WO2006125362A1 - A culture medium, preparation method thereof and method for culturing nisin - Google Patents

A culture medium, preparation method thereof and method for culturing nisin Download PDF

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Publication number
WO2006125362A1
WO2006125362A1 PCT/CN2006/000637 CN2006000637W WO2006125362A1 WO 2006125362 A1 WO2006125362 A1 WO 2006125362A1 CN 2006000637 W CN2006000637 W CN 2006000637W WO 2006125362 A1 WO2006125362 A1 WO 2006125362A1
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nisin
characterized
protein
medium
soy
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PCT/CN2006/000637
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French (fr)
Chinese (zh)
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Yanxin Shen
Youran Fan
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Yanxin Shen
Youran Fan
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/20Bacteria; Culture media therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00Preparation of peptides or proteins
    • C12P21/02Preparation 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 for producing nisin, preparation method thereof and method for culturing nisin. The preparation method comprises adding yeast protein, soy bean protein to a dosing jar, adding water and mixing, and then putting carbon source, sodium phosphate, magnesium sulfate in turn and mixing, adjusting pH to 6.2-7.2 and transferring into a culture jar, adding water, high pressure steaming, sterilizing, cooling inoculating food lactobacillus, culturing under the condition of pressure of 0.1 - 0.5 Mpa, 26-37°c for 24-72 hours, and stopping when the active of nisin is up to 4000-15000 IU/ml as measured by biological active testing.

Description

One kind of media, and methods for their preparation nisin culture BACKGROUND

The present invention relates to a culture medium, specifically relates to a method for producing a medium of nisin, its preparation method and the culture method for nisin. technical background

Nisin is a small peptide produced by certain Streptococcus lactis (Lactococcus Iactis), molecular weight of about 3510D a. Such bacteria many gram-positive bacteria, especially food spoilage caused by Bacillus, Clostridium, Staphylococcus, Listeria monocytogenes strong suppression of the killing effect, and in view of digestive enzymes may be degraded, a toxic to humans natural food preservative, it is widely used preservative dairy products, canned food, meat and beverages.

Institute of Microbiology, Chinese Academy of Sciences study of the use of lactic acid lactococcin Z yielding mutant strain Lactococcus lactis AL2 as production strain, peptone and other cheap materials instead of milk as a medium, the extraction process route after using, for industrial-scale production at 20 tons fermenter test, the average fermentation titer of 3009IU / ml, after extracting 60.9% overall yield.

Yang Bo, who in optimum conditions of streptozotocin Lactococcus lactis: Dalian Institute of Light Industry, Vol. 18, No. 1, 1999, when 365-69 for optimum fermentation conditions were streptozotocin Lactococcus lactis, the fermentation medium is: soy peptone 1.0%, 0.25% beef extract, yeast extract, 0. 25%, Na 2 HP0 4 · 12H 2 0 21. 5%, MgS0 4 · 7H 2 0 0. 012%, natural pH value; acid lactococcin titer assay medium was: 0.8% tryptone, yeast extract, 0. 5%, NaCl 0. 5% , NaHP0 4 · 12¾0 0. 2%, agar 1. 5%, pH6 8; used. Reagents are: Lactococcus lactis streptozotocin standard: Aplin & Barrett LTD production, tryptone: Oxoid LTD production; polypeptone: in Japan, other media for the Haidian District of microbiological culture media products factory.

Nisin culture prior art, a medium containing a nitrogen source is mostly used. Different nitrogen sources have a significant effect on the production of nisin, lactic acid bacteria to their picky. Nitrogen source organic nitrogen as well. Milk and yeast powder is commonly used two kinds of nitrogen sources. They do not have influence on the activity of the finished product, but slightly different color, a milky white, light brown or a grayish powder. Measuring the absorbance of 620nm of the solution, milk-based products due to nisin containing denatured milk components, compared with the absorbance value of the back yeast powder as raw material high, respectively, 1.0 and 0.3. Such as adding cottonseed meal as nitrogen source in a complex medium to increase the yield of nisin. De Vuyst found nisin biosynthesis depends on the sulfur-containing inorganic salts (MgS0 4, thiosulfate) or sulfur-containing amino acids (methionine, cysteine), or cystathionine these sulfur source. Serine, threonine and cysteine ​​is a precursor of nisin biosynthesis, may be adjusted to produce nisin, but does affect the final cell mass. No inhibition of N + Lactococcus or promotion.

