CN101999525A - Composite probiotics feed additive - Google Patents
Composite probiotics feed additive Download PDFInfo
- Publication number
- CN101999525A CN101999525A CN2010105278956A CN201010527895A CN101999525A CN 101999525 A CN101999525 A CN 101999525A CN 2010105278956 A CN2010105278956 A CN 2010105278956A CN 201010527895 A CN201010527895 A CN 201010527895A CN 101999525 A CN101999525 A CN 101999525A
- Authority
- CN
- China
- Prior art keywords
- bacillus subtilis
- lactobacillus plantarum
- feed additive
- yeast
- dilution
- 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.)
- Granted
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a composite probiotics feed additive which comprises probiotics, fillers and calcium polyglutamate, wherein the weight ratio of the calcium polyglutamate to a mixture of the probiotics and the fillers is 0.001-0.005:1. The probiotics is bacillus subtilis, lactobacillus plantarum and brewers' yeast. The calcium polyglutamate (gamma-PGA-Ga) is a high molecular weight calcium polymer formed by condensing alpha-amino and gamma-carboxyl of glutamic acid (Glu) and has excellent water-solubility and superstrong water-retaining property and adsorbability, not only can provide organic calcium easily absorbed by animals, but also can remarkably improve the survival rate of probiotics thallus, increase the adhering function between the additive and the feed, and improve the utilization ratio of the probiotics, thereby improving the disease-resistant capacity of the animals, promoting the animal growth, and increasing the economic benefit of breeding.
Description
Technical field
The present invention relates to a kind of feed addictive, relate in particular to a kind of composite prebiotics feed additive.
Background information:
In recent years, adopt antibiosis disease preventing and treating usually during animal husbandry is produced mostly, this not only makes the imbalance of animal intestinal normal flora, and occurs a large amount of medicament residues in animal products.Along with the enhancing with health perception of improving constantly of people's living standard, the residual side effect of the medicine of feeding antibiotic is subjected to people day by day and pays close attention to.Probiotic is by the repulsive interaction of animal alimentary canal biological competition, suppresses the growth of pernicious bacteria, forms dominant microflora or strengthens non-specific immune function to come prevent disease, thereby promoted growth of animal, improved the conversion ratio of feed.Prebiotics feed additive is a kind of natural biologically active agents, the characteristics that have nontoxic, the no resistance to the action of a drug, have no side effect, as safety, efficient, alternative feeding antibiotic again the product of no drug residue become a developing direction of additive agent for feeding.
Probiotic is to have active biologic product, and the key of using probiotic is after guaranteeing that these microorganisms are sneaked into feed, can keep active, until the relevant position breeding that enters animal intestinal.The factor that influences probiotic activity is a lot, and for the additive of solid drying, wherein to be stabilized in certain level most important for moisture.So both can keep its activity, even breed slightly, be unlikely to make its a large amount of breedings again and contention nutrition and the mutual antagonism of metabolin.And probiotic formulations requires thalline and feed addictive that certain adhesive capacity is arranged in process, so just is convenient to these viable bacterias and is searched for food by animal along with the adding of additive.
Animal is calcareous when not enough, can cause growth retardation, skeleton development bad, and crisp easy to break, the bending of the lighter's bone forms rickets, and weight person can develop into rickets, and its clinical symptoms is similar to vitamin D deficiency.If can in prebiotics feed additive, increase the organic calcium that animal is easy to absorb, then can when keeping the prebiotics feed additive advantage, give the animal body supplement calcium.
How to make and both be rich in the organic calcium that animal absorbs easily in the prebiotics feed additive, can make simultaneously the probiotic thalline keep stable activity again, strengthen and feed between adhesive effect and be easy to be searched for food by animal, improve the effect of prebiotics feed additive on the whole? resemble the calcium monohydrogen phosphate that adds in the Chinese patent application CN200810136835.4 prebiotics feed, only be simple supplement calcium, can not improve the utilization rate of prebiotics feed additive on the whole.
Summary of the invention
In view of this, in order to overcome the defective of prior art, the object of the present invention is to provide a kind of NEW TYPE OF COMPOSITE prebiotics feed additive, this additive not only is rich in the organic calcium that animal absorbs easily, simultaneously the probiotic thalline can keep stable activity, and the adhesive effect between feed is easy to be searched for food by animal by force, thus the utilization rate of raising probiotic, promote growth of animal, improve the culturing economic benefit.
Another object of the present invention provides the preparation method of described composite prebiotics feed additive.
A kind of composite prebiotics feed additive provided by the invention, described additive comprise probiotic, inserts and polyglutamic acid calcium, and the weight ratio of the weight of described polyglutamic acid calcium and probiotic and inserts mixture is 0.001-0.005: 1.
(γ-PGA-Ga) γ-PGA-Ga is polyglutamic acid (γ-PGA) close and generate with the calcium salt huge legendary turtle to polyglutamic acid calcium wherein.Polyglutamic acid is to be formed by the alpha-amido of glutamic acid (Glu) and γ-carboxyl condensation, molecular weight distribution at 100kDa between the 1000kDa.The production of γ-PGA can utilize natto to extract by biotechnology, also can be obtained by microbial fermentation technology, and it is manually synthetic to utilize biochemical process to carry out.Used polyglutamic acid is to get by Production by Microorganism Fermentation among the present invention.2007 the 28th volume the 5th phases " food research and development ", 18-20 page or leaf report has " Production by Microorganism Fermentation of gamma-polyglutamic acid-and sign initial analysis thereof ".
The present invention is by adding weight ratio 0.001-0.005 in composite prebiotics feed: 1 polyglutamic acid calcium, successful invention a kind of NEW TYPE OF COMPOSITE prebiotics feed additive, it not only is rich in the organic calcium that animal absorbs easily, again because of polyglutamic acid calcium good water-soluble and superpower water-retaining property and adsorptivity, can significantly improve the survival rate of probiotic thalline, increase the adhesive effect between additive and feed, improve the utilization rate of probiotic, thereby raising disease resistance of animals, improve the animal digestion function, promote growth of animal and improve the culturing economic benefit.
Further, described polyglutamic acid calcium purity is greater than 90%.
Further, the weight ratio of described probiotic dry powder and inserts is 1: 0.2～1.8.
