CN105087432A - Application of glutathione waste liquid in microorganism production and application method - Google Patents
Application of glutathione waste liquid in microorganism production and application method Download PDFInfo
- Publication number
- CN105087432A CN105087432A CN201510487030.4A CN201510487030A CN105087432A CN 105087432 A CN105087432 A CN 105087432A CN 201510487030 A CN201510487030 A CN 201510487030A CN 105087432 A CN105087432 A CN 105087432A
- Authority
- CN
- China
- Prior art keywords
- gsh
- tank
- waste liquid
- liquid
- controls
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses application of glutathione waste liquid in microorganism production and an application method and belongs to the field of microorganisms. The glutathione waste liquid is used as the effective constituent of a seeding tank culture medium and/or a fermentation tank culture medium to be applied to the microorganism production, good social and economic values are achieved, microorganism preparation with good performance can be obtained, and wide application value is achieved.
Description
Technical field
The present invention relates to microorganism field, the specifically application of gsh waste liquid in microorganisms producing and application method.
Background technology
Gsh, refer generally to reductive glutathione (Glutathione, GSH), it is a kind of biologically active substance be extensively present in human normal cell, be the content of synthesis naturally in human cell the abundantest containing the low molecule tripeptides of sulfydryl, clinical proof reductive glutathione is a kind of broad-spectrum small-molecule peptide active substance, there is important anti-oxidant and integration detoxification, for viral, drug toxicity, the liver damage that Alcoholism and other chemical substance cause, also be adjuvant therapy medicaments during cancer patients's chemicotherapy, to acute anemia, adult respiratory distress syndrome, septicemia combination therapy has positive effect.
At present, gsh preparation method mainly contains solvent-extraction process, chemical synthesis, Enzyme optrode, microbe fermentation method.Solvent-extraction process mainly utilizes wheatgerm, yeast is raw material, although industrialization, technique is backwardness comparatively, and industrial scale is little and yield poorly, and quality product is not high; Chemical synthesis mainly utilizes L-glutamic acid, halfcystine, glycine for raw material, technique is more ripe, but have that cost is high, reactions steps is many, the shortcoming of long reaction time, complicated operation, product is the levo form of gsh and the mixture of dextrorotatory form simultaneously, chemical resolution difficulty, cause product purity thus not high, and environmental pollution very seriously limits the application of the method; Enzyme optrode mainly with L-glutamic acid, semicanal propylhomoserin, glycine for substrate, utilize glutathione synthetase, add ATP synthesis, be in conceptual phase, although enzymatic production process is clear and definite, product purity is higher, complicated operation, and need substrate amino acid and expensive ATP, therefore production cost is very high.
Progress in Glutathione Production by Microbial Fermentation is mainly raw material with carbohydrate, utilize yeast and the metabolism of intestinal bacteria substance in vivo to obtain gsh, there is reaction conditions gentleness, cost is low, the advantages such as production efficiency is fast, its production process is: access seed liquor under aseptic condition, at a certain temperature, aerlbic culture, obtain the fermented liquid containing gsh, by fermented liquid centrifuging (1), obtain the thalline containing gsh, the thalline washed is made bacteria suspension, centrifuging (2), get filtrate and add appropriate material reaction generation precipitation, precipitation is made suspension, centrifuging (3), filtrate is decoloured, concentrated, crystallization, centrifugal (4) obtain gsh and to wet crystal, finally obtain gsh dry product with vacuum-drying.
The various waste water such as the raffinate after the equipment washing waste water in Progress in Glutathione Production by Microbial Fermentation process, fermented liquid are centrifugal, ground flushing waste water, centrifugal crystalline mother solution form gsh fermentative production waste liquid jointly.Various waste liquid directly discharges not only can serious environment pollution, and causes the wasting of resources.Traditional materializing strategy technology, biologic treating technique such as coacervation, evaporation concentration method, absorption method, activated sludge process, biological activated carbon method, biomembrance process etc. are utilized to carry out wastewater treatment, although can be up to standard, but initial cost and operation and maintenance cost high, complex process, castoff regenerative utilization ratio is low, easily produces secondary pollution.
Gsh effluent resource is the optimum strategy of gsh Sustainable Development of Enterprises, but correlative study achievement have not been reported.
Summary of the invention
Technical assignment of the present invention is for above-mentioned the deficiencies in the prior art, provides the application of gsh waste liquid in microorganisms producing.
The further technical assignment of the present invention is to provide the application method of above-mentioned application.
Containing a large amount of nutritive ingredients such as amino acid, carbohydrate, crude protein, nutritive salt in gsh waste liquid, obtain through applicant's lot of experiment validation, gsh waste liquid is applied in microorganisms producing, beyond thought technique effect can be reached.
In microorganisms producing, can use using gsh waste liquid as seed culture medium effective constituent.
In order to adapt to cultivate needs better, gsh waste liquid can be added pure water dilution 5 ~ 20 times, in pH5.5 ~ 7.2,121 DEG C ~ 125 DEG C, sterilizing 0.5 ~ 1h under 0.103MPa ~ 0.168MPa condition, uses as seed culture medium after being cooled to 27 ~ 36 DEG C.
When utilizing seed culture medium to carry out seed tank culture, the substratum charge amount of seeding tank controls at 50% ~ 75% of seeding tank nominal volume, preferably 60%, inoculum size controls 0.5% ~ 2.0% (ratio of the material liquid volume that the seed liquor volume in access seeding tank is in-built with seeding tank) in the actual charge amount of seeding tank, at proper temperature, pH value, air flow and stirring velocity bottom fermentation certain hour, obtain seed tank culture liquid.
The seed liquid used during seed tank culture, obtains by bacterial classification seed liquor preparation method commonly known in the art.Such as, first can carry out slant activation to preservation of bacteria strain, then triangular flask Spawn preparation be carried out to the bacterial classification after activation.When actication of culture, triangular flask Spawn preparation use the type of substratum, composition not to affect the realization of the technology of the present invention effect.
In microorganisms producing, can also use using gsh waste liquid as fermention medium effective constituent.
In order to adapt to cultivate needs better, gsh waste liquid can be added pure water dilution 5 ~ 20 times, in pH5.5 ~ 7.2,121 DEG C ~ 125 DEG C, sterilizing 0.5 ~ 1h under 0.103MPa ~ 0.168MPa condition, uses as fermention medium after being cooled to 27 ~ 36 DEG C.
When utilizing fermention medium to carry out fermentor cultivation, the substratum charge amount of fermentor tank controls at 50% ~ 75% of fermentor tank nominal volume, preferably 60%, the inoculum size of nutrient solution controls 3% ~ 10.0% (ratio of the material liquid volume that the seed liquor volume in access fermentor tank is in-built with fermentor tank) in the actual charge amount of fermentor tank, at proper temperature, pH value, air flow and stirring velocity bottom fermentation certain hour, obtain fermented liquid.
When utilizing above-mentioned seed culture medium and fermention medium to carry out seed tank culture and fermentor cultivation, temperature, pH value, air flow, stirring velocity and fermentation time can according to treating that organism of fermentation self-characteristic is determined, such as:
During bacillus licheniformis seed tank culture, leavening temperature controls at 28 ~ 31 DEG C, preferably 30 DEG C; PH controls 6.8 ~ 7.2, and preferably 7.0; Mixing speed controls at 160 ~ 180r/min, preferred 170r/min; Air flow controls at 1VVm ~ 2VVm, preferred 1.5VVm; Cultivate 18h ~ 26h, preferred 24h.
During bacillus licheniformis fermentor cultivation, the formula of fermentation culture and control condition are with lichens bacillus seed tank culture.
During Candida utilis bacterium seed tank culture, leavening temperature controls at 27 ~ 30 DEG C, preferably 28 DEG C; PH controls 5.8 ~ 6.2, and preferably 6.0; Mixing speed controls at 190 ~ 220r/min, preferred 200r/min; Air flow controls at 1VVm ~ 2VVm, preferred 1.5VVm; Cultivate 20h ~ 26h, preferred 24h.
During Candida utilis bacterium fermentor cultivation, the formula of fermentation culture and control condition are with Candida utilis bacterium seed tank culture.
