CN101530160B - A kind of preparation method of lactobacillus microecological preparation - Google Patents
A kind of preparation method of lactobacillus microecological preparation Download PDFInfo
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Abstract
The invention pertains to the field of microbial feed additives and relates to a preparation method of a microecological preparation of lactobacillus. The preparation method has the advantages of simple requirements on technological preparation equipment, easy and convenient operation, low production cost, stable product quality and the like and is applicable to large-scale mass production. The Prepared lactobacillus powder has higher endurance capacity than other common bacterium powder and can smoothly pass though stomach and enter the intestinal tract of animals to rapidly work. In the preparation method of the microecological preparation of lactobacillus, products produced by the production technology are pretty much the same as those produced by the currently common freeze-drying technology in respect of quality, but the production cost greatly reduces. Therefore, the preparation method is beneficial to the large-scale promotion and application of the microecological preparation of lactobacillus in the feed industry.
Description
Technical field
The invention belongs to the additive for microbe feedstuff field, relate to a kind of preparation method of microecological preparation of lactobacillus.
Background technology
Probiotics be with to the useful microbial safety bacterial classification of animal health and animal microecological balance etc. by bioengineering, concentrate, a kind of green safety that technology such as compound compatibility is developed, no medicine stress with the novel animal health agent of medicament residue, this kind preparation can promote the animal gastrointestinal tract microecological balance or strengthen non-specific immune function, thereby prevent disease, and improve food conversion ratio, promote the growth of animal effect.The development and use starting of China's probiotics is than later, although the rapid all desirable without comparison always product of development emerges.
The little ecological product major part of China all is a bacillus preparation, Bacillus acidi lactici is exactly a crucial class bacterium in this kind preparation, the materials such as lactic acid that this type of bacterium produces can suppress the breeding of enteron aisle spoilage organisms, pathogenic bacteria, keep the balance of intestinal environment, and have effect such as immunity resurrection.But general Bacillus acidi lactici is poor to the resistance of the poor environment factors such as drying, high temperature, hydrochloric acid in gastric juice and bile, and the kind and the quantity that can enter the active Bacillus acidi lactici of enteron aisle and maintenance are few, can not play due effect.Lactobacillus preparation is also mostly because of the resistance difference product proterties instability to poor environment, thereby do not having basic progress aspect antibiotic replacement.
At present, the production of China's feedstuff industry probiotics is except that the bacterial classification problem, and preparation technology influences another subject matter that product can't actually enter industrialization.It is the normal a kind of production method that adopts in the industry that solid fermentation method is produced probiotics, and the product stability that this method obtains is relatively poor.Adopt liquid high density fermentation and spray-dired cooperation technology can produce the viable count height, stay-in-grade product, but this kind technology only is fit to form the heat-resisting bacterial classification of gemma.Using at present more at the temperature-sensitive bacterial strain is to adopt freeze drying technology, and this technology can well guarantee that the activity of bacterial strain in dry process keeps, but production cost is higher.
Summary of the invention
The objective of the invention is to overcome current microecological preparation of lactobacillus and have problem of poor quality, that production cost is high, satisfy the all-round popularization of little ecological class feed addictive and use.Microecological preparation of lactobacillus preparation method of the present invention has advantages such as the requirement of preparation technology's equipment is simple, easy to operation, low production cost, constant product quality, be applicable to large-scale mass production, prepared bacterium powder is compared with other common bacteria powder has higher tolerance, can enter animal intestinal by stomach smoothly and play a role the growth of spoilage organisms in the inhibition enteron aisle rapidly.This production technology is being more or less the same qualitatively with the product that the freeze-dry process of using always at present makes, but production cost but significantly reduces, and helps microecological preparation of lactobacillus promoting the use of in the feed industry on a large scale.
