CN1306025C - Culture medium its rpeparation method and method for culturing lact-streptocin - Google Patents
Culture medium its rpeparation method and method for culturing lact-streptocin Download PDFInfo
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- CN1306025C CN1306025C CNB2005100719453A CN200510071945A CN1306025C CN 1306025 C CN1306025 C CN 1306025C CN B2005100719453 A CNB2005100719453 A CN B2005100719453A CN 200510071945 A CN200510071945 A CN 200510071945A CN 1306025 C CN1306025 C CN 1306025C
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- nisin
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- yeast
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Abstract
The present invention discloses a culture medium used for producing nisin and a preparation method thereof, and a method for culturing nisin. The preparation method for nisin comprises: yeast protein and soybean protein are put in a compounding tank, water is added into the mixture, and the mixture is uniformly stirred; then a carbon source, sodium phosphate and magnesium sulfate are orderly put into the compounding tank, the mixture is uniformly stirred, and after the pH is regulated to 6.2 to 7.2, the mixture is delivered into a culture tank; water is added to the mixture, high pressure steam is led into the mixture, the mixture is sterilized and cooled, food grade lactic acid bacteria are inoculated into the mixture, and the mixture is cultured for 24 to 72 hours under the conditions in the tank that the pressure is from 0.1MPa to 0.5MPa, and the temperature is from 26 DEG C to 37 DEG C; when nisin activity reaches 4000 to 15000 IU/ml through estimation of biological potency, the culture can be stopped. The culture medium is beneficial to the metabolism of lactococcus lactis, and has the advantages that the time for entering the log phase is shortened, the metabolism is vigorous, the consumption of sugar and nitrogen is accelerated, the decline phase is delayed, the nisin activity is greatly increased through the estimation of biological potency, and the output is greatly increased.
Description
Technical field
The present invention relates to a kind of substratum, specifically relate to a kind of substratum that is used to produce nisin, its preparation method and the method for cultivating nisin.
Technical background
Nisin is a kind of little peptide that is produced by some streptococcus acidi lactici (Lactococcus Iactis), and molecular weight is about 3510Da.This bacterium is to many gram-positive microorganisms, especially the genus bacillus that causes food spoilage, clostridium, staphylococcus, Listera etc. there is strong inhibitory or killing effect, again in view of can be by enzyme liberating in the digestive tube, be a kind of antiseptics for natural food nontoxic, therefore be widely used in the antisepsis antistaling agent of milk-product, tinned food, meat product and beverage human body.
Adopt the plain Z high productive mutant of Lactococcus lactis Lactococcus lactis AL2 for producing bacterial strain in Institute of Microorganism, Academia Sinica's research, replace milk to make substratum with cheap raw materials such as peptones, adopt back extraction process route, carry out the commercial scale production test at 20 tons of fermentor tanks, average fermentation is tired and is 3009IU/ml, and the back is extracted total recovery and reached 60.9%.
People such as Yang Bo are at the optimal conditions of fermentation of Lactococcus lactis element: the 18th the 1st phase of volume of Dalian Polytechnic College journal, when 1999,365-69 carried out the optimal conditions of fermentation of Lactococcus lactis element, fermention medium was: soy peptone 1.0%, extractum carnis 0.25%, yeast extract paste 0.25%, Na
2HPO
412H
2O21.5%, MgSO
47H
2O 0.012%, natural pH value; The Lactococcus lactis element is tired and is detected substratum and be: Tryptones 0.8%, and yeast extract paste 0.5%, NaCl 0.5%, NaHPO
412H
2O 0.2%, agar 1.5%, pH6.8; Agents useful for same is: the plain standard substance of Lactococcus lactis: Aplin ﹠amp; Barrett LTD produces, Tryptones: Oxoid LTD produces; Many peptones: Japan produces, and other substratum are Haidian District, Beijing City microbiological culture media products factory production.