During Lactobacterium acidophilum seed tank culture, leavening temperature controls at 35 ~ 37 DEG C, preferably 36 DEG C; PH controls 5.6 ~ 6.0, and preferably 5.8; Mixing speed controls at 135 ~ 160r/min, preferred 150r/min; Stuffiness, cultivates 23h ~ 36h, preferred 30h.
During Lactobacterium acidophilum fermentor cultivation, the formula of fermentation culture and control condition are with Lactobacterium acidophilum seed tank culture.
During subtilis seed tank culture, leavening temperature controls at 28 ~ 30 DEG C, preferably 29 DEG C; PH controls 7.0 ~ 7.2, and preferably 7.2; Mixing speed controls at 135 ~ 160r/min, preferred 150r/min; Air flow controls at 1VVm ~ 3VVm, preferred 1.5VVm; Cultivate 24h ~ 36h, preferred 30h.
When fermentation of bacillus subtilis tank is cultivated, the formula of fermentation culture and control condition are with subtilis seed tank culture.
During moral formula Bacterium lacticum subspecies bulgaricus seed tank culture, leavening temperature controls at 28 ~ 32 DEG C, preferably 30 DEG C; PH controls 6.2 ~ 6.5, and preferably 6.3; Mixing speed controls at 130 ~ 155r/min, preferred 150r/min; Stuffiness, cultivates 22h ~ 30h, preferred 28h.
During moral formula Bacterium lacticum subspecies bulgaricus fermentor cultivation, the formula of fermentation culture and control condition are with moral formula Bacterium lacticum subspecies bulgaricus seed tank culture.
During bacillus thuringiensis seed tank culture, leavening temperature controls at 35 ~ 37 DEG C, preferably 36 DEG C; PH controls 7.0 ~ 7.2, and preferably 7.2; Mixing speed controls at 135 ~ 160r/min, preferred 150r/min; Air flow controls at 1VVm ~ 3VVm, preferred 1.5VVm; Cultivate 24h ~ 36h, preferred 32h.
During bacillus thuringiensis fermentor cultivation, the formula of fermentation culture and control condition are with bacillus thuringiensis seed tank culture.
During bacillus amyloliquefaciens seed tank culture, leavening temperature controls at 35 ~ 37 DEG C, preferably 36 DEG C; PH controls 6.8 ~ 7.2, and preferably 7.0; Mixing speed controls at 140 ~ 160r/min, preferred 155r/min; Air flow controls at 1VVm ~ 3VVm, preferred 1.5VVm; Cultivate 24h ~ 36h, preferred 32h.
During bacillus amyloliquefaciens fermentor cultivation, the formula of fermentation culture and control condition are with bacillus amyloliquefaciens seed tank culture.
As preferably, described gsh waste liquid be in Progress in Glutathione Production by Microbial Fermentation process fermented liquid centrifugal after raffinate, the raffinate of the fermented liquid centrifuging (1) namely described in background technology.Its Major Nutrient material and content thereof are: nitrogen 1.48g/100mL ~ 1.55g/100mL; Phosphorus 0.096g/100mL ~ 0.12g/100mL; Potassium 3.0g/100m ~ 3.08g/100mL; Organic 40.35g/100mL ~ 41.0g/100mL; Total amino acid content 4.9 ~ 5.02g/100mL; Crude protein content 14.86 ~ 15.31g/100mL; Soluble solid content 38.0g/100mL ~ 38.86g/100mL, sugar degree 17.85g/100mL ~ 18.24g/100mL, pH5.0 ~ 5.5g/100mL; Moisture 53.50% ~ 55.20g/100mL.
In the described initial pH of seed tank culture base, the initial pH of fermentation tank culture medium, seed tank culture process, in material liquid pH, fermentor cultivation process, material liquid pH controls, by volumetric molar concentration be the sodium hydroxide solution of 1mol/L, volumetric molar concentration is that the hydrochloric acid soln of 1mol/L regulates.
Described bacillus licheniformis, Candida utilis bacterium, Lactobacterium acidophilum, subtilis, these five kinds of microorganisms of moral formula Bacterium lacticum subspecies bulgaricus, former bacterial classification comes from Chinese industrial Microbiological Culture Collection administrative center (CICC), they both can be used as agriculture microbe application, also can be used as livestock feed additive application, also can be used as environmental purification and use.
Described bacillus thuringiensis, these two kinds of microorganisms of bacillus amyloliquefaciens, former bacterial classification comes from Chinese agriculture Microbiological Culture Collection administrative center (ACCC), and they can be used as agriculture microorganism and use.
The application of gsh waste liquid of the present invention in microorganisms producing and application method compared with prior art have following outstanding beneficial effect:
(1) gsh waste liquid is utilized to produce microorganism, change the outdated ideas of gsh enterprise " namely waste is up to standard arranges ", formed " turning waste into wealth ", the ecological production new concept of " zero emission ", both microorganisms producing cost had been reduced, in turn save the high cost of wastewater treatment, energy-conserving and environment-protective, the production potential of material are given full play to, improve the utilization ratio of resource: gsh waste liquid becomes the raw material producing functional microorganism, enterprise is made not only to enjoy major product (gsh) economic benefit, also enjoy byproduct (functional microorganism) economic benefit simultaneously, improve market competitiveness of enterprises, enterprise is made to keep the development of sustainability.
(2) microorganism utilizing gsh waste liquid to produce is animal and plant nutrition regulating agents, it is again the fodder additives of bio-feritlizer and Almightiness type, the fields such as agricultural, livestock industry, environment protection can be widely used in, there is huge social benefit and economic benefit;
(3) in plant husbandry, the microorganism utilizing gsh waste liquid to produce can improve the output of farm crop; Improve quality of agricultural product; Strengthen disease-resistant, the anti-adversity ability of crop; Prevent continuous cropping from making crop continuous cultivation; Continuous use, increases soil fertility; Reducing the drug residue of agricultural-food, extend the shelf life, is the ideal chose building Organic food (OEDC) production base.
(4) in livestock industry, the microbial fermentation solution utilizing gsh waste liquid to produce contains a large amount of carbohydrates of multiple-microorganism synthesis, the physiologically active substance such as amino acid, VITAMIN; Containing multiple digestive ferment and metabolic enzyme, as fodder additives, the robust fibre in feed can be decomposed, improve the digestive utilization ratio of feed; Improve immunity of livestock, alleviate animal diseases; Improve livestock product quality; Improve livestock and poultry surviving rate, laying rate, rate of giving milk, shorten and deliver the cycle for sale; Eliminate feces of livestock and poultry stench, improve the ecological environment.
(5) environment protection aspect; utilize the microorganism that gsh waste liquid is produced; can be used as environmental purification agent; during for the treatment of domestic refuse; can remove refuse odor, purification rubbish seepage flow water quality, reduces the Flies population density of rubbish; also rubbish the like waste can be fermented into biological organic fertilizer, Developing Sustainable Agriculture.
(6) the present invention take functional microorganism as gordian technique point, utilize gsh waste liquid for raw material, by new technology, the novel process of low cost movement expense, the functional microorganism of production diversification is also applied to plant husbandry, livestock breeding industry and environmental protection industry, great induced effect will be produced to this three high pointes coproduction industry, significant.
Embodiment
Specifically to prepare embodiment, experimental example is described in detail below the application of gsh waste liquid of the present invention in microorganisms producing and application method.
In each embodiment, all regulate feed liquid or Medium's PH Value with the hydrochloric acid soln of the sodium hydroxide solution of 1mol/L or 1mol/L.
Preparation embodiment one:
[raw material]
In September, 2014, to take from the gsh waste liquid (fermented liquid namely during Progress in Glutathione Production by Microbial Fermentation centrifugal after raffinate) of Shandong Jin Cheng Bioceuticals Inc. for raw material.Detect through Shandong Academy of Agricultural Science, Institute of Agricultural Resources and Environment, its one-tenth is grouped into: nitrogen 1.51g/100mL; Phosphorus 0.10g/100mL; Potassium 3.05g/100mL; Organic 40.75g/100mL; Soluble solid content 38.4g/100mL, sugar degree 18g/100mL, pH5.11; Moisture 54.34%.