Further, described probiotic is bacillus subtilis Bacillus subtilis, brewer's yeast Saccharomyces cerevisiae and Lactobacillus plantarum Lactobacillus plantarum.
Further, the weight ratio of described three kinds of solid bacterium powder is: 1: 0.8-1.2: 0.8-1.2.
Further, described inserts is a zeolite.
The present invention also provides the preparation method of described composite prebiotics feed additive, comprising: (1) carries out preservation after the separation and purification with the probiotic bacterial classification, and the preservation bacterial classification is gone down to posterity; (2) to the actication of culture of preservation, shaking bottle earlier cultivates, cultivate through liquid fermentation tank again, carrying out solid fermentation then cultivates, the solid fermentation bacterial classification is dried to moisture 8%-15%, and (3) each dry bacterium powder mixes the inserts mixing that the back adds weight ratio 1: 0.2～1.8 in proportion, adds the polyglutamic acid calcium of weight ratio 1: 0.001-0.005 in the mixture again, (4) mix packing.
Further, described probiotic bacterial classification is bacillus subtilis (Bacillus subtilis), brewer's yeast (Saccharomyces cerevisiae) and Lactobacillus plantarum (Lactobacillus plantarum); Described bacillus subtilis (Bacillus subtilis) utilizes nutrient agar to carry out purifying, preservation and activation, brewer's yeast (Saccharomyces cerevisiae) utilizes soybean sprout juice culture medium purifying, preservation and activation, and Lactobacillus plantarum (Lactobacillus plantarum) utilizes MRS improved culture medium purifying, preservation and activation.
Wherein, nutrient agar is: beef extract 3.0 restrains, peptone 10 grams, and sodium chloride 5.0 grams, agar 15 grams, 1000 milliliters in water is adjusted the pH value to 7.2-7.6, and packing was sterilized 30 minutes for 121 ℃.The soybean sprout juice culture medium is: moyashi 100 grams, agar 15 grams, glucose 20 grams, 1000 milliliters in water; Clean moyashi, added water boil 30 minutes.Use filtered through gauze, add agar in the filtrate, put into sugar after the heating for dissolving, stir and to make its dissolving, supply moisture to 1000 milliliter, packing, 121 ℃ of sterilizations 20 minutes.The MRS improved culture medium is: peptone 10g, yeast extract 5g, beef extract 10g, glucose 20g, sodium acetate 5g, dibasic ammonium citrate 2g, Tween 80 1mL, magnesium sulfate 0.58g, manganese sulfate 0.05g, dipotassium hydrogen phosphate 2g, agar 15-17g, water 1000mL; PH transfers to 6.12-6.2 before the sterilization, sterilizes 20 minutes for 121 ℃.
Further, bacillus subtilis (Bacillus subtilis) is adopted broth bouillon in shaking the bottle cultivation, brewer's yeast (Saccharomyces cerevisiae) adopts improvement soybean sprout juice culture medium, Lactobacillus plantarum (Lactobacillus plantarum) adopts the MRS improved culture medium, shaking a bottle cultivation temperature is respectively: bacillus subtilis 31-33 ℃, brewer's yeast 28-30 ℃, Lactobacillus plantarum 36-38 ℃; Shaking speed is 140-150r/min; Cultivate and to be inoculated into liquid fermentation tank after a bacterium number reaches required viable count and to cultivate when shaking bottle, liquid fermentation medium is with to shake bottle culture medium identical, and the liquid fermentation and culture temperature is identical with the shaking table cultivation temperature.
Wherein broth bouillon is: beef extract 3.0 grams, and peptone 10 grams, sodium chloride 5.0 grams, 1000 milliliters in water is adjusted pH value to 7.2～7.6, and packing was sterilized 30 minutes for 121 ℃; Improvement soybean sprout juice medium culture base is: moyashi 120 grams, glucose 25 grams, 1000 milliliters in water is cleaned moyashi, added water boil 30 minutes, use filtered through gauze, add agar in the filtrate, put into sugar after the heating for dissolving, stirring makes its dissolving, supply moisture to 1000 milliliter, packing, 121 ℃ of sterilizations 20 minutes; MRS improved culture medium such as above-mentioned.
Usually, reach 10 when shaking bottle cultivation bacterium number
9Being inoculated into liquid fermentation tank behind the cfu/mL cultivates.
Further, when solid fermentation is cultivated, the zymotic fluids of described bacillus subtilis, brewer's yeast and three kinds of bacterium of Lactobacillus plantarum is inoculated in respectively and carries out solid fermentation on the solid medium, the used culture medium of solid fermentation is: add wheat bran in the urea liquid of weight percent concentration 0.20-0.15%, the weight ratio of described urea liquid and wheat bran is: 1: 1.1-1.3, sterilized 45-50 minute for 121 ℃, the solid fermentation temperature of three kinds of bacterium is all with to shake bottle cultivation temperature identical, regularly detect viable count, when reaching required viable count, fermentation ends; After described three kinds of bacterium dried bean noodles are dry by weight: 1: 0.8-1.2: 0.8-1.2 mixes.
After three kinds of bacterium dried bean noodles were dry, the yeast viable count should reach: 1.0 * 10
8More than the cfu/g (detection method is seen appendix C), the viable count of bacillus subtilis should reach: 1.0 * 10
9More than the cfu/g (detection method is seen appendix A), the viable count of Lactobacillus plantarum should reach: 1.0 * 10
7More than the cfu/g (detection method is seen appendix B).
Beneficial effect of the present invention is:
(1) adds polyglutamic acid calcium with microenvironment water retention (γ-PGA-Ga) in the probiotic additive by giving, and be the nutriment of main material with the wheat bran in the additive, make probio that moisture and the stable growing environment of nutrition can be arranged in storage life, improved the survival rate of thalline.
(2) (γ-PGA-Ga) be chelated with the organic calcium that animal is easy to absorb significantly improves the disease that animal causes because of calcium deficiency to the polyglutamic acid calcium that is added, and improves the resistance against diseases of cultivated animals, reduces the death rate, increases the number of heads of livestock for sale, and has improved culture benefit.