Above medium can guarantee the synthesis of basal metabolism of Lactococcus lactis and nisin, but milk and beef paste is animal protein, high cost of raw materials affected by origin, quality control is difficult, in the long-distance transport and storage often have to add some preservative, it will affect the normal fermentation of lactic acid streptococci. At the same time the finished product during the Kosher (Jewish) and other relevant certification will be when obstacles, difficult to pass a direct impact on product applications. Although soy peptone molecule is relatively small, but the trend of single nutrients, the manufacturing process also cause some loss of nutrients and trace elements such as carbon source. Thus yet media optimization, increased product yield increase to accommodate the request. The present invention employs yeasts protein and soy protein complexes applicable, overcoming the original process resulting in high unit cost, low yield of the defect. SUMMARY

Object of the present invention is to provide a process resulting in low unit cost and high yield of medium for the production of nisin.

The present invention provides a medium, comprising in weight percent was prepared from the following raw materials: Yeast Protein: 0. 5-2%, soy protein: 0. 1-09%, carbon: 1.0 -4. 0%, as well as water.

Preferably, in weight percent, comprising the following processing from raw materials preparation:

Yeast proteins: 0. 5-2%, soy protein: 0.1 - 0. 9% carbon: 1. 0- 4.0%, sodium: 0. 1-05%, magnesium 0. 1-0. 5%, further containing water.

Yeast protein is preferably a yeast powder, yeast extract.

Soy protein flour is preferably made of soybean and soybean protein isolates and soy hydrolyzate.

Carbon monosaccharides, disaccharides and polysaccharides.

The present invention also provides a method for producing the above-mentioned nisin medium:

The yeast-like protein, soy protein into a mixing tank, add water, stir, then a carbon source, phosphate, magnesium sequentially input, stir, with 29-31% hydrochloric acid or an edible food lye tone pH6. 2-7. after 2 into the culture tank, water was added into the saturated high pressure steam, maintaining the internal pressure 0. 09-0. HMPa, sterilization at a temperature of 118-121 ° C under conditions of 25-35 minutes, cooled with cooling water indirect cooling coil to 25- 35Ό Serve.

Above medium access food grade lactic acid bacteria inoculum of 0. 3-1. 0%. , Maintained in culture with sterile air tank pressure 0. 1-0. 5 MPa, a temperature of 24 to 72 hours culture conditions at 26-37 ° C, nisin activity was measured by the biological potency reaches 4000-15000IU / ml , to stop.

After the above-described medium, metabolism lactis advantageously, the number of times access to shorten, metabolism, accelerate the consumption of sugar to nitrogen, recession delay nisin activity was measured by the biological potency than the prior art 2000- 3000 IU / ml have greatly improved, so that the yield is greatly improved.

Above food grade lactic acid bacteria obtained from the Institute of Microbiology, Chinese Academy of Science, Culture Collection at the Institute Culture Collection, strain No. AL2. detailed description

The following Examples illustrate the use aspect of the invention, but not to limit the invention to the embodiment.

Unless otherwise indicated, used in the present invention:

Yeast powder provided by Shanghai Xian Chen Industrial Co., Ltd., lot number 0504036;

Yeast extract provided by the Guangdong Yipinxian company, lot number 05017;

Soy flour is made, i.e. soybeans soaked into the water after 12 hours it with a weak refiner to 150-300 mesh;

Soy protein isolate and soy hydrolyzate is purchased, isolated soy protein supplier Shandong Yu Wang Group Co., Ltd., lot number 0504011; soybean hydrolyzate suppliers Harbin High-Tech Soybean Food Co., Ltd., lot number 20050315;

Monosaccharides glucose, supplied by the United States Food Co., Ltd. Zhejiang Bein, lot number 20050313; disaccharide is sucrose, provided by the Guangdong Donghua Sugar Corporation, lot number 20050120; polysaccharide is maltose, provided by the Tiantai Yi sugar processing plant, lot number 20050305; Example 1

Material comprising the following percentages by weight:

Yeast extract: 0.5%, soybean meal: 0.9%, glucose 1.0%, the rest is deionized water. Yeast and soybean powder into a mixing tank, mix deionized water, adjusted with 29% hydrochloric acid consumption PH6. After 2 into the culture tank, deionized water, into a high-pressure saturated steam, to maintain the internal pressure 0 . Lompa, sterilized 35 minutes at a temperature of 118 ° C under conditions of indirect cooling with cooling water to 25 ° C via cooling coils Serve.