Detect through Ministry of Agriculture's urban agriculture (north) key lab calendar L-8900 high speed automatic analyzer for amino acids, its 18 seed amino acid and crude protein content analysis in table 1:
18 seed amino acids in table 1 gsh waste liquid and crude protein content analysis
[bacterial classification source]
Bacillus licheniformis (providing form: ampoul tube lyophilized powder) is bought to Chinese industrial Microbiological Culture Collection administrative center, numbering CICC23584.
[fermented liquid preparation]
1) bacillus licheniformis actication of culture
Prepared by slant medium: peptone 0.5%, beef leaching thing 0.3%, NaCl1.5%, and agar 1.5%, adjust substratum to pH7.0,121 DEG C, 0.103MPa, sterilizing 30min, prepare slant tube under aseptic condition.
Activation method: under aseptic condition, by the bacillus licheniformis strain inoculation in freezing pipe in inclined-plane, activates 48h under 30 DEG C of conditions, obtains slant strains or strain inclined plane.
2) bacillus licheniformis triangular flask seed liquor preparation
Prepared by triangular flask liquid nutrient medium: peptone 0.5%, beef leaching thing 0.3%, NaCl1.5%, MnSO
4h
2o5ppm, adjusts substratum to initial pH7.0,121 DEG C ~ 125 DEG C, 0.103MPa ~ 0.168MPa, sterilizing 30min.
Triangular flask seed liquor preparation method: under aseptic condition, get 1 ~ 2 ring through step 1) the bacillus licheniformis slant strains that activates, access loading amount is in the 250mL triangular flask of 30mL liquid nutrient medium, be placed in 170r/min shaking table, 30 DEG C of constant temperature culture 24h, obtain bacillus licheniformis triangular flask seed liquor.
3) bacillus licheniformis seed tank culture
Prepared by seeding tank liquid nutrient medium: gsh waste liquid pure water dilutes 10 times, and pH controls 7.0 (regulating with the hydrochloric acid soln of the sodium hydroxide solution of 1mol/L or 1mol/L), and 121 DEG C, 0.103MPa, sterilizing 1h, be cooled to 30 DEG C, for subsequent use.
Seed tank culture process: the volume of seeding tank is 200L, the substratum charge amount of seeding tank controls at 60% of seeding tank nominal volume, namely the charge amount of seeding tank is 120L, aseptically, to learn from else's experience step 2) bacillus licheniformis triangular flask seed liquor (the seed liquor 1.5L altogether of 50 triangular flasks for preparing, namely inoculum size is 1.25% of seeding tank charge amount) access in seeding tank, leavening temperature is made to control at 30 DEG C, in fermenting process, pH controls 7.0, mixing speed controls at 170r/min, air flow controls [to refer to unit time (min) unit seed liquor volume (m at 1.5VVm
3) volume of air (m under the standard state that passes into
3)], cultivate 24h, obtain bacillus licheniformis seed tank culture liquid, at the end of cultivation, recording spore forming rate is 83%, and viable count is 10.37 × 10
8cFU/mL, bacterial contamination rate is 0.26%.
4) ferment tank
Fermentor liquid medium preparing: gsh waste liquid pure water dilutes 10 times, pH7.0,121 DEG C, 0.103MPa, sterilizing 0.5h is cooled to 30 DEG C, for subsequent use.
Fermentor cultivation method: the volume of fermentor tank is 5m
3, the actual charge amount of fermentor tank is 3m
3aseptically, to learn from else's experience step 3) the bacillus licheniformis seed tank culture liquid prepared all accesses in fermentor tank, as calculated, 4% of the actual charge amount volume of inoculum size volume fermentor tank, leavening temperature controls at 30 DEG C, in fermenting process, pH controls 7.0, and mixing speed controls at 170r/min, and air flow controls at 1.5VVm, keep fermentation 24h, obtain fermented liquid.
Fermented liquid index: pH7.02; Bacillus licheniformis bacteria containing amount 12.61 × 10
8cFU/mL; Spore content 73%, heavy metal 2.3mg/Kg; Fungi count 8CFU/mL; Intestinal bacteria 4CFU/100mL; Salmonellas is without detecting.
Preparation embodiment two:
[raw material]
With preparation embodiment one.
[bacterial classification source]
Lactobacterium acidophilum (providing form: ampoul tube lyophilized powder) is bought to Chinese industrial Microbiological Culture Collection administrative center, numbering CICC6074).
[fermented liquid preparation]
1) Lactobacillus acidophilus species's activation
Prepared by slant medium: casein peptone 1%, extractum carnis 1%, yeast powder 0.5%, glucose 0.5%, sodium acetate 0.5%, dibasic ammonium citrate 0.2%, Tween800.1%, K
2hPO
40.2%, MgSO
47H
2o0.02%, CaCO
32%, agar 1.5%, adjust the pH5.8 of substratum, 121 DEG C, 0.103MPa, sterilizing 30min, prepare slant tube under aseptic condition.
Activation method: under aseptic condition, is inoculated in inclined-plane by the Lactobacterium acidophilum in freezing pipe, activates 36h, obtain slant strains or strain inclined plane under 36 DEG C of conditions.
2) Lactobacterium acidophilum triangular flask seed liquor preparation
Prepared by triangular flask liquid nutrient medium: casein peptone 1%, extractum carnis 1%, yeast powder 0.5%, glucose 0.5%, sodium acetate 0.5%, dibasic ammonium citrate 0.2%, Tween800.1%, K
2hPO
40.2%, MgSO
47H
2o0.02%, CaCO
32%, pH5.8,121 DEG C, 0.103MPa, sterilizing 30min.
Triangular flask seed liquor preparation method: under aseptic condition, get a ring through step 1) the Lactobacterium acidophilum bacterium slant strains that activates, access loading amount is in the 500mL triangular flask of 100mL liquid nutrient medium, be placed in 150r/min shaking table, 30h is cultivated in 36 DEG C of sealings, obtains Lactobacterium acidophilum bacterium triangular flask seed liquor.
3) Lactobacterium acidophilum seed tank culture
Prepared by seeding tank liquid nutrient medium: gsh waste liquid pure water dilutes 8 times, pH5.8,121 DEG C, 0.103MPa, and sterilizing 1h is cooled to 36 DEG C, for subsequent use.
Seed tank culture process: the volume of seeding tank is 250L, the charge amount of seeding tank controls at 60% of seeding tank nominal volume, namely the charge amount of seeding tank is 150L, to learn from else's experience step 2) the Lactobacterium acidophilum triangular flask seed liquor (the seed liquor 1.2L altogether of 12 triangular flasks) prepared accesses in seeding tank, namely inoculum size is 0.8% of the actual charge amount of seeding tank, leavening temperature controls at 36 DEG C, pH controls 5.8, mixing speed controls at 150r/min, stuffiness, cultivate 30h, obtain Lactobacterium acidophilum seed tank culture liquid, recording viable count at the end of cultivation is 20.64 × 10
8cFU/mL, bacterial contamination rate is 0.37%.
4) ferment tank
Fermentor liquid medium preparing: gsh waste liquid pure water dilutes 8 times, pH5.8,121 DEG C, 0.103MPa, sterilizing 1h is cooled to 36 DEG C, for subsequent use.
Fermentor cultivation method: the volume of fermentor tank is 5m
3, charge amount is 3m
3aseptically, to learn from else's experience step 3) the Lactobacterium acidophilum seed tank culture liquid prepared all accesses in fermentor tank, and as calculated, inoculum size is 5% of fermentor tank charge amount, leavening temperature controls at 36 DEG C, pH controls 5.8, and mixing speed controls at 150r/min, stuffiness, cultivate 30h, obtain fermented liquid.
Fermented liquid index: pH5.76; Lactobacterium acidophilum bacteria containing amount 25.86 × 10
8cFU/mL; Heavy metal 1.67mg/Kg; Fungi count 11CFU/mL; Intestinal bacteria 7CFU/100mL; Salmonellas is without detecting; Outward appearance, smell have the dark brown liquid of sour fragrance.