(3) (Ultrastrength adhesive of γ-PGA-Ga) have has strengthened the adhesive effect between probio and feed to the polyglutamic acid calcium that is added, and has improved the utilization rate of additive.
The specific embodiment
Further illustrate the present invention in the following embodiments, this does not limit the scope of the invention.
Embodiment 1:
(Bacillus subtilis) utilizes nutrient agar to carry out purifying, preservation and activation to bacillus subtilis, brewer's yeast (Saccharomyces cerevisiae) utilizes soybean sprout juice culture medium purifying, preservation and activation, and Lactobacillus plantarum (Lactobacillus plantarum) utilizes MRS improved culture medium purifying, preservation and activation.Further shaking bottle cultivates, bacillus subtilis (Bacillus subtilis) is adopted broth bouillon, brewer's yeast (Saccharomyces cerevisiae) adopts improvement soybean sprout juice culture medium, Lactobacillus plantarum (Lactobacillus plantarum) adopts the MRS improved culture medium, shaking a bottle cultivation temperature is respectively: 33 ℃ of bacillus subtilises, 28 ℃ of brewer's yeasts, 37 ℃ of Lactobacillus plantarums; Shaking speed is 140-150r/min; Cultivate the bacterium number when medicine bottle and reach 10
9Be inoculated into the 50L liquid fermentation tank during cfu/mL and cultivate, liquid fermentation medium is with to shake bottle culture medium identical, and the liquid fermentation and culture temperature is identical with the shaking table cultivation temperature.
With above-mentioned bacillus subtilis, the zymotic fluid of brewer's yeast and three kinds of bacterium of Lactobacillus plantarum is inoculated in respectively and carries out solid fermentation on the solid medium, culture medium is: weight percent concentration is to add wheat bran in 0.2% the urea liquid, the weight ratio of urea liquid and wheat bran is: 1: 1.3, sterilized 45-50 minute for 121 ℃, the solid fermentation temperature of three kinds of bacterium is all with to shake bottle cultivation temperature identical, regularly detect viable count, when reaching required viable count, fermentation ends, it is 13.5% that the laboratory is dried to moisture with 58 ℃ of blowings of drying box, obtains solid bacterium powder 100kg separately.
After three kinds of solid bacterium powder were mixed according to 1: 1: 1 ratio, respectively to three bacterial classifications countings, the yeast viable count was: 2.0 * 10 with the dilution rubbing method
8Cfu/g (detection method is seen appendix C), the viable count of bacillus subtilis is: 2.0 * 10
9Cfu/g (detection method is seen appendix A), the viable count of Lactobacillus plantarum is: 1.6 * 10
7Cfu/g (detection method is seen appendix B).With the ratio adding 300kg zeolite powder of mixed solid bacterium powder according to 1: 1, add purity again and be 90% polyglutamic acid calcium (available from Zijin port, Haining, Jiaxing City, Zhejiang Province bio tech ltd) 3.0kg, mix, obtain composite prebiotics feed additive of the present invention.
Embodiment 2
Substantially the same manner as Example 1, institute's difference is that bacillus subtilis, brewer's yeast and three kinds of bacterium 50L of Lactobacillus plantarum zymotic fluid are inoculated in the 500L liquid fermentation tank as first order seed respectively in 10% ratio.With above-mentioned bacillus subtilis, the 500L zymotic fluid of brewer's yeast and three kinds of bacterium of Lactobacillus plantarum is inoculated in respectively and carries out solid fermentation on the solid medium, culture medium is: weight percent concentration is to add wheat bran in 0.2% the urea liquid, the weight ratio of urea liquid and wheat bran is: 1: 1.2,121 ℃ of single pot of horizontal all steel 700 type retort that adopt the powerful machinery plant in Zhucheng to produce, sterilized 50 minutes, after inoculating separately again, change the solid fermentation pond over to, fermentation vat is solid aerated koji making pond, the fermentation vat volume is: wide 1.2m * long 3m * high 1.5m, solid fermentation finishes, the DGS2000 type low-temperature drier that adopts the light big chemical mechanical equipment factory of Wuxi City to produce is dried to moisture 13.0% for 58 ℃, obtains 1 ton in solid bacterium powder separately.
After three kinds of solid bacterium powder were mixed according to 1: 1: 1 ratio, respectively to three bacterial classifications countings, the yeast viable count was: 1.8 * 10 with the dilution rubbing method
8Cfu/g (detection method is seen appendix C), the viable count of bacillus subtilis is: 1.5 * 10
9Cfu/g (detection method is seen appendix A), the viable count of Lactobacillus plantarum is: 1.2 * 10
7Cfu/g (detection method is seen appendix B) is with mixed solid bacterium powder 3 tons of zeolite powders of ratio adding according to 1: 1, add purity again and be 90% polyglutamic acid calcium (available from Zijin port, Haining, Jiaxing City, Zhejiang Province bio tech ltd) 12kg, the model of producing with grain and oil conveying machinery Dezhou, Dongtai general headquarters is that the mixer of DSH/SLH 6 mixes, obtain the composite prebiotics preparation, packing.
Embodiment 3:
With the product among the embodiment 2 with according to method preparation among the embodiment 2 but the prebiotics feed additive control group that does not add polyglutamic acid calcium is stored period sampling measuring thalline viable count down at 18 ℃.Bacillus subtilis adopts the nutrient agar dilution to cultivate counting method (seeing appendix A), and Lactobacillus plantarum adopts the MRS dilution to cultivate counting method (seeing appendix B), and brewer's yeast adopts the rose-bengal dilution to cultivate counting method and measures viable count (seeing appendix C).The results are shown in following table:
The result shows, the survival rate of three kinds of bacterium is with the preservation time lengthening, adds one group of polyglutamic acid calcium apparently higher than control group.This has proved that also polyglutamic acid calcium has the effect that improves survival rate to probiotic.
Embodiment 4:
With product among the embodiment 2 and the check analysis but the prebiotics feed additive control group that does not add polyglutamic acid calcium is fed according to method preparation among the embodiment 2.
Material: test is big bone chicken with chicken breed.Grouping: 1500 baby chicks buying simultaneously are divided into three groups: first and second each 500 baby chicks of group are test group, feed according to the probiotic of the adding polyglutamic acid calcium of the method among the embodiment 1 preparation, the 3rd group of 500 baby chicks are control group, feed according to the method among the embodiment 1 preparation but do not add the probiotic of polyglutamic acid calcium.Feeding method is: 0.5% ratio and normal diet mixing according to daily ration are fed chicken.