The objective of the invention is to be achieved through the following technical solutions:
The invention provides a kind of preparation method of microecological preparation of lactobacillus, comprise the steps:
(1) expansion of Bacillus acidi lactici is cultivated
1. one-level is cultivated: adopt conventional MRS culture medium as slant medium, preparation first order seed inclined-plane, was cultivated 24-30 hour by 37 ℃;
2. secondary Liquid Culture: use fermentation medium, under 37 ℃, pH4.5-6.0,150-180rpm shaken cultivation 16-18 hour;
3. three grade fermemtation is cultivated: adopt the used fermentation medium of secondary Liquid Culture, cultivate in fermentation tank, inoculum concentration is 0.5-3%, liquid amount 60-80%, rotating speed 80-120rpm, ventilation is than 1: 0.2-1: 0.5,37 ℃ of temperature, pH4.5-5.0 cultivated 18-24 hour, got zymotic fluid;
(2) lactobacillus fermentation liquid concentrates
Zymotic fluid is undertaken centrifugal by tube centrifuge, rotating speed is 16000-20000rpm, and charging rate is 0.5-1kg/min, is lower than 1 * 10 to the zymotic fluid viable count
5Cfu/ml, when water content is 75-95%, stop centrifugal, bacterium mud;
(3) granulation of Bacillus acidi lactici
In bacterium mud, add carrier protecting agent, be divided into 30%-50% to bacterium muddy water, bacterium mud mixture, this mixture by rotation wet granulator extruder grain, is prepared the Bacillus acidi lactici particle, and control particle aperture is 0.5-0.8mm, particle length is 1-3mm, uniform particles degree 85%-100%;
(4) drying of Bacillus acidi lactici particle
The Bacillus acidi lactici particle of step (3) is carried out drying by the low temperature fluid bed handles, EAT 60-80 ℃, leaving air temp 30-40 ℃, to pellet moisture be 5%-12%, dried product is the Bacillus acidi lactici pulvis;
(5) composite
The Bacillus acidi lactici pulvis is mixed with feed vector, promptly get microecological preparation of lactobacillus.
Wherein, the prescription of described fermentation medium is: glucose 1-5%, yeast extract 0.05-3%, soy peptone 0.05-3%, ammonium sulfate 0.05-4%, calcium carbonate 0.2-5%.
The carrier protecting agent that adds in the granulation process of Bacillus acidi lactici can be starch and/or starch derivatives, and it is that 30%-50% selects that addition mixes the back moisture content of material by control.
Feed vector described in the present invention comprises solvable type carrier and/or insoluble type carrier, and solvable type carrier comprises: any in glucose, the soluble starch or two kinds; Insoluble type carrier comprises: in corn core material, albumen powder, wheatfeed, oily chaff, rice bran, the zeolite powder any, two kinds or multiple mixture.
Microecological preparation of lactobacillus preparation method of the present invention has advantages such as the requirement of preparation technology's equipment is simple, easy to operation, low production cost, constant product quality, is applicable to large-scale mass production; Compare with the bacterium powder that other common methods makes by the prepared bacterium powder of this preparation method and to have higher tolerance, can enter animal intestinal by stomach smoothly also plays a role rapidly, suppress the growth of spoilage organisms in the enteron aisle, can promote growth of animal and improve food conversion ratio, can reach the purpose that replaces antibiotic or steroids product, thereby solve the serious problem of livestock products medicament residue, improve breeding environment; This preparation technology is not having too big difference qualitatively with the product that the freeze-dry process of using always at present makes, but production cost but significantly reduces, and helps microecological preparation of lactobacillus promoting the use of in the feed industry on a large scale.
, it should be understood that described embodiment only is for the present invention is described, rather than limit the scope of the invention by any way more specific description the present invention by the following example.