Preparation embodiment three:
[raw material]
In December, 2014, to take from the gsh waste liquid (fermented liquid namely during Progress in Glutathione Production by Microbial Fermentation centrifugal after raffinate) of Shandong Jin Cheng Bioceuticals Inc. for raw material.Detect through Shandong Academy of Agricultural Science, Institute of Agricultural Resources and Environment, its one-tenth is grouped into: nitrogen 1.53g/100mL; Phosphorus 0.097g/100mL; Potassium 3.04g/100mL; Organic 40.97g/100mL; Soluble solid content 38.33g/100mL, sugar degree 17.78g/100mL, pH5.22; Moisture 53.94%.
Detect through Ministry of Agriculture's urban agriculture (north) key lab calendar L-8900 high speed automatic analyzer for amino acids, its 18 seed amino acid and crude protein content analysis in table 2:
18 seed amino acids in table 2 gsh waste liquid and crude protein content analysis
[bacterial classification source]
Candida utilis bacterium (providing form: ampoul tube lyophilized powder) is bought to Chinese industrial Microbiological Culture Collection administrative center, numbering CICC31188).
[fermented liquid preparation]
1) Candida utilis bacterium actication of culture
Prepared by slant medium: 5 ° of B é worts, agar 1.5%, natural pH, 121 DEG C, 0.103MPa, sterilizing 30min, prepare slant tube under aseptic condition.
Activation method: under aseptic condition, is inoculated in inclined-plane by the Candida utilis bacterium in freezing pipe, activates 48h, obtain slant strains or strain inclined plane under 28 DEG C of conditions.
2) Candida utilis bacterium triangular flask seed liquor preparation
Prepared by triangular flask liquid nutrient medium: 5 ° of B é worts, natural pH, 121 DEG C, 0.103MPaMPa, sterilizing 30min.
Triangular flask seed liquor preparation method: under aseptic condition, get a ring through step 1) the Candida utilis bacterium slant strains that activates, access loading amount is in the 250mL triangular flask of 50mL liquid nutrient medium, be placed in 200r/min shaking table, 28 DEG C of constant temperature culture 24h, obtain Candida utilis bacterium triangular flask seed liquor.
3) Candida utilis bacterium seed tank culture
Prepared by seeding tank liquid nutrient medium: gsh waste liquid pure water dilutes 12 times, pH6.0,121 DEG C, 0.103MPa, and sterilizing 1h is cooled to 28 DEG C, for subsequent use.
Seed tank culture process: seeding tank volume 250L, the charge amount of seeding tank controls at 60% of seeding tank nominal volume, namely the charge amount of seeding tank is 150L, aseptically, to learn from else's experience step 2) the Candida utilis bacterium triangular flask seed liquor (the seed liquor 3L altogether of 60 triangular flasks) prepared, namely inoculum size is 2% of seeding tank charge amount, in access seeding tank, leavening temperature controls at 28 DEG C, pH controls 6.0, mixing speed controls at 200r/min, air flow controls at 1.5VVm, cultivate 24h, obtain Candida utilis bacterium seeding tank nutrient solution, recording viable count at the end of cultivation is 5.33 × 10
8cFU/mL, bacterial contamination rate is 0.36%.
4) ferment tank
Fermentor liquid medium preparing: gsh waste liquid pure water dilutes 12 times, pH6.0,121 DEG C, 0.103MPa, sterilizing 1h is cooled to 28 DEG C, for subsequent use.
Fermentor cultivation method: the volume of fermentor tank is 5m
3, charge amount is 3m
3, aseptically, step 3 of learning from else's experience) and the Candida utilis bacterium seeding tank nutrient solution prepared all accesses in fermentor tank, and as calculated, inoculum size is 5% of fermentor tank charge amount, and leavening temperature controls at 28 DEG C, and pH controls 6.0.Leavening temperature controls at 28 DEG C, and pH controls 6.0, and mixing speed controls at 200r/min, and air flow controls at 1.5VVm, cultivates 24h.
Fermented liquid index: pH6.04; Candida utilis bacterium bacteria containing amount 7.93 × 10
8cFU/mL; Heavy metal 0.4mg/Kg; Fungi count 6CFU/mL; Intestinal bacteria 7CFU/100mL; Salmonellas is without detecting.
Preparation embodiment four:
[raw material]
With preparation embodiment three.
[bacterial classification source]
Subtilis (providing form: ampoul tube lyophilized powder) is bought to Chinese industrial Microbiological Culture Collection administrative center, numbering CICC20872).
[fermented liquid preparation]
1) Bacillus subtilis strain activation
Prepared by slant medium: peptone 0.5%, and beef leaching thing 0.3%, NaCl0.5%, agar 1.5%, pH7.0,121 DEG C, 0.103MPa, sterilizing 30min, prepare slant tube under aseptic condition.
Activation method: under aseptic condition, is inoculated in inclined-plane by the subtilis in freezing pipe, activates 24h, obtain slant strains or strain inclined plane under 29 DEG C of conditions.
2) subtilis triangular flask seed liquor preparation
Prepared by triangular flask liquid nutrient medium: peptone 0.5%, beef leaching thing 0.3%, NaCl0.5%, MnSO
4h
2o5ppm, pH7.2,121 DEG C, 0.103MPa, sterilizing 30min.
Triangular flask seed liquor preparation method: under aseptic condition, get a ring through step 1) the subtilis slant strains that activates, access loading amount is in the 250mL triangular flask of 50mL liquid nutrient medium, be placed in 150r/min shaking table, 29 DEG C of constant temperature culture 30h, obtain subtilis triangular flask seed liquor.
3) subtilis seed tank culture
Prepared by seeding tank liquid nutrient medium: gsh waste liquid pure water dilutes 12 times, pH7.2,121 DEG C, 0.103MPaMPa, and sterilizing 1h is cooled to 29 DEG C, for subsequent use.
Seed tank culture method: seeding tank volume 250L, the charge amount of seeding tank controls at 60% of seeding tank nominal volume, namely the charge amount of seeding tank is 150L, aseptically, to learn from else's experience step 2) the subtilis triangular flask seed liquor (the seed liquor 1.5L altogether of 30 triangular flasks) prepared accesses in seeding tank, namely inoculum size is 1% of the actual charge amount of seeding tank, leavening temperature controls at 29 DEG C, pH controls 7.2, mixing speed controls at 150r/min, air flow controls at 1.5VVm, cultivate 30h, obtain subtilis seed tank culture liquid, recording spore forming rate at the end of cultivation is 93.2%, bacteria containing amount is 11.71 × 10
8cFU/mL, bacterial contamination rate is 0.2%.
4) ferment tank
Fermentor liquid medium preparing: gsh waste liquid pure water dilutes 12 times, pH7.2,121 DEG C, 0.103MPaMPa, sterilizing 1h is cooled to 29 DEG C, for subsequent use.
Fermentor cultivation method: the volume of fermentor tank is 5m
3, charge amount is 3m
3aseptically, to learn from else's experience step 3) the bacillus licheniformis seed tank culture liquid prepared all accesses in fermentor tank, as calculated, inoculum size controls in 5% of the actual charge amount of fermentor tank, and leavening temperature controls at 29 DEG C, pH controls 7.2, mixing speed controls at 150r/min, and air flow controls at 1.5VVm, cultivates 30h.
Fermented liquid index: recording spore forming rate is 92.8%, subtilis bacteria containing amount 12.77 × 10
8cFU/mL; Heavy metal 0.20mg/Kg; Fungi count 10CFU/mL; Intestinal bacteria 6CFU/100mL; Salmonellas is without detecting.
Preparation embodiment five:
[raw material]
In May, 2015, to take from the gsh waste liquid (fermented liquid namely during Progress in Glutathione Production by Microbial Fermentation centrifugal after raffinate) of Shandong Jin Cheng Bioceuticals Inc. for raw material.Detect through Shandong Academy of Agricultural Science, Institute of Agricultural Resources and Environment, its one-tenth is grouped into: nitrogen 1.49g/100mL; Phosphorus 0.102g/100mL; Potassium 3.0g/100mL; Organic 40.51g/100mL; Soluble solid content 38.56g/100mL, sugar degree 18.18g/100mL, pH5.12; Moisture 55.04%.