In the process of the test, under technical staff's guidance, detail record advances situations such as chicken number, item number, dead number of elements, feed consumption rate respectively, so that take statistics analysis.
Test results and analysis: go through 120 days feeding experiment result, find that the death rate of test group is obviously low than control group.
Find out that from last table numeral 3 groups are compared, the death rate of control group is the highest.From then on can think, utilize novel prebiotics feed additive raising chicken, can reduce the death rate of chicken, increase the number of heads of livestock for sale, directly increase raise benefit.
Embodiment 5:
The feed that adds product among the embodiment 2 is done the effect checking with the feed control group that does not add this product.By product among the embodiment 2 of interpolation 0.35% in educating swine rations, pig production performance such as following table:
| Handle | Control group | Experimental group |
| A test piglet number | 200 | 200 |
| Average daily gain (g/d) | 940.3 | 1098.5 |
| Average daily ingestion amount (g/d) | 3002.8 | 3218.4 |
| Feedstuff-meat ratio | 3.2 | 2.9 |
Application result shows: feed addictive of the present invention can significantly improve the utilization rate of feed, has saved feed resource, has reduced aquaculture cost, has improved culture benefit.
Although by reference some preferred embodiment of the present invention, invention has been described, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
Appendix A
The mensuration of bacillus subtilis sum
A.1 principle
After sample dilution 10, in 80 ℃ of waters bath with thermostatic control, kill heat labile cell, be diluted to suitable concentration again, with specific culture medium, aerobic cultivation is 72 ± 3 hours under 30 ± 1 ℃, and the clump count that grows on the counting flat board calculates the bacillus subtilis sum in every gram sample.
A.2 reagent
A.2.1 nutrient agar
A.2.2 sterilized water
A.3 instrument and equipment
A.3.1 high-pressure sterilizing pot
A.3.2 constant incubator
A.3.3 vibrating machine
A.3.4 thermostat water bath
A.3.5 test glass apparatus.
A.4 operating procedure
A.4.1 the preparation of nutrient agar and sterilized water
A.4.1.1 nutrient agar
Beef extract 3g, peptone 10g, sodium chloride 5g, agar 15-20g distilled water 1000mL
Compound method: each composition beyond the agar is dissolved in the distilled water, adds 15% the about 2mL correction of sodium hydroxide solution pH to 7.2-7.4.Add agar and boil, agar is dissolved.The packing test tube, 121 ℃ of autoclaving 20min take out standby.
A.4.1.2 sterilized water
225mL distilled water is packed in the 500mL triangular flask, refill 9mL distilled water to dilution in vitro, 121 ℃ of autoclaving 20min take out standby.
A.4.2 sampling
Press GC/T 14699.1 samplings.
A.4.3 sample dilution and cultivation
A.4.3.1 the aseptic sample 25.0g that takes by weighing puts into the triangular flask (bottle is put into the bead of some in advance) that fills the 225mL sterilized water.Be placed on the 15min that vibrates on the vibrating machine, triangular flask is put into 80 ℃ of thermostat water baths, accurately timing 5min makes 1: 10 dilution.
A.4.3.2 with 1: 10 dilution 1mL of 1mL sterilization pipette, extract, inject in vitro (the attention pipette tip does not touch in-line dilution liquid) of containing the 9mL sterilized water, make 1: 100 dilution along tube wall.
A.4.3.3 get a pipette in addition, the dilution above the pressure-vaccum ten times is pressed the aforesaid operations order, does 10 times and increases progressively dilution, so whenever increases progressively once, promptly changes a pipette.
A.4.3.4 choose 1: 10
6, 1: 10
7, 1: 10
8Three dilution ranks when doing to increase progressively dilution, are promptly moved the 1mL dilution in the sterilization plate with the pipette of drawing this dilution level respectively, and each dilution rank is made two plates.
A.4.3.5 after dilution moves into plate, should in time the nutrient agar that dries in the air to 46 ± 1 ℃ (putting into 46 ℃ of water-baths after molten in advance the opening) be injected the about 15mL of plate, rotate plate and make sample and the abundant mixing of culture medium.To pouring into the culture medium, the time can not surpass 20min from the dilution sample.
A.4.3.6 after treating that agar solidifies, plate is inverted in cultivation 72 ± 3h. in 30 ± 1 ℃ of constant temperature culture
A.4.4 method for counting colonies
When A.4.4.1 making plate count, the bacterium colony that can detect by an unaided eye is in case of necessity by lens examination, in case omit.
A.4.4.2 other selection of dilution level
A.4.4.3 the dilution level with 20-300 clump count occurring on the flat board serves as that effectively counting is dull and stereotyped.
When having only a dilution rank, when its average drops between the 20-300, then multiply by its extension rate with this average clump count.
A.4.4.4 if there are two other averages of dilution level to drop between the 20-300, should decide by the ratio of both total plate counts, if its ratio should be counted both mean value less than 2, if greater than 2 less total plate counts of counting dilution wherein.
A.4.4.5 if three other average bacterium colony number averages of dilution level not between 20-300, then multiply by extension rate with immediate 30 or 300 average clump count.
A.5 calculate
Statistics is calculated the bacterium colony average on two plates of same dilution rank.
The bacillus subtilis sum/(CFU/g)=bacterium colony average * extension rate
Appendix B
The mensuration of Lactobacillus plantarum sum
B.1 principle
Sample is diluted to suitable concentration, and with specific culture medium, anaerobism is cultivated 48 ± 3h under specified temp, and the clump count that grows on the counting flat board calculates the Lactobacillus plantarum sum in every gram sample.
B.2 reagent MRS culture medium
B.3 instrument and equipment
B.3.1 high-pressure sterilizing pot
B.3.2 constant incubator
B.3.3 vibrating machine
B.3.4 thermostat water bath
B.3.5 test glass apparatus.