The specific embodiment
The preparation of 1 one kinds of insoluble type microecological preparation of lactobacillus of embodiment
(1) expansion of Bacillus acidi lactici is cultivated
1. one-level is cultivated: adopt conventional MRS culture medium as slant medium, preparation first order seed inclined-plane, was cultivated 26 hours by 37 ℃;
2. the secondary Liquid Culture adopts fermentation medium, and 37 ℃, ph4.5-6.0, shaken cultivation is 18 hours under the 150rpm;
The prescription of fermentation medium is: glucose 3.2%, yeast extract 1.7%, soy peptone 2.1%, ammonium sulfate 2.7%, calcium carbonate 3.1%;
3. three grade fermemtation cultivation adopts secondary to cultivate used fermentation medium, in fermentation tank, cultivates, and inoculum concentration 1%, liquid amount 60%, rotating speed 80rpm, ventilation ratio 1: 0.2,37 ℃ of temperature, pH4.5-5.0 cultivated 22 hours, got zymotic fluid;
Viable count in the zymotic fluid is 4.5 * 10 at this moment
9Cfu/ml;
(2) lactobacillus fermentation liquid concentrates
Zymotic fluid is undertaken centrifugal by tube centrifuge, rotating speed is 16000rpm, and charging rate is 0.5kg/min, is lower than 1 * 10 to the zymotic fluid viable count
5Cfu/ml, water content is 80% o'clock, stop centrifugal, bacterium mud;
(3) granulation of Bacillus acidi lactici
In bacterium mud, add starch, be divided into 45% to bacterium muddy water, bacterium mud mixture, with this mixture by rotation wet granulator extruder grain, preparation Bacillus acidi lactici particle, control particle aperture is 0.8mm, particle length is 1-3mm;
(4) drying of Bacillus acidi lactici particle
The Bacillus acidi lactici particle of step (3) is carried out drying by the low temperature fluid bed handles, 60 ℃ of EATs, 40 ℃ of leaving air temps, to pellet moisture be 5%-8%, dried product is the Bacillus acidi lactici pulvis;
(5) composite
Bacillus acidi lactici pulvis and zeolite powder were carried out composite according to 1: 1, promptly get a kind of insoluble type microecological preparation of lactobacillus.
This probiotics is applicable to that meat/laying hen feeds
The preparation of 2 one kinds of solvable type microecological preparation of lactobacillus of embodiment
(1) expansion of Bacillus acidi lactici is cultivated
1. one-level is cultivated: adopt conventional MRS culture medium as slant medium, preparation first order seed inclined-plane, was cultivated 30 hours by 37 ℃;
2. the secondary Liquid Culture adopts fermentation medium, and 37 ℃, ph4.5-6.0, shaken cultivation is 18 hours under the 170rpm;
The prescription of fermentation medium is: glucose 5%, yeast extract 3%, soy peptone 3%, ammonium sulfate 4%, calcium carbonate 5%;
3. three grade fermemtation cultivation adopts secondary to cultivate used fermentation medium, in fermentation tank, cultivates, and inoculum concentration 0.5%, liquid amount 80%, rotating speed 120rpm, ventilation ratio 1: 0.5,37 ℃ of temperature, pH4.5-5.0 cultivated 24 hours, got zymotic fluid;
Viable count in the zymotic fluid is 3.9 * 10 at this moment
9Cfu/ml;
(2) lactobacillus fermentation liquid concentrates
Zymotic fluid is undertaken centrifugal by tube centrifuge, rotating speed is 18000rpm, and charging rate is 0.8kg/min, is lower than 1 * 10 to the zymotic fluid viable count
5Cfu/ml, water content is 95% o'clock, stop centrifugal, bacterium mud;
(3) granulation of Bacillus acidi lactici
In bacterium mud, add soluble starch, be divided into 50% to bacterium muddy water, bacterium mud mixture, with this mixture by rotation wet granulator extruder grain, preparation Bacillus acidi lactici particle, control particle aperture is 0.5-0.8mm, particle length is 1-3mm;
(4) drying of Bacillus acidi lactici particle
The Bacillus acidi lactici particle of step (3) is carried out drying by the low temperature fluid bed handles, 80 ℃ of EATs, leaving air temp 30-40 ℃, to pellet moisture be 5%-12%, dried product is the Bacillus acidi lactici pulvis;
(5) composite
Bacillus acidi lactici pulvis and glucose, soluble starch were carried out composite according to 1: 0.5: 0.5, promptly get a kind of solvable type microecological preparation of lactobacillus.
This preparation is applicable to that sow in lactation feeds.