Cultivate in the prior art of streptococcus acidi lactici, adopt the substratum that contains nitrogenous source mostly.Production has remarkably influenced to different nitrogen sources to nisin, and milk-acid bacteria is fastidious unusually to it.Nitrogenous source with organonitrogen for well.Milk and yeast powder are two kinds of nitrogenous sources using always.The two is to the not influence of activity of finished product, but color and luster is slightly variant, and one is oyster white, and one is light brown or slightly canescent powder.Measure the light absorption value of its solution 620nm, milk be the nisin product of raw material because of containing the sex change milk constituents, its light absorption value is anti-to be the height of raw material with the yeast powder, is respectively 1.0 and 0.3.As in complex medium, adding cottonseed meal will improve nisin as nitrogenous source output.De Vuyst finds that the nisin biosynthesizing depends on sulfur-bearing inorganic salt (MgSO
4, Sulfothiorine) or sulfur-containing amino acid (methionine(Met), halfcystine) or these sulphur sources of cystathionine.Serine, Threonine and halfcystine are the precursors in the nisin biosynthesizing, can regulate the generation of nisin, but do not influence the final cell amount.Do not find NH
4 +Restraining effect or promoter action to Lactococcus lactis.
Though above-mentioned substratum can guarantee the basal metabolism of Lactococcus lactis and synthesizing of nisin, but milk and extractum carnis etc. are animal protein, the cost height, raw material is influenced by the place of production, the quality control difficulty is big, in long-distance transportation and storage, often must add some antisepsis antistaling agents, will influence the normal fermentation of streptococcus acidi lactici.Simultaneously finished product will be hindered when carrying out Kosher relevant authentications such as (Jew), be difficult to by, directly influence the product application scope.Though the soy peptone molecular ratio is less, the nutritive ingredient trend is single, and manufacturing processed also causes the loss of some nutrient as trace element and carbon source.Therefore substratum waits to optimize, improve, to adapt to the requirement that improves the product productive rate.The present invention adopts suitable yeast proteinoid and soybean protein compound, has overcome original technology and has caused the unit cost height, the defective that productive rate is low.
Summary of the invention
The purpose of this invention is to provide a kind of technology and cause the substratum that is used to produce nisin that unit cost is low, productive rate is high.
The invention provides a kind of substratum, comprise that by weight percentage following raw material processing and preparing forms: yeast proteinoid: 0.5-2%, soybean protein: 0.1-0.9%, carbon source: 1.0-4.0% also has water.
Preferably, comprise that by weight percentage following raw material processing and preparing forms:
Yeast proteinoid: 0.5-2%, soybean protein: 0.1-0.9%, carbon source: 1.0-4.0%, sodium phosphate: 0.1-0.5%, sal epsom 0.1-0.5% also contains water.
The yeast proteinoid is preferably yeast powder, yeast extract.
Soybean protein is preferably soybean and made powder, protein isolate and the soybean hydrolyzate of dregs of beans thereof.
Carbon source is monose, disaccharide and polysaccharide.
The present invention also provides the above-mentioned preparation method who is used to produce the substratum of nisin:
Yeast proteinoid, soybean protein are dropped in the material-compound tank, adding water stirs evenly, then carbon source, sodium phosphate, sal epsom are dropped into successively, stir evenly,, add water with sending in the culture tank behind 29-31% edible hydrochloric acid or the edible alkali lye accent pH6.2-7.2, feed saturated high pressure steam, keeping a jar internal pressure 0.09-0.11MPa, is sterilization 25-35 minute under the 118-121 ℃ of condition in temperature, is cooled to 25-35 ℃ with water coolant indirectly through spiral coil cooling tube.
It is 0.3-1.0 ‰ that above-mentioned substratum inserts food grade lactobacillus inoculum amount, keeping culture tank internal pressure 0.1-0.5MPa, temperature with sterile air is to cultivate 24-72 hour under the 26-37 ℃ of condition, the nisin activity reaches 4000-15000IU/ml through estimation of biological potency, can stop.
After using above-mentioned substratum, favourable to the metabolism of streptococcus acidi lactici, enter logarithmic phase shortening in period, metabolism is vigorous, and sugared nitrogen consumption is accelerated, and the paracme postpones, the nisin activity has very big raising through estimation of biological potency than the 2000-3000IU/ml of prior art, thereby output is improved greatly.
Above-mentioned food grade milk-acid bacteria obtains from Institute of Microorganism, Academia Sinica, and bacterial classification is in this DSMZ's preservation, and bacterial classification number is AL2.
Embodiment
With embodiment technical scheme of the present invention is described below, but does not limit the present invention with embodiment.