B.4 operating procedure
B.4.1MRS the preparation of agar and sterilized water
B.4.1.1MRS agar
Casein peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, glucose 5.g, sodium acetate 5.0g, citric acid diamines 2.0g, soil temperature 80 1.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.2g, manganese sulfate 0.05g, agar 15-20g distilled water 1000mL
Compound method: each composition beyond the agar is dissolved in the distilled water, regulates pH to 6.7-6.9, add agar and boil, agar is dissolved.The packing triangular flask, 121 ℃ of autoclaving 20min take out standby.
B.4.1.2 sterilized water
225mL distilled water is packed in the 500mL triangular flask, refill 9mL distilled water to dilution in vitro, 121 ℃ of autoclaving 20min take out standby.
B.4.2 sampling
Press GB/T 14699.1 samplings.
B.4.3 sample dilution and cultivation
B.4.3.1 the aseptic sample 25.0g that takes by weighing puts into the triangular flask (bottle is put into the bead of some in advance) that fills the 225mL sterilized water.Be placed on the 15min that vibrates on the vibrating machine, triangular flask is put into 80 ℃ of thermostat water baths, accurately timing 5min makes 1: 10 dilution.
B.4.3.2 with 1: 10 dilution 1mL of 1mL sterilization pipette, extract, inject in vitro (the attention pipette tip does not touch in-line dilution liquid) of containing the 9mL sterilized water, make 1: 100 dilution along tube wall.
B.4.3.3 get a pipette in addition, the dilution above the pressure-vaccum ten times is pressed the aforesaid operations order, does 10 times and increases progressively dilution, so whenever increases progressively once, promptly changes a pipette.
B.4.3.4 choose 1: 10
6, 1: 10
7, 1: 10
8Three dilution ranks when doing to increase progressively dilution, are promptly moved the 1mL dilution in the sterilization plate with the pipette of drawing this dilution level respectively, and each dilution rank is made two plates.
B.4.3.5 after dilution moves into plate, should rotate plate and make sample and the abundant mixing of culture medium in time with drying in the air to the about 15mL of the aseptic injection plate of MRS culture medium of 46 ± 1 ℃ (putting into 46 ℃ of water-baths after molten in advance the opening).To pouring into the culture medium, the time can not surpass 20min from the dilution sample.
B.4.3.6 after treating that agar solidifies, plate is inverted in cultivation 72 ± 3h. in 37 ± 1 ℃ of constant temperature culture
B.4.4 method for counting colonies
When B.4.4.1 making plate count, the bacterium colony that can detect by an unaided eye is in case of necessity by lens examination, in case omit.
B.4.4.2 other selection of dilution level
B.4.4.3 the dilution level with 20-300 clump count occurring on the flat board serves as that effectively counting is dull and stereotyped.
When having only a dilution rank, when its average drops between the 20-300, then multiply by its extension rate with this average clump count.
B.4.4.4 if there are two other averages of dilution level to drop between the 20-300, should decide by the ratio of both total plate counts, if its ratio should be counted both mean value less than 2, if greater than 2 less total plate counts of counting dilution wherein.
B.4.4.5 if three other average bacterium colony number averages of dilution level not between 20-300, then multiply by extension rate with immediate 30 or 300 average clump count.
B.5 calculate
Statistics is calculated the bacterium colony average on two plates of same dilution rank.
The Lactobacillus plantarum sum/(BFU/g)=bacterium colony average * extension rate
Appendix C
The mensuration of saccharomyces cerevisiae sum
C.1 principle
Sample is diluted to suitable concentration, selects culture medium with yeast, cultivate 72 ± 3h under specified temp, the clump count that grows on the counting flat board calculates the saccharomyces cerevisiae sum in every gram sample.
C.2 reagent rose bengal medium
C.3 instrument and equipment
C.3.1 high-pressure sterilizing pot
C.3.2 constant incubator
C.3.3 vibrating machine
C.3.4 thermostat water bath
C.3.5 test glass apparatus.
C.4 operating procedure
C.4.1 the preparation of rose bengal medium and sterilized water
C.4.1.1 add the Meng as red culture medium
Peptone: 5g, glucose: 10g, potassium dihydrogen phosphate: 1g, magnesium sulfate (MgSO47H2O): 0.5g, agar: 15-20g/, 1/3000 rose-bengal solution: 00mL, distilled water: 1000mL, chloramphenicol: 0.1g.Compound method: after the dissolving, add rose-bengal solution again in above-mentioned each composition adding distilled water.With small amount of ethanol dissolved chlorine mycin, add in the culture medium in addition, after the packing, 121 ℃ of sterilization 20min take out standby.
C.4.1.2 sterilized water
225mL distilled water is packed in the 500mL triangular flask, refill 9mL distilled water to dilution in vitro, 121 ℃ of autoclaving 20min take out standby.
C.4.2 sampling
Press GC/T 14699.1 samplings.
C.4.3 sample dilution and cultivation
C.4.3.1 the aseptic sample 25.0g that takes by weighing puts into the triangular flask (bottle is put into the bead of some in advance) that fills the 225mL sterilized water.Be placed on the 15min that vibrates on the vibrating machine, triangular flask is put into 80 ℃ of thermostat water baths, accurately timing 5min makes 1: 10 dilution.
C.4.3.2 with 1: 10 dilution 1mL of 1mL sterilization pipette, extract, inject in vitro (the attention pipette tip does not touch in-line dilution liquid) of containing the 9mL sterilized water, make 1: 100 dilution along tube wall.
C.4.3.3 get a pipette in addition, the dilution above the pressure-vaccum ten times is pressed the aforesaid operations order, does 10 times and increases progressively dilution, so whenever increases progressively once, promptly changes a pipette.
C.4.3.4 choose 1: 10
5, 1: 10
6, 1: 10
7Three dilution ranks when doing to increase progressively dilution, are promptly moved the 1mL dilution in the sterilization plate with the pipette of drawing this dilution level respectively, and each dilution rank is made two plates.
C.4.3.5 after dilution moves into plate, should rotate plate and make sample and the abundant mixing of culture medium in time with drying in the air to the about 15mL of the aseptic injection plate of rose bengal medium of 46 ± 1 ℃ (putting into 46 ℃ of water-baths after molten in advance the opening).To pouring into the culture medium, the time can not surpass 20min from the dilution sample.