The preparation of 3 one kinds of solvable type microecological preparation of lactobacillus of embodiment
(1) expansion of Bacillus acidi lactici is cultivated
1. one-level is cultivated: adopt conventional MRS culture medium as slant medium, preparation first order seed inclined-plane, was cultivated 28 hours by 37 ℃;
2. the secondary Liquid Culture adopts fermentation medium, and 37 ℃, ph4.5-6.0, shaken cultivation is 18 hours under the 180rpm;
The prescription of fermentation medium is: glucose 1%, yeast extract 0.05%, soy peptone 0.05%, ammonium sulfate 0.05%, calcium carbonate 0.2%;
3. three grade fermemtation cultivation adopts secondary to cultivate used fermentation medium, in fermentation tank, cultivates, and inoculum concentration 3%, liquid amount 70%, rotating speed 90rpm, ventilation ratio 1: 0.4,37 ℃ of temperature, pH4.5-5.0 cultivated 18 hours, got zymotic fluid;
Viable count in the zymotic fluid is 2.8 * 10 at this moment
9Cfu/ml;
(2) lactobacillus fermentation liquid concentrates
Zymotic fluid is undertaken centrifugal by tube centrifuge, rotating speed is 20000rpm, and charging rate is 1kg/min, is lower than 1 * 10 to the zymotic fluid viable count
5Cfu/ml, water content is 85% o'clock, stop centrifugal, bacterium mud;
(3) granulation of Bacillus acidi lactici
In bacterium mud, add maltodextrin, be divided into 30% to bacterium muddy water, bacterium mud mixture, with this mixture by rotation wet granulator extruder grain, preparation Bacillus acidi lactici particle, control particle aperture is 0.5-0.8mm, particle length is 1-3mm;
(4) drying of Bacillus acidi lactici particle
The Bacillus acidi lactici particle of step (3) is carried out drying by the low temperature fluid bed handles, 70 ℃ of EATs, 40 ℃ of leaving air temps, to pellet moisture be 5%-12%, dried product is the Bacillus acidi lactici pulvis;
(5) composite
Bacillus acidi lactici pulvis and glucose were carried out composite according to 1: 1, promptly get a kind of solvable type microecological preparation of lactobacillus.
This microecological preparation of lactobacillus is applicable to that newborn piglet feeds.
The preparation of 4 one kinds of insoluble type microecological preparation of lactobacillus of embodiment
(1) expansion of Bacillus acidi lactici is cultivated
1. one-level is cultivated: adopt conventional MRS culture medium as slant medium, preparation first order seed inclined-plane, was cultivated 26 hours by 37 ℃;
2. the secondary Liquid Culture adopts fermentation medium, and 37 ℃, ph4.5-6.0, shaken cultivation is 18 hours under the 150rpm;
The prescription of fermentation medium is: glucose 3.2%, yeast extract 1.7%, soy peptone 2.1%%, ammonium sulfate 2.7%, calcium carbonate 3.1%;
3. three grade fermemtation cultivation adopts secondary to cultivate used fermentation medium, in fermentation tank, cultivates, and inoculum concentration 1%, liquid amount 60%, rotating speed 80rpm, ventilation ratio 1: 0.2,37 ℃ of temperature, pH4.5-5.0 cultivated 22 hours, got zymotic fluid;
Viable count in the zymotic fluid is 4.5 * 10 at this moment
9Cfu/ml;
(2) lactobacillus fermentation liquid concentrates
Zymotic fluid is undertaken centrifugal by tube centrifuge, rotating speed is 16000rpm, and charging rate is 0.5kg/min, to water content be 80% o'clock, stop centrifugal, bacterium mud;
(3) granulation of Bacillus acidi lactici
In bacterium mud, add sour modified starch, be divided into 30%-50% to bacterium muddy water, bacterium mud mixture, this mixture by rotation wet granulator extruder grain, is prepared the Bacillus acidi lactici particle, and control particle aperture is 0.5-0.8mm, particle length is 1-3mm, uniform particles degree 85%-100%;
(4) drying of Bacillus acidi lactici particle
The Bacillus acidi lactici particle of step (3) is carried out drying by the low temperature fluid bed handles, 60 ℃ of EATs, 30 ℃ of leaving air temps, to pellet moisture be 5%-12%, dried product is the Bacillus acidi lactici pulvis;
(5) composite
Bacillus acidi lactici particle and albumen powder, zeolite powder were carried out composite according to 1: 0.2: 0.8, promptly get a kind of insoluble type probiotics.
This preparation is applicable to the chick nursing.
Embodiment 5 microecological preparation of lactobacillus are to the influence of piglet production performance
Experimental animal: select 35 ages in days wean for use, the length of body weight about 9kg is white * and Da Bai hybridization piglet is as experimental animal, and totally 120, test pig is from the horse's mouth, and a collection of choosing is neat, and the date of birth differs and is no more than 7 days.