Detect through Ministry of Agriculture's urban agriculture (north) key lab calendar L-8900 high speed automatic analyzer for amino acids, its 18 seed amino acid and crude protein content analysis in table 3:
18 seed amino acids in table 3 gsh waste liquid and crude protein content analysis
[bacterial classification source]
Lactobacillus delbruockii subspecies bulgaricus (providing form: ampoul tube lyophilized powder) is bought to Chinese industrial Microbiological Culture Collection administrative center, numbering CICC6100).
[fermented liquid preparation]
1) lactobacillus delbrueckii subspecies bulgaricus actication of culture
Prepared by slant medium: casein peptone (trysinization) 10g, broth extract 10g, yeast powder 5g, glucose 20g, tween 80 1g, K
2hPO
42g, sodium-acetate 5g, dibasic ammonium citrate 2g, MgSO
47H
2o0.2g, MnSO4H
2o0.05g, agar 15g, pH6.3,121 DEG C, 0.103MPa, sterilizing 30min, prepare slant tube under aseptic condition.
Activation method: under aseptic condition, is inoculated in inclined-plane by the lactobacillus delbruockii subspecies bulgaricus in freezing pipe, activates 28h, obtain slant strains or strain inclined plane under 30 DEG C of conditions.
2) lactobacillus delbruockii subspecies bulgaricus triangular flask seed liquor preparation
Prepared by triangular flask liquid nutrient medium: casein peptone (trysinization) 10g, broth extract 10g, yeast powder 5g, glucose 20g, tween 80 1g, K
2hPO
42g, sodium-acetate 5g, dibasic ammonium citrate 2g, MgSO
47H
2o0.2g, MnSO4H
2o0.05g, pH6.3,121 DEG C, 0.103MPa, sterilizing 30min.
Triangular flask seed liquor preparation method: under aseptic condition, get a ring through step 1) the lactobacillus delbruockii subspecies bulgaricus slant strains that activates, access loading amount is in the 250mL triangular flask of 50mL liquid nutrient medium, be placed in 150r/min shaking table, 30 DEG C of constant temperature culture 28h, obtain lactobacillus delbruockii subspecies bulgaricus triangular flask seed liquor.
3) lactobacillus delbruockii subspecies bulgaricus seed tank culture
Prepared by seeding tank liquid nutrient medium: gsh waste liquid pure water dilutes 15 times, pH6.3,121 DEG C, 0.103MPa, and sterilizing 1h is cooled to 30 DEG C, for subsequent use.
Seed tank culture process: the volume of seeding tank is 250L, the charge amount of seeding tank controls at 60% of seeding tank nominal volume, namely the charge amount of seeding tank is 150L, to learn from else's experience step 2) the lactobacillus delbruockii subspecies bulgaricus triangular flask seed liquor (the seed liquor 2.25L altogether of 45 triangular flasks) prepared accesses in seeding tank, as calculated, inoculum size controls in 1.5% of the actual charge amount of seeding tank, leavening temperature controls at 30 DEG C, pH controls 6.3, mixing speed controls at 150r/min, stuffiness, cultivate 28h, obtain lactobacillus delbruockii subspecies bulgaricus seed tank culture liquid, recording viable count at the end of cultivation is 18.44 × 108CFU/mL, bacterial contamination rate is 0.5%.
4) ferment tank
Fermentor liquid medium preparing: gsh waste liquid pure water dilutes 15 times, pH6.3,121 DEG C, 0.103MPa, sterilizing 1h is cooled to 30 DEG C, for subsequent use.
Fermentor cultivation method: the volume of fermentor tank is 7.5m
3, charge amount is 4.5m
3aseptically, to learn from else's experience step 3) the lactobacillus delbruockii subspecies bulgaricus nutrient solution prepared all accesses in fermentor tank, and as calculated, inoculum size controls in 3% of the actual charge amount of fermentor tank, leavening temperature controls at 30 DEG C, pH controls 6.3, and mixing speed controls at 150r/min, stuffiness, cultivate 28h, obtain fermented liquid.
Fermented liquid index: pH6.22; Lactobacillus delbruockii subspecies bulgaricus bacteria containing amount 22.31 × 10
8cFU/mL; Heavy metal 2mg/Kg; Fungi count 10CFU/mL; Intestinal bacteria 5CFU/100mL; Salmonellas is without detecting; Outward appearance, smell have the dark brown liquid of sour fragrance.
Preparation embodiment six:
[raw material]
With preparation embodiment five.
[bacterial classification source]
Bacillus thuringiensis (providing form: ampoul tube lyophilized powder) is bought to Chinese industrial Microbiological Culture Collection administrative center, numbering CICC21298.
[fermented liquid preparation]
1) bacillus thuringiensis actication of culture
Prepared by slant medium: yeast extract 0.5g, N.F,USP MANNITOL 20.0g, KH2PO40.2g, K2HPO40.8g, MgSO47H2O0.2g, CaSO42H2O0.1g, FeCl3 trace, Na2MoO42H2O trace, agar 15.0g, distilled water 1.0L, pH7.2,121 DEG C, 0.103MPa, sterilizing 30min, prepares slant tube under aseptic condition.
Activation method: under aseptic condition, by the bacillus thuringiensis strain inoculation in freezing pipe in inclined-plane, activates 32h under 36 DEG C of conditions, obtains slant strains or strain inclined plane.
2) bacillus thuringiensis triangular flask seed liquor preparation
Prepared by triangular flask liquid nutrient medium: yeast extract 0.5g, N.F,USP MANNITOL 20.0g, KH2PO40.2g, K2HPO40.8g, MgSO47H2O0.2g, CaSO42H2O0.1g, FeCl3 trace, Na2MoO42H2O trace, distilled water 1.0L, pH7.2,121 DEG C, 0.103MPa, sterilizing 30min.
Triangular flask seed liquor preparation method: under aseptic condition, get 1 ~ 2 ring through step 1) the bacillus licheniformis slant strains that activates, access loading amount is in the 500mL triangular flask of 100mL liquid nutrient medium, be placed in 170r/min shaking table, control pH7.2,36 DEG C of constant temperature culture 32h, obtain bacillus thuringiensis triangular flask seed liquor.
3) bacillus thuringiensis seed tank culture
Prepared by seeding tank liquid nutrient medium: gsh waste liquid pure water dilutes 10 times, and pH controls 7.2,121 DEG C, 0.103MPa, and sterilizing 1h is cooled to 30 DEG C, for subsequent use.
Seed tank culture process: the volume of seeding tank is 250L, the charge amount of seeding tank controls at 60% of seeding tank nominal volume, namely the charge amount of seeding tank is 150L, aseptically, to learn from else's experience step 2) the bacillus thuringiensis triangular flask seed liquor (the seed liquor 3L altogether of 30 triangular flasks) prepared accesses in seeding tank, namely the seed liquor volume accessed is 2% of seeding tank charge amount volume, leavening temperature controls at 36 DEG C, in fermenting process, pH controls 7.2, mixing speed controls at 150r/min, air flow controls at 1.5VVm, cultivate 24h, obtain bacillus thuringiensis seed tank culture liquid, at the end of cultivation, recording spore forming rate is 80%, viable count is 9.42 × 10
8cFU/mL, bacterial contamination rate is 0.43%.
4) ferment tank
Fermentor liquid medium preparing: gsh waste liquid pure water dilutes 10 times, pH7.2,121 DEG C, 0.103MPa, sterilizing 0.5h is cooled to 36 DEG C, for subsequent use.
Fermentor cultivation method: the volume of fermentor tank is 3m
3, the actual charge amount of fermentor tank is 1.8m
3aseptically, to learn from else's experience step 3) the bacillus thuringiensis seed tank culture liquid prepared all accesses in fermentor tank, and as calculated, the seed liquor volume namely accessed is 8.5% of fermentor tank charge amount volume, leavening temperature controls at 36 DEG C, in fermenting process, pH controls 7.2, and mixing speed controls at 150r/min, and air flow controls at 1.5VVm, keep fermentation 32h, obtain fermented liquid.
Fermented liquid index: pH7.18; Bacillus thuringiensis bacteria containing amount 11.27 × 10
8cFU/mL; Spore content 76.3%, heavy metal 3mg/Kg; Fungi count 6CFU/mL; Intestinal bacteria 5CFU/100mL; Salmonellas is without detecting.