Other has except the explanation, and is used in the present invention:
Yeast powder is provided by Shanghai Industrial Co., Ltd. in celestial morning, and lot number is 0504036;
Yeast extract is provided by the bright company of Guangdong one product, and lot number is 05017;
Soyflour is self-control, and soon soybean is put into and is milled to the 150-300 order with paste roller mill after the water immersion made it feel like jelly in 12 hours;
Soybean protein isolate and soybean hydrolyzate are outsourcing, and soybean protein isolate supplier is a Shandong king Yu Group Co.,Ltd, and lot number is 0504011; Soybean hydrolyzate supplier is a Ha Gaoke soybean Food Co., Ltd, and lot number is 20050315;
Monose is glucose, is provided because of tasteable company limited by the Zhejiang shellfish, and lot number is 20050313;
Disaccharide is sucrose, and magnificent sugar industry company provides by east, Guangdong, and lot number is 20050120;
Polysaccharide is a maltose, and making a gift of sugared source mill by the Zhejiang balcony provides, and lot number is 20050305;
Embodiment 1
Comprise following raw material with weight percent:
Yeast powder: 0.5%, soyflour: 0.9%, glucose 1.0%, all the other are deionized water.
Yeast powder and soyflour are dropped in the material-compound tank, adding deionized water stirs evenly, with sending in the culture tank behind the 29% edible hydrochloric acid accent pH6.2, add deionized water, feed saturated high pressure steam, keeping a jar internal pressure 0.10MPa, is sterilization 35 minutes under 118 ℃ of conditions in temperature, is cooled to 25 ℃ with water coolant indirectly through spiral coil cooling tube.
It is 0.3 ‰ that above-mentioned substratum inserts food grade lactobacillus inoculum amount.Keeping culture tank internal pressure 0.5MPa, temperature with sterile air is to cultivate 72 hours under 26 ℃ of conditions, and the nisin activity reaches 4000IU/ml through estimation of biological potency, can stop.
Above-mentioned yeast powder is provided by Shanghai Industrial Co., Ltd. in celestial morning, and lot number is 0504036;
Soyflour is self-control, and soon soybean is put into and is milled to the 150-300 order with paste roller mill after the water immersion made it feel like jelly in 12 hours;
Glucose is provided because of tasteable company limited by the Zhejiang shellfish, and lot number is 20050313.
Embodiment 2
Comprise following raw material with weight percent:
Yeast powder: 0.5%, soybean protein isolate: 0.5%, sucrose: 3.0%, sodium phosphate: 0.5%, all the other are well water.
Yeast powder and soybean protein isolate are dropped in the material-compound tank, adding well water stirs evenly, add sodium phosphate, with sending in the culture tank behind the 31% edible alkali lye accent pH7.0, add well water, feed saturated high pressure steam, keep a jar internal pressure 0.10MPa, in temperature is sterilization 30 minutes under 121 ℃ of conditions, is cooled to 30 ℃ with water coolant indirectly through spiral coil cooling tube.
It is 0.5 ‰ that above-mentioned substratum inserts food grade lactobacillus inoculum amount.Keeping culture tank internal pressure 0.1MPa, temperature with sterile air is to cultivate 24 hours under 37 ℃ of conditions, and the nisin activity reaches 10000IU/ml through estimation of biological potency, can stop.
Above-mentioned yeast powder is provided by Shanghai Industrial Co., Ltd. in celestial morning, and lot number is 0504036;
Soybean protein isolate is outsourcing, and supplier is a Shandong king Yu Group Co.,Ltd, and lot number is 0504011;
Sucrose, magnificent sugar industry company provides by east, Guangdong, and lot number is 20050120.
Embodiment 3
Comprise following raw material with weight percent:
Yeast extract: 1%, the soybean hydrolyzate: 0.3%, maltose: 1.5%, sodium phosphate: 0.1%, sal epsom 0.5%, all the other are tap water.
Yeast extract and soybean hydrolyzate are dropped in the material-compound tank, adding tap water stirs evenly, add sodium phosphate and sal epsom, with sending in the culture tank behind the 30% edible alkali lye accent pH7.2, add tap water, feed saturated high pressure steam, keep a jar internal pressure 0.09MPa, in temperature is sterilization 35 minutes under 120 ℃ of conditions, is cooled to 35 ℃ with water coolant indirectly through spiral coil cooling tube.
It is 1.0 ‰ that above-mentioned substratum inserts food grade lactobacillus inoculum amount.Keeping culture tank internal pressure 0.3MPa, temperature with sterile air is to cultivate 48 hours under 30 ℃ of conditions, and the nisin activity reaches 15000IU/ml through estimation of biological potency, can stop.
Above-mentioned yeast extract is provided by the bright company of Guangdong one product, and lot number is 05017;
The soybean hydrolyzate is provided by Ha Gaoke soybean Food Co., Ltd, and lot number is 20050315;
Maltose, making a gift of sugared source mill by the Zhejiang balcony provides, and lot number is 20050305.