Test material: microecological preparation of lactobacillus (viable count 〉=2 * 10 of getting the embodiment of the invention 3
9Cfu/g);
Basal diet: control group all uses the identical creep feed of trophic level, weaning pigs and piggy to expect the mixed feed of preparation with the test group pig;
Test method: test pig is divided into control group and experimental group at random according to the close principle of average weight.Control group weanling pig and piggy do not add any additives.All do not add the antibiotics growth accelerator in the test group I feed, add 0.2% Bacillus acidi lactici pulvis.Test group II adds the antibiotics growth accelerator routinely.All each was organized experimental period and is 42 days with antibiotic drinking-water prevention diarrhoea and breathing problem in 5-7 days to wean back in wean for test group and control group, and it is identical that each organizes the raising condition, and feeding and management is carried out according to the normal feeding and management system in pig farm.
Test index and method: on an empty stomach pig is only weighed morning in on-test with when finishing, be respectively that unit carries out full group and weighs, calculates indexs such as feed consumption rate, daily gain, feedstuff-meat ratio, diarrhea rate with the repeating groups, and carry out otherness and significantly check.
Result of the test: the production performance record analysis table of test piglet
| Group | Beginning counterpoise (kg) | End counterpoise (kg) | Equal daily gain (g/ head) | Average feed consumption (the g/ head. day) | Feedstuff-meat ratio | Diarrhea rate (%) |
| Control group | 8.8 | 23.82 | 501 | 891 | 1.78 | 11.5 |
| Test group I | 8.7 | 24.33 | 520 | 844 | 1.62 | 4.3 |
| Test group II | 8.9 | 24.40 | 517 | 873 | 1.69 | 5.9 |
Test result analysis: show that according to last table test group I increases day by day, reduces and have a clear superiority in than control group and test group II aspect feedstuff-meat ratio, the diarrhea rate improving piglet, and the test group I piglet of find adding " microecological preparation of lactobacillus " that the present invention produces in experimental observation is added that antibiotic test group II and control group piglet show that the state of mind is good, honey stomach, ill number of times is few, fur is smooth, and institute's ight soil stink that draws reduces.Illustrate that " microecological preparation of lactobacillus " that adopt the present invention to produce not only can improve the production performance of piglet, reduces aquaculture cost, and can improve the feeding environment on piglet health status and pig house and pig farm.
Claims (2)
1. the preparation method of a microecological preparation of lactobacillus is characterized in that step is as follows:
(1) expansion of Bacillus acidi lactici is cultivated
1. one-level is cultivated: adopt conventional MRS culture medium as slant medium, preparation first order seed inclined-plane, was cultivated 24-30 hour by 37 ℃;
2. secondary Liquid Culture: use fermentation medium, under 37 ℃, pH4.5-6.0,150-180rpm shaken cultivation 16-18 hour;
3. three grade fermemtation is cultivated: adopt the used fermentation medium of secondary Liquid Culture, cultivate in fermentation tank, inoculum concentration is 0.5-3%, liquid amount 60-80%, rotating speed 80-120rpm, ventilation is than 1: 0.2-1: 0.5,37 ℃ of temperature, pH4.5-5.0 cultivated 18-24 hour, got zymotic fluid;
(2) lactobacillus fermentation liquid concentrates
Zymotic fluid is undertaken centrifugal by tube centrifuge, rotating speed is 16000-20000rpm, and charging rate is 0.5-1kg/min, is lower than 1 * 10 to the zymotic fluid viable count
5Cfu/ml, when water content is 75-95%, stop centrifugal, bacterium mud;
(3) granulation of Bacillus acidi lactici
In bacterium mud, add carrier protecting agent, be divided into 30%-50% to bacterium muddy water, bacterium mud mixture, this mixture by rotation wet granulator extruder grain, is prepared the Bacillus acidi lactici particle, and control particle aperture is 0.5-0.8mm, particle length is 1-3mm, uniform particles degree 85%-100%;
(4) drying of Bacillus acidi lactici particle
The Bacillus acidi lactici particle of step (3) is carried out drying by the low temperature fluid bed handles, EAT 60-80 ℃, leaving air temp 30-40 ℃, to pellet moisture be 5%-12%, dried product is the Bacillus acidi lactici pulvis;
(5) composite
The Bacillus acidi lactici pulvis is mixed with feed vector, promptly get microecological preparation of lactobacillus;
Wherein, the prescription of described fermentation medium is: glucose 1-5%, yeast extract 0.05-3%, soy peptone 0.05-3%, ammonium sulfate 0.05-4%, calcium carbonate 0.2-5%; Described carrier protecting agent is starch and/or starch derivatives.