Above medium access food grade lactic acid in an amount of 0.3% inoculum. . Culture with sterile air to keep the internal pressure 0. 5 MPa, 72 hours at a temperature of 26 ° C conditions, Nisin activity was determined by biological potency reached 4000IU / ml, can be stopped.

Limited by the above-described yeast Shanghai Industrial Xian Chen, lot number 0504036; soy flour is made, i.e. placed in water for 12 hours soybean milled to 150-300 mesh it with the refiner weak;

Glucose, supplied by the United States Food Co., Ltd. Zhejiang Bein, lot number 20050313. Example 2

Material comprising the following percentages by weight:

Yeast extract: 0.5%, isolated soy protein: 0.5%, sugar: 3.0%, phosphate: 0.5%, the rest is well water.

The yeast and soy protein isolate into a mixing tank, add stir well water, sodium phosphate, adjusted with 31% caustic consumption P into the culture tank after H7. 0, add well water, high pressure steam into saturated, maintaining the tank pressure 0. lOMPa, sterilization conditions of a temperature of 121 ° C for 30 minutes, cooled indirectly with cooling water to 30 ° C via cooling coils Serve.

Above medium access food grade lactic acid bacteria inoculum was 0.5%. . Culture with sterile air to keep the internal pressure 0. 1 MPa, the temperature is incubated for 24 hours 37 ° C for conditions of nisin activity by measuring biological potency reaches 10000IU / ml, can be stopped.

Above yeast powder provided by Shanghai Xian Chen Industrial Co., Ltd., lot number 0504036; soy protein isolate as outsourcing, supplier Shandong Yu Wang Group Co., Ltd., lot number 0504011; sucrose, provided by the Guangdong Donghua Sugar Corporation, lot number 20,050,120. Example 3

Material comprising the following percentages by weight:

Yeast extract: 1% soy hydrolyzate: 0.3%, maltose: 1.5%, phosphate: 0.1%, 0.5% magnesium, the balance being water.

The yeast extract and soy hydrolyzate into a mixing tank, add water, stir, sodium phosphate, and magnesium sulfate was added, adjusted with 30% caustic consumption pH7. After 2 into the culture tank, add water, into a high-pressure saturated steam, to maintain the internal pressure 0. 09MPa, sterilization at a temperature of 120 ° C under the conditions of 35 minutes, it is cooled indirectly with cooling water to 35 ° C via cooling coils Serve.

Above medium access food grade lactic acid bacteria inoculum was 1.0%. . Culture with sterile air to keep the internal pressure 0. 3MPa, cultured for 48 hours at a temperature of 30 ° C conditions, Nisin activity was determined by biological potency reaches 15000IU / ml, can be stopped.

The above-described yeast extract provided by the Guangdong Yipinxian company, lot number 05017;

Soy hydrolyzate provided by Harbin High-Tech Soybean Food Co., Ltd., lot number 20050315; maltose, provided by the Tiantai Yi sugar processing plant, lot number 20050305.

Example 4

Material comprising the following percentages by weight:

Yeast extract: 2%, isolated soy protein: 0.9%, maltose: 4.0%, sodium phosphate - 0.3%, magnesium sulfate 0.1%, the remainder being mineral water.

The yeast extract and soy protein isolate into a mixing tank, add mineral mix, sodium phosphate, and magnesium sulfate, with 30% hydrochloric acid, pH adjusted to eat into the culture tank after 6.5, plus mineral water, into saturated high pressure steam, maintaining the internal pressure 0. lOMPa, sterilization at a temperature of 119 ° C under the conditions of 28 minutes, with cooling water 25 ° C Serve indirect cooling via a cooling coil.