Embodiment 4
Comprise following raw material with weight percent:
Yeast extract: 2%, soybean protein isolate: 0.9%, maltose: 4.0%, sodium phosphate: 0.3%, sal epsom 0.1%, all the other are mineral water.
Yeast extract and soybean protein isolate are dropped in the material-compound tank, stir evenly with mineral water, add sodium phosphate and sal epsom, with sending in the culture tank behind the 30% edible hydrochloric acid accent pH 6.5, with mineral water, feed saturated high pressure steam, keep a jar internal pressure 0.10MPa, in temperature is sterilization 28 minutes under 119 ℃ of conditions, with water coolant through indirectly 25 ℃ of coolings of spiral coil cooling tube.
It is 0.9 ‰ that above-mentioned substratum inserts food grade lactobacillus inoculum amount.Keeping culture tank internal pressure 0.2MPa, temperature with sterile air is to cultivate 60 hours under 35 ℃ of conditions, and the nisin activity reaches 8000IU/ml through estimation of biological potency, can stop.
Above-mentioned yeast extract is provided by the bright company of Guangdong one product, and lot number is 05017;
Soybean protein isolate is outsourcing, and supplier is a Shandong king Yu Group Co.,Ltd, and lot number is 0504011;
Maltose, making a gift of sugared source mill by the Zhejiang balcony provides, and lot number is 20050305.
Embodiment 5
Comprise following raw material with weight percent:
Yeast powder: 1.5%, soybean protein isolate: 0.7%, glucose: 3.0%, sodium phosphate: 0.4%, sal epsom 0.4%, all the other are distilled water.
Yeast powder and soybean protein isolate are dropped in the material-compound tank, adding distil water stirs evenly, add sodium phosphate and sal epsom, with sending in the culture tank behind the 31% edible alkali lye accent pH6.8, adding distil water feeds saturated high pressure steam, keeps a jar internal pressure 0.09MPa, in temperature is sterilization 33 minutes under 120 ℃ of conditions, is cooled to 28 ℃ with water coolant indirectly through spiral coil cooling tube.
It is 0.8 ‰ that above-mentioned substratum inserts food grade lactobacillus inoculum amount.Keeping culture tank internal pressure 0.4MPa, temperature with sterile air is to cultivate 70 hours under 30 ℃ of conditions, and the nisin activity reaches 10000IU/ml through estimation of biological potency, can stop.
Above-mentioned yeast powder is provided by Shanghai Industrial Co., Ltd. in celestial morning, and lot number is 0504036;
Soybean protein isolate is outsourcing, and supplier is a Shandong king Yu Group Co.,Ltd, and lot number is 0504011;
Glucose is provided because of tasteable company limited by the Zhejiang shellfish, and lot number is 20050313.
Embodiment 6
Comprise following raw material with weight percent:
Yeast extract: 0.5%, soybean hydrolyzate 1.2%, glucose: 3.5%, sodium phosphate: 0.4%, sal epsom 0.4%, all the other are pure water.
Yeast extract and soybean hydrolyzate are dropped in the material-compound tank, adding pure water stirs evenly, add sodium phosphate and sal epsom, with sending in the culture tank behind the 31% edible alkali lye accent pH7.0, add pure water, feed saturated high pressure steam, keep a jar internal pressure 0.10MPa, in temperature is sterilization 30 minutes under 120 ℃ of conditions, is chilled to but 29 ℃ with water coolant indirectly through spiral coil cooling tube.
It is 0.5 ‰ that above-mentioned substratum inserts food grade lactobacillus inoculum amount.Keeping culture tank internal pressure 0.4MPa, temperature with sterile air is to cultivate 30 hours under 30 ℃ of conditions, and the nisin activity reaches 12000IU/ml through estimation of biological potency, can stop.
Above-mentioned yeast extract is provided by the bright company of Guangdong one product, and lot number is 05017;
The soybean hydrolyzate is provided by Ha Gaoke soybean Food Co., Ltd, and lot number is 20050315;
Glucose is provided because of tasteable company limited by the Zhejiang shellfish, and lot number is 20050313.