2. the preparation method of microecological preparation of lactobacillus as claimed in claim 1 is characterized in that described feed vector comprises solvable type carrier and/or insoluble type carrier, and solvable type carrier comprises: any in glucose, the soluble starch or two kinds; Insoluble type carrier comprises: in corn core material, albumen powder, wheatfeed, oily chaff, rice bran, the zeolite powder any, two kinds or multiple mixture.
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| CN102524570B (en) * | 2011-12-30 | 2013-04-03 | 青岛赛博迪科技有限公司 | Method for preparing flavored pig cultivation feed |
| CN102696883B (en) * | 2012-05-23 | 2015-01-14 | 浙江大学 | Feed additive and application of feed additive |
| CN102987055B (en) * | 2012-10-17 | 2014-04-23 | 李雪平 | Compound encapsulated lactic acid bacteria product and preparation method thereof |
| CN103405479B (en) * | 2013-06-26 | 2016-02-24 | 浙江省农业科学院 | A kind of Lemonal for prevention and control cultivation later stage shrimp pond vibrios |
| TWI486323B (en) * | 2013-07-31 | 2015-06-01 | Advanced Green Biotechnology Inc | Manufacturing method of a granular microbial fertilizer |
| CN104664055B (en) * | 2015-03-23 | 2017-11-10 | 四川高福记生物科技有限公司 | A kind of feeding lactobacillus micropill |
| CN104642746B (en) * | 2015-03-23 | 2017-09-15 | 四川高福记生物科技有限公司 | A kind of preparation method of feeding lactobacillus micropill |
| CN107006720A (en) * | 2017-04-12 | 2017-08-04 | 湖北华扬科技发展有限公司 | A kind of probiotics and its application |
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Effective date of registration: 20180115 Address after: 100080 Zhongguancun street, Haidian District, Zhongguancun building, No., layer 27, 14 Co-patentee after: SHANDONG FENGWO XINNONG AGRICULTURAL AND ANIMAL HUSBANDRY SCIENCE AND TECHNOLOGY CO.,LTD. Patentee after: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. Address before: 100083, Zhongguancun building, No. 27 Zhongguancun street, Beijing, Haidian District, China, 14 floor Patentee before: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. |
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Effective date of registration: 20191212 Address after: 363502, Fujian, Zhangzhou province Zhaoan County gold industrial concentration zone Co-patentee after: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. Patentee after: FUJIAN DABEINONG FISHERIES SCIENCE & TECHNOLOGY Co.,Ltd. Co-patentee after: SHANDONG FENGWO XINNONG AGRICULTURAL AND ANIMAL HUSBANDRY SCIENCE AND TECHNOLOGY CO.,LTD. Address before: 100080, Zhongguancun building, No. 27 Zhongguancun street, Beijing, Haidian District, China, 14 floor Co-patentee before: SHANDONG FENGWO XINNONG AGRICULTURAL AND ANIMAL HUSBANDRY SCIENCE AND TECHNOLOGY CO.,LTD. Patentee before: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. |
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Address after: 363502 Jindu Industrial Concentration Zone, Zhaoan County, Zhangzhou City, Fujian Province Patentee after: Fujian Dabei nonghuayou Aquatic Technology Group Co.,Ltd. Patentee after: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. Patentee after: SHANDONG FENGWO XINNONG AGRICULTURAL AND ANIMAL HUSBANDRY SCIENCE AND TECHNOLOGY CO.,LTD. Address before: 363502 Jindu Industrial Concentration Zone, Zhaoan County, Zhangzhou City, Fujian Province Patentee before: FUJIAN DABEINONG FISHERIES SCIENCE & TECHNOLOGY Co.,Ltd. Patentee before: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. Patentee before: SHANDONG FENGWO XINNONG AGRICULTURAL AND ANIMAL HUSBANDRY SCIENCE AND TECHNOLOGY CO.,LTD. |