C.4.3.6 after treating that agar solidifies, plate is inverted in cultivation 72 ± 3h. in 37 ± 1 ℃ of constant temperature culture
C.4.4 method for counting colonies
When C.4.4.1 making plate count, the bacterium colony that can detect by an unaided eye is in case of necessity by lens examination, in case omit.
C.4.4.2 other selection of dilution level
C.4.4.3 the dilution level with 20-300 clump count occurring on the flat board serves as that effectively counting is dull and stereotyped.
When having only a dilution rank, when its average drops between the 20-300, then multiply by its extension rate with this average clump count.
C.4.4.4 if there are two other averages of dilution level to drop between the 20-300, should decide by the ratio of both total plate counts, if its ratio should be counted both mean value less than 2, if greater than 2 less total plate counts of counting dilution wherein.
C.4.4.5 if three other average bacterium colony number averages of dilution level not between 20-300, then multiply by extension rate with immediate 30 or 300 average clump count.
C.5 calculate
Statistics is calculated the bacterium colony average on two plates of same dilution rank.
Saccharomyces cerevisiae bacterium sum/(CFU/g)=bacterium colony average * extension rate.
Claims (10)
1. a composite prebiotics feed additive is characterized in that, described additive comprises probiotic, inserts and polyglutamic acid calcium, and the weight ratio of the weight of described polyglutamic acid calcium and probiotic and inserts mixture is 0.001-0.005: 1.
2. according to the described composite prebiotics feed additive of claim 1, it is characterized in that described polyglutamic acid calcium purity is greater than 90%.
3. according to the described composite prebiotics feed additive of claim 1, it is characterized in that the weight ratio of described probiotic and inserts is 1: 0.2～1.8.
4. according to the described composite prebiotics feed additive of claim 1, it is characterized in that described probiotic is bacillus subtilis Bacillus subtilis, brewer's yeast Saccharomyces cerevisiae and Lactobacillus plantarum Lactobacillus plantarum.
5. according to the described composite prebiotics feed additive of claim 4, it is characterized in that the weight ratio of described three kinds of solid bacterium powder is: 1: 0.8-1.2: 0.8-1.2.
6. according to the described composite prebiotics feed additive of claim 1, it is characterized in that described inserts is a zeolite.
7. according to the preparation method of the described composite prebiotics feed additive of claim 1, it is characterized in that described preparation method comprises: (1) carries out preservation after the separation and purification with the probiotic bacterial classification, and the preservation bacterial classification is gone down to posterity; (2) to the actication of culture of preservation, shaking bottle earlier cultivates, cultivate through liquid fermentation tank again, carrying out solid fermentation then cultivates, the solid fermentation bacterial classification is dried to moisture 8%-15%, and (3) each dry bacterium powder mixes the inserts mixing that the back adds weight ratio 1: 0.2～1.8 in proportion, adds the polyglutamic acid calcium of weight ratio 1: 0.001-0.005 in the mixture again, (4) mix packing.
8. according to the preparation method of the described composite prebiotics feed additive of claim 7, it is characterized in that described probiotic bacterial classification is bacillus subtilis (Bacillus subtilis), brewer's yeast (Saccharomycescerevisiae) and Lactobacillus plantarum (Lactobacillus plantarum); Described bacillus subtilis (Bacillus subtilis) utilizes nutrient agar to carry out purifying, preservation and activation, brewer's yeast (Saccharomyces cerevisiae) utilizes soybean sprout juice culture medium purifying, preservation and activation, and Lactobacillus plantarum (Lactobacillus plantarum) utilizes MRS improved culture medium purifying, preservation and activation.
9. according to the preparation method of the described composite prebiotics feed additive of claim 7, it is characterized in that, bacillus subtilis (Bacillus subtilis) is adopted broth bouillon in shaking the bottle cultivation, brewer's yeast (Saccharomyces cerevisiae) adopts improvement soybean sprout juice culture medium, Lactobacillus plantarum (Lactobacillus plantarum) adopts the MRS improved culture medium, shaking a bottle cultivation temperature is respectively: bacillus subtilis 31-33 ℃, brewer's yeast 28-30 ℃, Lactobacillus plantarum 36-38 ℃; Shaking speed is 140-150r/min; Cultivate and to be inoculated into liquid fermentation tank after a bacterium number reaches required viable count and to cultivate when shaking bottle, liquid fermentation medium is with to shake bottle culture medium identical, and the liquid fermentation and culture temperature is identical with the shaking table cultivation temperature.