Claims (12)
1, a kind of method of producing nisin is characterized in that:
Substratum inserts DSMZ of Institute of Microorganism, Academia Sinica, bacterial classification number is the food grade milk-acid bacteria of AL2, in the jar internal pressure is that 0.1-0.5MPa, temperature are to cultivate 24-72 hour under the 26-37 ℃ of condition, the nisin activity reaches 4000-15000IU/ml through estimation of biological potency, can stop.Wherein said substratum comprises that by weight percentage following raw material processing and preparing forms:
Yeast proteinoid: 0.5-2%, soybean protein: 0.1-0.9%, carbon source: 1.0-4.0% also contains water.
2. according to the method for the production nisin of claim 1, it is characterized in that inoculum size is 0.3-1.0 ‰.
3,, it is characterized in that keeping the culture tank internal pressure with sterile air according to the method for the production nisin of claim 1.
4,, it is characterized in that described substratum comprises also that by weight percentage following raw material processing and preparing forms according to the described a kind of method of producing nisin of claim 1:
Sodium phosphate: 0.1-0.5%, sal epsom 0.1-0.5%.
5,, it is characterized in that the yeast proteinoid is a yeast extract according to the described a kind of method of producing nisin of claim 1.
6,, it is characterized in that soybean protein is the made powder of soybean and dregs of beans thereof, soybean protein isolate, soybean hydrolyzate according to the described a kind of method of producing nisin of claim 1.
7,, it is characterized in that carbon source is monose, disaccharide and polysaccharide according to the described a kind of method of producing nisin of claim 1.
8, according to the described a kind of method of producing nisin of claim 4, it is characterized in that the preparation method of described substratum, comprise the steps:
Yeast proteinoid, soybean protein are dropped in the material-compound tank, add water and stir evenly, then carbon source, sodium phosphate, sal epsom are dropped into successively, stir evenly, send in the culture tank behind the accent pH6.2-7.2, add water, feed high pressure steam, sterilization, cooling.
9,, it is characterized in that with 29-31% edible hydrochloric acid or edible alkali lye adjust pH according to the described a kind of method of producing nisin of claim 8.
10,, it is characterized in that saturated high pressure steam pressure is 0.09-0.11Mpa according to the described a kind of method of producing nisin of claim 8.
11, according to the described a kind of method of producing nisin of claim 8, temperature is 118-121 ℃ when it is characterized in that sterilizing, and sterilization time is 25-35 minute.
12,, it is characterized in that water coolant is cooled to 25-35 ℃ indirectly through spiral coil cooling tube according to the described a kind of method of producing nisin of claim 8.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100719453A CN1306025C (en) | 2005-05-26 | 2005-05-26 | Culture medium its rpeparation method and method for culturing lact-streptocin |
| PCT/CN2006/000637 WO2006125362A1 (en) | 2005-05-26 | 2006-04-11 | A culture medium, preparation method thereof and method for culturing nisin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100719453A CN1306025C (en) | 2005-05-26 | 2005-05-26 | Culture medium its rpeparation method and method for culturing lact-streptocin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1687401A CN1687401A (en) | 2005-10-26 |
| CN1306025C true CN1306025C (en) | 2007-03-21 |
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| CNB2005100719453A Active CN1306025C (en) | 2005-05-26 | 2005-05-26 | Culture medium its rpeparation method and method for culturing lact-streptocin |
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|---|---|
| CN (1) | CN1306025C (en) |
| WO (1) | WO2006125362A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103781898B (en) * | 2011-06-29 | 2016-03-02 | 雪印种苗株式会社 | Novel lactic acid bacteria express and use its silage or the preparation method of fermented feed |
| CN103642709B (en) * | 2013-08-28 | 2015-10-28 | 浙江工业大学 | One strain streptococcus acidi lactici and the application prepared at fermentable in fodder additives |
| CN109627300B (en) * | 2019-02-18 | 2022-08-05 | 浙江新银象生物工程有限公司 | Nisin solution stabilizer development and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1273237A1 (en) * | 2001-07-02 | 2003-01-08 | CSK Food Enrichment B.V. | Nisin-producing starter cultures for fermented food products |
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| IES70514B2 (en) * | 1995-04-12 | 1996-12-11 | Teagasc Agric Food Dev Authori | Bacteriocins |
| CN1225551C (en) * | 2002-07-16 | 2005-11-02 | 李盛学 | Liquid-solid combined fermentation process of probiotics by using bifidobacteria as main component |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1273237A1 (en) * | 2001-07-02 | 2003-01-08 | CSK Food Enrichment B.V. | Nisin-producing starter cultures for fermented food products |
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| CN1687401A (en) | 2005-10-26 |
| WO2006125362A1 (en) | 2006-11-30 |
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