Preparation embodiment seven:
[raw material]
With preparation embodiment five.
[bacterial classification source]
Bacillus amyloliquefaciens (providing form: ampoul tube lyophilized powder) is bought to Chinese agriculture Microbiological Culture Collection administrative center, numbering ACCC19745.
[fermented liquid preparation]
1) bacillus amyloliquefaciens actication of culture
Prepared by slant medium: peptone 5.0g, and beef leaching thing 3.0g, NaCl5.0g, agar 15.0g, distilled water 1.0L, pH7.0,121 DEG C, 0.103MPa, sterilizing 30min, prepare slant tube under aseptic condition.
Activation method: under aseptic condition, by the bacillus amyloliquefaciens strain inoculation in freezing pipe in inclined-plane, activates 32h under 36 DEG C of conditions, obtains slant strains or strain inclined plane.
2) bacillus amyloliquefaciens triangular flask seed liquor preparation
Prepared by triangular flask liquid nutrient medium: peptone 5.0g, beef leaching thing 3.0g, NaCl5.0g, agar 15.0g, distilled water 1.0L, pH7.0,121 DEG C, 0.103MPa, sterilizing 30min.
Triangular flask seed liquor preparation method: under aseptic condition, get 1 ~ 2 ring through step 1) the bacillus licheniformis slant strains that activates, access loading amount is in the 250mL triangular flask of 60mL liquid nutrient medium, be placed in 155r/min shaking table, control pH7.0,36 DEG C of constant temperature culture 32h, obtain bacillus amyloliquefaciens triangular flask seed liquor.
3) bacillus amyloliquefaciens seed tank culture
Prepared by seeding tank liquid nutrient medium: gsh waste liquid pure water dilutes 10 times, and pH controls 7.0,121 DEG C, 0.103MPa, and sterilizing 1h is cooled to 36 DEG C, for subsequent use.
Seed tank culture process: the volume of seeding tank is 300L, the charge amount of seeding tank controls at 60% of seeding tank nominal volume, namely the charge amount of seeding tank is 180L, aseptically, to learn from else's experience step 2) (seed liquor of 30 triangular flasks altogether 1.8L accesses in seeding tank for the bacillus amyloliquefaciens triangular flask seed liquor prepared, namely inoculum size volume is 1% of seeding tank charge amount volume, leavening temperature controls at 36 DEG C, in fermenting process, pH controls 7.0, mixing speed controls at 155r/min, air flow controls at 1.5VVm, cultivate 32h, obtain bacillus amyloliquefaciens seed tank culture liquid, at the end of cultivation, recording spore forming rate is 80%, viable count is 8.36 × 10
8cFU/mL, bacterial contamination rate is 0.54%.
4) ferment tank
Fermentor liquid medium preparing: gsh waste liquid pure water dilutes 10 times, pH7.0,121 DEG C, 0.103MPa, sterilizing 0.5h is cooled to 36 DEG C, for subsequent use.
Fermentor cultivation method: the volume of fermentor tank is 5m
3, the actual charge amount of fermentor tank is 3m
3aseptically, to learn from else's experience step 3) the bacillus amyloliquefaciens seed tank culture liquid prepared all accesses in fermentor tank, and as calculated, inoculum size is 6% of fermentor tank charge amount volume, leavening temperature controls at 36 DEG C, in fermenting process, pH controls 7.0, and mixing speed controls at 155r/min, and air flow controls at 1.5VVm, keep fermentation 32h, obtain fermented liquid.
Fermented liquid index: pH7.04; Bacillus amyloliquefaciens bacteria containing amount 10.02 × 10
8cFU/mL; Spore content 81.2%, heavy metal 4.3mg/Kg; Fungi count 9CFU/mL; Intestinal bacteria 8CFU/100mL; Salmonellas is without detecting.
Preparation embodiment eight (preparation of compound liquid microbe bacterial manure)
1) mixing of fermented liquid
By embodiment one, embodiment seven step 4) the lichen bacillus ferments liquid of preparing, bacillus amyloliquefaciens fermented liquid carry out the mixing of 1:1 equal-volume;
2) preparation of compound microbial bacterial manure
By 1) mixed fermented liquid squeezes into hold-up vessel, add pure water, dilute 10 times, after filtering, (filling machine 120 barrels/min, 5L/ bucket is washed finally by full-automatic filling production lines, 10 barrels/case), last 1 hour 45 minutes, obtain liquid-type barreled functional microorganism product 1250 case, product viable count concentration is 2.3 × 10
8cFU/mL, the quality guaranteed period is 1 year, inspection bacterial classification survival rate 81.34% when checking bacterial classification survival rate to be 85.79%, 1 year when 6 months.
Preparation embodiment nine (preparation of compound liquid microbe fodder additives):
1) ferment tank
Fermentor liquid medium preparing: gsh waste liquid pure water dilutes 10 times, pH6.2,121 DEG C, 0.103MPa, sterilizing 1h is cooled to 30 DEG C, for subsequent use.
Fermentor cultivation method: the volume of fermentor tank is 15m
3charge amount is 9000L, namely the volume of charge amount accounts for 60% of fermenter volume, aseptically, get preparation embodiment two, three, four step 3) the Lactobacterium acidophilum seed tank culture liquid 150L, Candida utilis bacterium seeding tank nutrient solution 150L, the subtilis seed tank culture liquid 150L that prepare, amount to 450L and access in fermentor tank, namely the cumulative volume of three seed tank culture liquid accounts for 5% of fermentor tank charge amount volume, and leavening temperature controls at 30 DEG C, and pH controls 6.2.When fermenting beginning, closing simultaneously and stirring and all valves, after 24h, opening purging valve, the switch amplitude of purging valve is regulated to control fermentor tank tank pressure 0.025MPa, within every 4 hours later, open simultaneously and stir and ventilation, stirring and aeration time are 30min, and purging valve is open mode always, by regulating the switch amplitude of purging valve, fermentor tank tank pressure is remained on 0.025MPa, and air flow controls at 1.5VVm, and mixing speed controls at 200r/min, keep fermentation 36h, obtain fermented liquid.Fermented liquid index: pH5.72; Lactobacterium acidophilum bacteria containing amount 26.01 × 108CFU/mL; Candida utilis bacterium bacteria containing amount 8.07 × 108CFU/mL; Subtilis bacteria containing amount 12.84 × 108CFU/mL; Total viable count 46.92 × 108CFU/mL; Heavy metal 0.5mg/Kg; Fungi count 8CFU/mL; Intestinal bacteria 4CFU/100mL; Salmonellas is without detecting; Outward appearance, smell have the dark brown liquid of sour fragrance;
2) through 1) composite bacteria liquid that ferments squeezes in hold-up vessel, 20 times are diluted with pure water, obtain the bacterium liquid after 18.9 tons of dilutions, by blending and mixing, filter after through full-automatic filling production lines (washing filling machine 150 bottles/min, 2L/ bottle, 12 bottles/case), obtaining the total viable count of liquid-type bottled functional microorganism product is 2.4 × 108CFU/mL, quality guaranteed period is 1 year, inspection bacterial classification survival rate 82.67% when checking bacterial classification survival rate to be 86.14%, 1 year when 6 months.