10. according to the preparation method of the described composite prebiotics feed additive of claim 7, it is characterized in that, when solid fermentation is cultivated with described bacillus subtilis, the zymotic fluid of brewer's yeast and three kinds of bacterium of Lactobacillus plantarum is inoculated in respectively and carries out solid fermentation on the solid medium, the used culture medium of solid fermentation is: add wheat bran in the urea liquid of weight percent concentration 0.2-0.15%, the weight ratio of described urea liquid and wheat bran is: 1: 1.1-1.2, sterilized 45-50 minute for 121 ℃, the solid fermentation temperature of three kinds of bacterium is all with to shake bottle cultivation temperature identical, regularly detect viable count, when reaching required viable count, fermentation ends; After described three kinds of bacterium dried bean noodles are dry by weight: 1: 0.8-1.2: 0.8-1.2 mixes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105278956A CN101999525B (en) | 2010-11-02 | 2010-11-02 | Composite probiotics feed additive |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105278956A CN101999525B (en) | 2010-11-02 | 2010-11-02 | Composite probiotics feed additive |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101999525A true CN101999525A (en) | 2011-04-06 |
| CN101999525B CN101999525B (en) | 2012-11-21 |
Family
ID=43807414
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010105278956A Expired - Fee Related CN101999525B (en) | 2010-11-02 | 2010-11-02 | Composite probiotics feed additive |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101999525B (en) |
Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102423015A (en) * | 2011-11-29 | 2012-04-25 | 天津中裕维特生物科技有限公司 | Special synbioticmbiotic for broiler |
| CN102488100A (en) * | 2011-12-22 | 2012-06-13 | 山东轻工业学院 | Compound-probiotics-containing feed additive and preparation method thereof |
| CN103421720A (en) * | 2013-08-20 | 2013-12-04 | 沈阳金科丰牧业科技有限公司 | Complex microbial inoculant for broiler chickens and application thereof to improvement of broiler chicken productivity and immunity |
| CN103416584A (en) * | 2013-08-20 | 2013-12-04 | 沈阳科丰牧业科技有限公司 | Method for improving broiler chicken quality by microecologics |
| CN103416585A (en) * | 2013-08-20 | 2013-12-04 | 沈阳科丰牧业科技有限公司 | Method for improving boiler chicken production performance and chicken quality by microecologics |
| CN103416628A (en) * | 2013-08-20 | 2013-12-04 | 沈阳金科丰牧业科技有限公司 | Application of complex microbial inoculant for broiler chickens to preparation of products for improving immunity of broiler chicken |
| CN103431226A (en) * | 2013-08-20 | 2013-12-11 | 沈阳科丰牧业科技有限公司 | Method for improving production performance of broiler chickens by using microecologics for broiler chickens |
| CN103478443A (en) * | 2013-08-20 | 2014-01-01 | 沈阳金科丰牧业科技有限公司 | Micro-ecological preparation and application thereof in improvement of production performance, immune performance as well as chicken quality of broiler |
| CN103535520A (en) * | 2012-07-10 | 2014-01-29 | 汪秀红 | Application of polyaspartic acid and salts thereof as animal feed additive |
| CN104686842A (en) * | 2013-08-20 | 2015-06-10 | 沈阳金科丰牧业科技有限公司 | Application of complex bacteria agent for broiler chickens in preparation of boiler chicken immunity enhancing products |
| CN104839444A (en) * | 2015-05-28 | 2015-08-19 | 河南双成生物科技有限公司 | Production method of functional protein feed rich in three essential amino-acids |
| CN104904994A (en) * | 2015-07-14 | 2015-09-16 | 谷实农牧集团股份有限公司 | Three major types of bacteria mixed liquid fermentation, solid yeast feed additive and preparation method thereof |
| CN105010757A (en) * | 2015-06-30 | 2015-11-04 | 西南大学 | Application of calcium poly-malate in the preparation of feed acidifier |
| CN107183379A (en) * | 2017-06-05 | 2017-09-22 | 时运昌 | A kind of bacteria agent pig feed additive |
| KR20180050093A (en) * | 2016-11-04 | 2018-05-14 | 주식회사 바이오리더스 | Composition for Prebiotics Containing Poly-Gamma-Glutamate |
| CN110024913A (en) * | 2019-04-09 | 2019-07-19 | 南京师范大学 | One primary yeast tunning and the compound of polyglutamic acid and the preparation method and application thereof |
| CN112501154A (en) * | 2020-11-02 | 2021-03-16 | 河北科技大学 | Extraction reagent, extraction method and kit for RNA in yeast cells |
| CN117186395A (en) * | 2023-09-13 | 2023-12-08 | 南京轩凯生物科技股份有限公司 | Preparation method and application of calcium polyglutamate |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101061867A (en) * | 2005-05-16 | 2007-10-31 | 东海生物科技股份有限公司 | Gamma-polyglutamic acid (gamma-PGA, H form) and gamma-polyglutamate used as nutrition supplement in dietary product |
| CN101371682A (en) * | 2007-08-21 | 2009-02-25 | 上海创博生态工程有限公司 | Microorganism formulation for eliminating nutrilit-resistance function and preparation method |
| CN101401921A (en) * | 2008-10-30 | 2009-04-08 | 河南省龙腾高科实业有限公司 | Microbial ecological traditional Chinese medicine preparation for livestock and poultry from fermentation production of multiple bacterials and fermentation method thereof |
-
2010
- 2010-11-02 CN CN2010105278956A patent/CN101999525B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101061867A (en) * | 2005-05-16 | 2007-10-31 | 东海生物科技股份有限公司 | Gamma-polyglutamic acid (gamma-PGA, H form) and gamma-polyglutamate used as nutrition supplement in dietary product |
| CN101371682A (en) * | 2007-08-21 | 2009-02-25 | 上海创博生态工程有限公司 | Microorganism formulation for eliminating nutrilit-resistance function and preparation method |
| CN101401921A (en) * | 2008-10-30 | 2009-04-08 | 河南省龙腾高科实业有限公司 | Microbial ecological traditional Chinese medicine preparation for livestock and poultry from fermentation production of multiple bacterials and fermentation method thereof |
Cited By (29)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102423015A (en) * | 2011-11-29 | 2012-04-25 | 天津中裕维特生物科技有限公司 | Special synbioticmbiotic for broiler |
| CN102488100A (en) * | 2011-12-22 | 2012-06-13 | 山东轻工业学院 | Compound-probiotics-containing feed additive and preparation method thereof |
| CN103535520A (en) * | 2012-07-10 | 2014-01-29 | 汪秀红 | Application of polyaspartic acid and salts thereof as animal feed additive |
| CN103416585B (en) * | 2013-08-20 | 2015-09-30 | 沈阳科丰牧业科技有限公司 | Broiler chicken probiotics is utilized to improve the method for meat chicken production performance and chicken meat quality |
| CN105029080A (en) * | 2013-08-20 | 2015-11-11 | 沈阳科丰牧业科技有限公司 | Method for improving production performance and chicken quality of broiler chicken by means of fungicide |
| CN103416628A (en) * | 2013-08-20 | 2013-12-04 | 沈阳金科丰牧业科技有限公司 | Application of complex microbial inoculant for broiler chickens to preparation of products for improving immunity of broiler chicken |
