 2.33 | 2.08 |
| 49 age in days survival rates (%) | 95.0 | 98.0 |
Embodiment 6
Test is carried out on Wu Pingxian pig farm, Fujian, and big weanling pig, test begin to finish with the 3rd week of back of extremely weaning from wean, and the date is on August 5,15 days to 2006 July in 2006.
Table 5 weanling pig test arrangement and result
| Project | The antibiotic group | Sterilization enzyme combination group |
| A piglet number | 100 | 100 |
| Neomycinsulphate (g/ ton complete feed) | 200 | 0 |
| 10% Kitasamycin (g/ ton complete feed) | 300 | 0 |
| Zinc oxide (g/ ton complete feed) | 3000 | 0 |
| Sterilization enzyme combination (g/ ton complete feed) | 0 | 50 |
| Medicine cost (unit/ton complete feed) | 96 | 70 |
| The animal number of having loose bowels | 0 | 0 |
| Other disease incident | 0 | 0 |
Annotate: carrying out twice similarly test altogether on same pig farm, is the medicine that removes in the feed for the first time, keeps zinc oxide; Be to remove medicine and zinc oxide for the second time, all less than having loose bowels and other disease.
Claims (3)
1. an enzyme makes up biocide preparation, it is characterized in that: in the content alive of enzyme in the preparation, press lysozyme 400～1000U/g, β-1, the proportioning of 3-dextranase 20000～80000U/g, 'beta '-mannase 5000～10000U/g and neutral proteinase 80000～140000U/g is formulated as the combination enzyme, adopt the proportioning of trehalose 30%～50%, xanthans 20%～40%, sodium carboxymethylcellulose 15%～30% and pullulan 5%～10% to be formulated as coating agent, will make up enzyme and be packaged as enzyme combination biocide preparation.
2. the preparation method of the described enzyme combination of a claim 1 biocide preparation is characterized in that:
1) preparation of combination enzyme: be converted into quality according to the enzyme of the used enzyme content of living, take by weighing lysozyme, the β-1 of respective quality by the proportioning of combination enzyme, 3-dextranase, 'beta '-mannase and neutral proteinase, mixing then is the combination enzyme;
2) coated for protecting of enzyme: take by weighing trehalose, xanthans, sodium carboxymethylcellulose and pullulan by the coating agent proportioning; mix and be coating agent; steeped 3-5 hour with 30-50 ℃ the water logging that coating agent weight 8-10 doubly measures; add operation 1) gained combination enzyme; grind twice with colloid mill; adopt white carbon absorption to be prepared into powder particle, or adopt spray-drying to be prepared into microcapsules.
3. the application of the enzyme of claim 1 combination biocide preparation is characterized in that being used for feed: the combination enzyme adds directly use in the feed to powder type, addition: enzyme combination biocide preparation/complete feed is the 40-50g/ ton; Or be used for food preservation: after will making up the dissolving of enzyme water, filter with quick filter paper again, get filtrate use, enzyme combination biocide preparation solution concentration is 0.03%-0.05%.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA200710135415XA CN101199319A (en) | 2007-11-08 | 2007-11-08 | Enzyme composite bactericide with broad-spectrum high-efficient bactericidal action and its preparation method |
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| CNA200710135415XA CN101199319A (en) | 2007-11-08 | 2007-11-08 | Enzyme composite bactericide with broad-spectrum high-efficient bactericidal action and its preparation method |
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| CN103396886A (en) * | 2013-07-26 | 2013-11-20 | 湖北中烟工业有限责任公司 | Compound microcapsule wall material and application thereof in essence microcapsules for cigarettes |
| CN104782661A (en) * | 2015-04-01 | 2015-07-22 | 李�杰 | Efficient biological disinfectant for swimming pool and preparation method of efficient biological disinfectant |
| CN105802936A (en) * | 2016-05-26 | 2016-07-27 | 湖南南北旺生物技术有限公司 | Preparation method of high-temperature resistant neutral phytase, high-temperature resistant neutral phytase and application of high-temperature resistant neutral phytase |
| CN106561716A (en) * | 2016-11-02 | 2017-04-19 | 厦门华厦学院 | Agricultural antibacterial complex enzyme preparation and preparation method thereof |
| CN106819430A (en) * | 2016-12-28 | 2017-06-13 | 新昌县赛因斯生物科技有限公司 | Improve the feed formula of goat resistance against diseases |
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- 2007-11-08 CN CNA200710135415XA patent/CN101199319A/en active Pending
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| CN103396886A (en) * | 2013-07-26 | 2013-11-20 | 湖北中烟工业有限责任公司 | Compound microcapsule wall material and application thereof in essence microcapsules for cigarettes |
| CN104782661A (en) * | 2015-04-01 | 2015-07-22 | 李�杰 | Efficient biological disinfectant for swimming pool and preparation method of efficient biological disinfectant |
| CN105802936A (en) * | 2016-05-26 | 2016-07-27 | 湖南南北旺生物技术有限公司 | Preparation method of high-temperature resistant neutral phytase, high-temperature resistant neutral phytase and application of high-temperature resistant neutral phytase |
| CN105802936B (en) * | 2016-05-26 | 2019-10-29 | 湖南南北旺生物技术有限公司 | The preparation method and high temperature resistant neutral phytase of a kind of high temperature resistant neutral phytase and its application |
| CN106561716A (en) * | 2016-11-02 | 2017-04-19 | 厦门华厦学院 | Agricultural antibacterial complex enzyme preparation and preparation method thereof |
| CN106819430A (en) * | 2016-12-28 | 2017-06-13 | 新昌县赛因斯生物科技有限公司 | Improve the feed formula of goat resistance against diseases |
| CN106819365A (en) * | 2016-12-28 | 2017-06-13 | 新昌县赛因斯生物科技有限公司 | Promote production and use domestic animal feed additive |
| CN106822880A (en) * | 2016-12-30 | 2017-06-13 | 江阴市本特塞缪森生命科学研究院有限公司 | A kind of antimicrobial compositions and its application |
| CN108634111A (en) * | 2018-04-12 | 2018-10-12 | 西南民族大学 | The special preparation method for licking brick of Adult Yak warm season |
| CN113317327A (en) * | 2020-02-28 | 2021-08-31 | 中粮营养健康研究院有限公司 | Bactericidal virucidal enzyme preparation composition and application thereof |
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