Preparation embodiment ten (preparation of compound pulvis microorganism feed addictive):
1) horizontal-type biaxial paddle mixer model SSHJ-4, full volumetric 4m
3, often criticizing combined amount is 2 tons, 1.7 tons, the wheat bran (water content 8.5%) wherein loaded, fermented liquid 0.3 ton (preparation embodiment nine step one gained), mixing time 60s is 22% from mixing machine material moisture out, subtilis bacteria containing amount 2.69 × 10
8cFU/mL; Candida utilis bacterium bacteria containing amount 2.88 × 10
8cFU/mL; Lactobacterium acidophilum bacteria containing amount 4.61 × 10
8cFU/mL; Total viable count 10.18 × 10
8cFU/mL;
2) load in pallet by from mixing machine material out, the material depth that each pallet fills is 4cm, delivered to by pallet on drying shed stereoscopic frame, drying shed 12m × 12m × 3m, every 1h turning over materials once, drying shed temperature controls at 37 DEG C, drying time 20h, obtain 1.7 tons of dried solid functional microorganism products, outward appearance is the powder product of sour fragrance, moisture is 8.5%, subtilis bacteria containing amount 2.17 × 10
8cFU/mL; Candida utilis bacterium bacteria containing amount 2.38 × 10
8cFU/mL; Lactobacterium acidophilum bacteria containing amount 4.31 × 10
8cFU/mL; Total viable count 8.86 × 10
8cFU/mL; Before comparing oven dry, total viable count reduces by 12.97%, heavy metal 0.3mg/Kg; Mould 6CFU/mL; Intestinal bacteria 4CFU/100mL; Salmonellas is without detecting.Through Full-automatic quantitative Production line of packing machine, 200 bags/minute, 2Kg/ bag, 12 bags/case, the quality guaranteed period is 1 year, inspection bacterial classification survival rate 86.23% when checking bacterial classification survival rate to be 92.58%, 1 year when 6 months.
Experimental example one;
That produces with other companies of preparing embodiment eight gained compound liquid microbe bacterial manure and commercially available purchase carries out simultaneous test.
Test site: be located at Jinan City's Feature of Agricultural New High Technology Industry Demonstration Garden romaine lettuce booth
Test period: on October 08th, 2014 to December 25 and on March 7th, 2015 were to May 18.On October 08th, 2014 and on March 7th, 2015 carry out seeding and seedling raising respectively; On November 2nd, 2014 and on March 31st, 2015 carry out field planting respectively, and plantation density is row, strain 20 ~ 25 centimetres; On December 23rd, 2014 and on May 21st, 2015 gather respectively.
For examination romaine lettuce kind: the large fast-growing of the U.S..
For examination bacterial manure: (strain combination is bacillus amyloliquefaciens, Bacillus licheniformis to the compound liquid microbe bacterial manure of the embodiment of the present invention eight gained, and total viable count is 2.3 × 10 after testing
8cUF/mL); (strain combination is bacillus amyloliquefaciens, Bacillus licheniformis to China's rain liquid microbe bacterial manure, and total viable count is 2.27 × 10 after testing
8cUF/mL), from Hua Yu bio tech ltd, Cangzhou.
Test design:
A, B represent respectively for examination bacterial manure for the compound liquid microbe bacterial manure of the embodiment of the present invention eight gained, magnificent 2 kinds, rain liquid microbe bacterial manure, often kind of bacterial manure arranges 3 concentration for the treatment of, be respectively 1:100,1:200,1:500 ratio of the water yield (the bacterial manure dosage with), with clear water in contrast (CK), amount to 7 process, each process setting refers to table 4.Each process plot area 30m
2, random district group arranges, and repeats for 3 times.Before nursery, seed is respectively with each process bacterial manure concentration seed soaking 1.5h, and after seedling is delayed in romaine lettuce field planting, start foliage-spray microbial-bacterial fertilizer process, spray once every 1 week, the time of spraying is point in afternoon 3, sprays 5 times altogether.Spray bacterial manure and adopt atomizer spraying method, the bacterial manure of every community adapted 15 kilograms of stand-by concentration, then evenly sprays to romaine lettuce blade.
Table 4 respectively processes title
Mensuration project and method:
1) mensuration of romaine lettuce morphological index and output
The mensuration of romaine lettuce overground part growth indexes: after planting 3d starts the seedling rate of the romaine lettuce recording the process of different microorganisms bacterial manure; Within after field planting 45 days, get 30 strain romaine lettuce at random, clean with distilled water flushing, suck surface-moisture, measure its overground part growth indexes, comprise plant height, leaf area, overground part fresh weight, overground part dry weight, per mu yield.Plant height is that root Lian Chu grows apogee distance to time slice; Overground part and underground part complete respectively at 105 DEG C, and 75 DEG C dry to constant weight, and measure dry mass.
2) mensuration of lettuce quality index
Collection period, often process and get 30 strain romaine lettuce at random, yellow for rotten leaf leaf is removed, other blades is shredded mixing, measures the content of soluble sugar, soluble proteins, vitamins C, total free aminoacids.The mensuration Bian anthrone colorimetry of soluble sugar content; The mensuration of soluble protein adopts Coomassie brilliant G-250 staining; Vitamins C adopts spectrophotometer method to measure; Total free aminoacids adopts ninhydrin solution determination of color.
Test-results:
1) in the 10th after planting day, the seedling rate of two kinds of each concentration of bacterial manure all reaches more than 85%, shows that the gas permeability of soil after microbial-bacterial fertilizer process and water-retentivity are all conducive to emerging of romaine lettuce seed, effectively can shorten seedling raise period; The porosity of A2 (the compound liquid microbe bacterial manure 1:200 clear water dilution with embodiment eight gained) is maximum, and ventilation property is best, so have the greatest impact to nursery, its seedling rate is up to 98.53%, improves 4.5% compared with clear water control group; The overall porosity of B2 (magnificent rain liquid microbe bacterial manure 1:200 clear water dilution) takes second place, corresponding seedling rate lower than A2 (the compound liquid microbe bacterial manure 1:200 clear water dilution with embodiment eight gained), but also improves 0.58% than clear water control group.
The impact of two kinds of microbial-bacterial fertilizer different concns process on Growth of Lettuce has certain difference, compared with clear water control group, process A2, B2 all significantly can increase the plant height of greenhouse romaine lettuce, leaf area, overground part fresh weight, overground part dry weight, per mu yield, and facilitation effect shows as A2 > B2, wherein A2 (concentration with the compound liquid microbe bacterial manure 1:200 of embodiment eight gained) treatment effect is best, make plant height respectively, overground part fresh weight, overground part dry weight, per mu yield, leaf area ratio clear water control group adds 27.21%, 22.53%, 61.49%, 23.70%, 15.88%, B2 (magnificent rain liquid microbe bacterial manure 1:200 clear water dilution) treatment effect takes second place, make plant height respectively, overground part fresh weight, overground part dry weight, per mu yield, leaf area ratio clear water control group adds 23.0%, 19.87%, 52.06%, 20.44%, 13.19%, result difference compared with clear water control group of other concentration is not remarkable.
Experimental example two:
The compound microbial powder feed additive obtained to prepare embodiment ten carries out contrast experiment.
Test method: term, parity, kind is identical, the colour of skin is close 50 age in days DLY three way cross weanling pig 75 are selected in test, test period 125 days, be divided into three groups at random, often organize 25, be equally divided into three circles to raise, the pig weight no significant difference (P > 0.05) often organized.Three groups of feeding piglets are in same building pig house, and envrionment conditions is identical, raise two weeks phases in advance, raise in advance in the phase and complete castration, expelling parasite, immunization inocultation; Microbial feed additive powder agent product (purchased from Harbin He Mu Science and Technology Ltd.) three process is herded, control group: complete diet pellet by the compound microbial powder feed additive treatment group that contrast is established in test, interpolation preparation embodiment ten obtains, interpolation China; Test group: microbial feed additive powder agent product is herded by complete diet pellet+0.3% China;
Free choice feeding, measures quantity-unlimiting, and automatic drinking bowl is drunk water.
Indicator-specific statistics: accurate recording day weight gain, feed-weight ratio, protein digestibility, organic matter digestibility, Incidence of Diarrhea, diarrhoea recurrence rate, compares testing data row.
Effect: the compound microbial powder feed additive effect that complete diet pellet+0.3% preparation embodiment ten obtains is best, complete diet pellet+0.3% China herds microbial feed additive powder agent product and takes second place, the compound microbial powder feed additive day weight gain that wherein preparation embodiment ten obtains improves 8.25% than control group, and feed-weight ratio test group reduces by 5.32% than control group; Incidence of Diarrhea is starkly lower than control group, low by 23.1%, and Incidence of Diarrhea 0%, and control group recurrence rate is 23%.Difference is P < (0.01) all extremely significantly; Protein digestibility improves 7% than control group, and organic matter digestibility improves 6.32% than control group.