| CN103431226A (en) * | 2013-08-20 | 2013-12-11 | 沈阳科丰牧业科技有限公司 | Method for improving production performance of broiler chickens by using microecologics for broiler chickens |
| CN103478443A (en) * | 2013-08-20 | 2014-01-01 | 沈阳金科丰牧业科技有限公司 | Micro-ecological preparation and application thereof in improvement of production performance, immune performance as well as chicken quality of broiler |
| CN103416584A (en) * | 2013-08-20 | 2013-12-04 | 沈阳科丰牧业科技有限公司 | Method for improving broiler chicken quality by microecologics |
| CN103478443B (en) * | 2013-08-20 | 2014-12-03 | 沈阳金科丰牧业科技有限公司 | Micro-ecological preparation and application thereof in improvement of production performance, immune performance as well as chicken quality of broiler |
| CN104686842A (en) * | 2013-08-20 | 2015-06-10 | 沈阳金科丰牧业科技有限公司 | Application of complex bacteria agent for broiler chickens in preparation of boiler chicken immunity enhancing products |
| CN104738340A (en) * | 2013-08-20 | 2015-07-01 | 沈阳科丰牧业科技有限公司 | Method for improving chicken quality of broiler chicken by employing micro-ecological preparation for broiler chicken |
| CN105255783B (en) * | 2013-08-20 | 2018-10-26 | 沈阳金科丰牧业科技有限公司 | Composite bacteria agent and its application in improving meat chicken production performance and broiler chicken chicken meat quality |
| CN105255783A (en) * | 2013-08-20 | 2016-01-20 | 沈阳金科丰牧业科技有限公司 | Complex inoculum and application of complex inoculum in improving production property and immunity property of broiler chickens |
| CN103421720A (en) * | 2013-08-20 | 2013-12-04 | 沈阳金科丰牧业科技有限公司 | Complex microbial inoculant for broiler chickens and application thereof to improvement of broiler chicken productivity and immunity |
| CN103421720B (en) * | 2013-08-20 | 2015-12-23 | 沈阳金科丰牧业科技有限公司 | Broiler chicken composite fungus agent and the application in raising meat chicken production performance and immune performance thereof |
| CN103416585A (en) * | 2013-08-20 | 2013-12-04 | 沈阳科丰牧业科技有限公司 | Method for improving boiler chicken production performance and chicken quality by microecologics |
| CN103431226B (en) * | 2013-08-20 | 2015-11-18 | 沈阳科丰牧业科技有限公司 | Broiler chicken probiotics is utilized to improve the method for meat chicken production performance |
| CN105146205A (en) * | 2013-08-20 | 2015-12-16 | 沈阳科丰牧业科技有限公司 | Method for improving broiler chicken production performance by means of microecological preparations for broiler chickens |
| CN104839444A (en) * | 2015-05-28 | 2015-08-19 | 河南双成生物科技有限公司 | Production method of functional protein feed rich in three essential amino-acids |
| CN105010757A (en) * | 2015-06-30 | 2015-11-04 | 西南大学 | Application of calcium poly-malate in the preparation of feed acidifier |
| CN105010757B (en) * | 2015-06-30 | 2018-05-11 | 西南大学 | Application of the polymalic acid calcium in fodder acidulant is prepared |
| CN104904994A (en) * | 2015-07-14 | 2015-09-16 | 谷实农牧集团股份有限公司 | Three major types of bacteria mixed liquid fermentation, solid yeast feed additive and preparation method thereof |
| KR20180050093A (en) * | 2016-11-04 | 2018-05-14 | 주식회사 바이오리더스 | Composition for Prebiotics Containing Poly-Gamma-Glutamate |
| KR102440573B1 (en) | 2016-11-04 | 2022-09-06 | 주식회사 비엘 | Composition for Prebiotics Containing Poly-Gamma-Glutamate |
| CN107183379A (en) * | 2017-06-05 | 2017-09-22 | 时运昌 | A kind of bacteria agent pig feed additive |
| CN110024913A (en) * | 2019-04-09 | 2019-07-19 | 南京师范大学 | One primary yeast tunning and the compound of polyglutamic acid and the preparation method and application thereof |
| CN112501154A (en) * | 2020-11-02 | 2021-03-16 | 河北科技大学 | Extraction reagent, extraction method and kit for RNA in yeast cells |
| CN117186395A (en) * | 2023-09-13 | 2023-12-08 | 南京轩凯生物科技股份有限公司 | Preparation method and application of calcium polyglutamate |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101999525B (en) | 2012-11-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101999525B (en) | Composite probiotics feed additive | |
| CN109251874A (en) | A kind of probiotics preparation and its preparation method and application | |
| CN101919494B (en) | Complex microorganism additive as well as preparation method and application thereof | |
| CN103988977B (en) | Feeding micro-ecological preparation and preparation method thereof | |
| CN101278702B (en) | Novel microbial feed additive and method of producing the same | |
| CN105483050B (en) | One lactobacillus plantarum and its application in fermented feed | |
| CN108013248A (en) | One boar fermentable fiber feed and preparation method thereof | |
| CN106260504B (en) | Method for producing microbial fermentation wet feed by using beer yeast paste | |
| CN104012803B (en) | A kind of preparation method of the fermented feed that can prevent mastitis for milk cows | |
| CN103088010A (en) | Biological health padding and preparation method thereof | |
| CN108208335A (en) | A kind of technique for improving cotton dregs Forage Nutritive Value using microbe fermentation method | |
| CN101971920B (en) | Porcine lactobacillus reuteri lyophilized preparation and preparation method thereof | |
| CN105432935A (en) | Production method for functional amino-acid humic-acid microecological preparation for aquatic animal and poultry | |
| CN100584209C (en) | Feed level microbe additive and its preparation and application | |
| CN109022333A (en) | A kind of preparation method and applications of composite microbial fermentation bacteria agent | |
| CN110447765A (en) | A kind of method and its application preparing bafillus natto culture using distillers ' grains | |
| CN103053791A (en) | Grain-saving and environmentally-friendly microbial fermented feed and manufacturing method thereof | |
| CN107801837A (en) | A kind of fodder utilizes the bio-fermentation process of chicken manure | |
| CN105543131A (en) | Compound bacteria/cottonseed meal fermented feed and preparation method thereof | |
| CN106962594A (en) | A kind of Selenium-enriched fermentation dregs of beans, preparation method and application | |
| CN103184174A (en) | Production method of bacillus subtilis biological agent used for sodium humate-containing feed in medium | |
| CN113215051A (en) | Method for preparing feed probiotics by using lactobacillus through rice flour wastewater and passion fruit peel | |
| CN110269146A (en) | A kind of probiotics applied on feed | |
| CN105132311B (en) | Utilize the method for glutathione waste liquid production functional microorganism | |
| CN101933946A (en) | Preparation of bacillus subtilis preparation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20121121 Termination date: 20131102 |