The compound microbial powder feed additive that the embodiment of the present invention ten obtains is nontoxic, have no side effect, pollution-free, safe and reliable, add in Diet For Finishing Pig.On the one hand, this additive effectively can improve the utilization ratio of feed, day weight gain can be increased, this is except solid type functional microorganism product itself is containing a large amount of viable bacterias, utilized by animal consumption, play outside the material making in feed part not easily absorb degraded, also relevant with the amount of activated nutritive substance in solid type functional microorganism product; On the other hand, this additive has the obvious livestock and poultry body that strengthens to the effect of the resistibility of epidemic disease, particularly intestinal bacteriosis, this may define dominant microflora due to probiotics in solid type functional microorganism product together with the probiotics in enteron aisle, amount reproduction, by competitive inhibition, the breeding of harmful bacteria is suppressed, prevents the generation of bacterial epidemic disease, thus reach the effect strengthening disease resistance.
Claims (10)
1. the application of gsh waste liquid in microorganisms producing.
2. application according to claim 1, is characterized in that: described gsh waste liquid be in Progress in Glutathione Production by Microbial Fermentation process fermented liquid centrifugal after raffinate.
3. the application method of gsh waste liquid in microorganisms producing, is characterized in that: in microorganisms producing, uses using gsh waste liquid as seed culture medium effective constituent.
4. application method according to claim 3, it is characterized in that: gsh waste liquid is added pure water dilution 5 ~ 20 times, in pH5.5 ~ 7.2,121 DEG C ~ 125 DEG C, sterilizing 0.5 ~ 1h under 0.103MPa ~ 0.168MPa condition, uses as seed culture medium after being cooled to 27 ~ 36 DEG C.
5. application method according to claim 4, it is characterized in that: when utilizing seed culture medium to carry out seed tank culture, the substratum charge amount of seeding tank controls at 50% ~ 75% of seeding tank nominal volume, inoculum size controls in 0.5% ~ 2% of the actual charge amount of seeding tank, at proper temperature, pH value, air flow and stirring velocity bottom fermentation certain hour, obtain seed tank culture liquid.
6. the application method of gsh waste liquid in microorganisms producing, is characterized in that: in microorganisms producing, uses using gsh waste liquid as fermention medium effective constituent.
7. application method according to claim 6, it is characterized in that: gsh waste liquid is added pure water dilution 5 ~ 20 times, in pH5.5 ~ 7.2,121 DEG C ~ 125 DEG C, sterilizing 0.5 ~ 1h under 0.103MPa ~ 0.168MPa condition, uses as fermention medium after being cooled to 27 ~ 36 DEG C.
8. application method according to claim 7, it is characterized in that: when utilizing fermention medium to carry out fermentor cultivation, the substratum charge amount of fermentor tank controls at 50% ~ 75% of fermentor tank nominal volume, inoculum size controls in 3% ~ 6.0% of the actual charge amount of fermentor tank, at proper temperature, pH value, air flow and stirring velocity bottom fermentation certain hour, obtain fermented liquid.
9. the application method according to claim 3,4,5,6,7 or 8, is characterized in that, described gsh waste liquid be in Progress in Glutathione Production by Microbial Fermentation process fermented liquid centrifugal after raffinate.
10. application method according to claim 9, it is characterized in that, described microorganism comprises bacillus licheniformis, Candida utilis bacterium, Lactobacterium acidophilum, subtilis, moral formula Bacterium lacticum subspecies bulgaricus, bacillus thuringiensis, bacillus amyloliquefaciens.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510487030.4A CN105087432A (en) | 2015-08-10 | 2015-08-10 | Application of glutathione waste liquid in microorganism production and application method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510487030.4A CN105087432A (en) | 2015-08-10 | 2015-08-10 | Application of glutathione waste liquid in microorganism production and application method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105087432A true CN105087432A (en) | 2015-11-25 |
Family
ID=54568768
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510487030.4A Pending CN105087432A (en) | 2015-08-10 | 2015-08-10 | Application of glutathione waste liquid in microorganism production and application method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105087432A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105434321A (en) * | 2015-12-22 | 2016-03-30 | 温州任和教育科技有限责任公司 | Whitening freckle-removing mask and preparation method thereof |
| CN112430163A (en) * | 2020-11-26 | 2021-03-02 | 宁夏大学 | Biological fertilizer for relieving continuous cropping obstacle of watermelon with pressed sand |
| CN113637624A (en) * | 2021-09-01 | 2021-11-12 | 珠海瑞德林生物有限公司 | Recombinant bacillus subtilis, application thereof and method for producing tetrahydropyrimidine by using waste water generated in glutathione synthesis by enzyme method |
-
2015
- 2015-08-10 CN CN201510487030.4A patent/CN105087432A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 刘娟等: "发酵法生产谷胱甘肽的研究进展", 《微生物学报》 * |
| 秦麟源: "《新编废水生物处理》", 30 September 2011 * |
| 陈坚等: "微生物发酵法生产谷胱甘肽", 《无锡轻工大学学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105434321A (en) * | 2015-12-22 | 2016-03-30 | 温州任和教育科技有限责任公司 | Whitening freckle-removing mask and preparation method thereof |
| CN112430163A (en) * | 2020-11-26 | 2021-03-02 | 宁夏大学 | Biological fertilizer for relieving continuous cropping obstacle of watermelon with pressed sand |
| CN113637624A (en) * | 2021-09-01 | 2021-11-12 | 珠海瑞德林生物有限公司 | Recombinant bacillus subtilis, application thereof and method for producing tetrahydropyrimidine by using waste water generated in glutathione synthesis by enzyme method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103404370B (en) | The method of edible fungus species is cultivated with pelelith | |
| CN103173371B (en) | Production of saccharomyces cerevisiae and lactobacillus acidophilus composite microbe preparation used for feed | |
| CN101444170A (en) | Strain separation method of apricot ormer mushroom and cultivating method thereof | |
| CN105684727B (en) | The culture medium and cultural method for the black collybia albuminosa of cultivation expected based on ferment bacteria residue | |
| CN101914445B (en) | Indigenous probiotic microorganism solid fungicide and preparation method and application thereof | |
| CN105132311B (en) | Utilize the method for glutathione waste liquid production functional microorganism | |
| CN105432935A (en) | Production method for functional amino-acid humic-acid microecological preparation for aquatic animal and poultry | |
| CN103305434A (en) | Microecological preparation with dual functions of probiotics and organic selenium and preparation method of microecological preparation | |
| CN103988977A (en) | Feed microecological preparation and preparation method thereof | |
| CN103184174B (en) | Production method of bacillus subtilis biological agent used for sodium humate-containing feed in medium | |
| CN101088362B (en) | Fish and shrimp phagostimulant of fermented product adhesion protein and its preparation process | |
| CN109022333A (en) | A kind of preparation method and applications of composite microbial fermentation bacteria agent | |
| CN106754463A (en) | One plant of tool dissolving P capacity Burkholderia bacterium NJAU B8 and its microbial manure of development | |
| CN110122188A (en) | Edible mushroom cultivation nutrition promotor and its application | |
| CN109549025A (en) | One seed shrimp feed addictive and preparation method thereof | |
| CN107853478A (en) | A kind of feed additive of Chinese herbal medicine containing enzyme for reducing ox discharge of methane | |
| CN103300209A (en) | Marsh rhodopseudomonas activation preparation and preparation method thereof | |
| CN101836688B (en) | Preparation method of antibiotic-free microbial fermentation feed | |
| CN104621357A (en) | Novel liquid feed additive and preparation method thereof | |
| CN108633626A (en) | The method for preparing White mushroom cultivation base as primary raw material using cattle pen bedding and padding | |
| CN106387317A (en) | Microorganism feed additive capable of protecting pig livers and preparation method of microorganism feed additive | |
| CN105087432A (en) | Application of glutathione waste liquid in microorganism production and application method | |
| CN106747839A (en) | A kind of culture medium of edible fungus, its preparation method and application | |
| CN109123076A (en) | A kind of production method of livestock and poultry vitamin B2 auxotype probiotics | |
| CN100372814C (en) | Organic fertilizer prepared by using waste blackfungus culture medium and its preparation method and use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151125 |
|
| RJ01 | Rejection of invention patent application after publication |