Above medium access food grade lactic acid bacteria inoculum was 0.9%. . Culture with sterile air to keep the internal pressure 0. 2MPa, for 60 hours at a temperature of 35 ° C conditions, Nisin activity was determined by biological potency reached 8000IU / ml, can be stopped.

The above-described yeast extract provided by the Guangdong Yipinxian company, lot number 05017;

Soy protein isolate is purchased, the supplier Shandong Yu Wang Group Co., Ltd., lot number 0504011;

Maltose, provided by the Tiantai Yi sugar processing plant, lot number 20050305. Example 5

Material comprising the following percentages by weight:

Yeast extract: 1.5%, isolated soy protein: 0.7%, glucose: 3.0%, phosphate: 0.4%, 0.4% magnesium, the balance being distilled water.

The yeast and soy protein isolate into a mixing tank, add distilled water, stir, sodium phosphate, and magnesium sulfate, the alkali consumption of 8 tone pH6. Fed with 31% culture tank, add distilled water, into a high-pressure saturated steam, maintaining the tank pressure 0. 09MPa, sterilization at a temperature of 12Q ° C under the conditions of 33 minutes, it is cooled indirectly with cooling water to 28 ° C via cooling coils Serve.

Above medium access food grade lactic acid bacteria inoculum was 0.8%. . Culture with sterile air to keep the internal pressure 0. 4MPa, temperature 70 hours under culture conditions 30Ό, Nisin activity was determined by biological potency reaches 10000IU / ml, can be stopped.

Above yeast Co., Ltd. by the Shanghai Xian Chen Industrial Co., lot number 0504036; soy protein isolate as outsourcing, supplier Shandong Yu Wang Group Co., Ltd., lot number 0504011;

Glucose, supplied by the United States Food Co., Ltd. Zhejiang Bein, lot number 20050313. Example 6

Material comprising the following percentages by weight:

Yeast extract: 0.5%, 1.2% soybean hydrolyzate, glucose: 3.5%, phosphate: 0.4%, 0.4% magnesium, the remainder being purified water.

The yeast extract and soy hydrolyzate into a mixing tank, add pure water, stir, sodium phosphate and magnesium sulfate, with 31% consumption of lye adjusted pH7. 0 into the culture tank, add purified water, passed through saturated high pressure steam, maintaining the internal pressure 0. lOMPa, at a temperature of 120 ° C for sterilization for 30 minutes, cooled with a cooling water pipe plate was indirectly cooled to 29 ° C Serve.

Above medium access food grade lactic acid bacteria inoculum was 0.5%. . Culture with sterile air to keep the internal pressure 0. 4MPa, for 30 hours at a temperature of 30 ° C conditions, Nisin activity was determined by biological potency reaches 12000IU / ml, can be stopped.

The above-described yeast extract provided by the Guangdong Yipinxian company, lot number 05017;

Soy hydrolyzate provided by Harbin High-Tech Soybean Food Co., Ltd., lot number 20050315; glucose, supplied by the United States Food Co., Ltd. Zhejiang Bein, lot number 20050313.

Claims

Rights request
I, one kind of medium for producing nisin, including yeasts and soy proteins, which is characterized in that the preparation comprises in weight percent from the following raw materials: Yeast Protein: 0. 5-2%, soy protein: 0. 1-09% carbon source: 1. 0- 4.0%, further containing water.
, The medium according to claim 1 for producing nisin claim, wherein further comprising, in weight percent was prepared from the following raw materials:
Sodium: 0. 1-05% magnesium sulfate 0.5 1-05%.
3, according to one of claim 1 or claim 2 for the production of nisin medium, characterized in that the yeast extract is a yeast-like protein.
4, according to one or more of claim 1, wherein the medium for the production of nisin, characterized in that the protein is soy flour and soy meal made of, isolated soy protein, soy hydrolyzate.
5, for the culture medium according to claim nisin claim 1 or 2, characterized in that the carbon source is a monosaccharide, disaccharides and polysaccharides.
6, the method 1 or the medium of claim 2 for the preparation, comprising the steps of:
The yeast-like protein, soy protein into a mixing tank, add water, stir, then a carbon source, phosphate, magnesium sequentially input, stir, adjusting pH 6. 2-7. 2 into the culture tank, water was added through into the high pressure steam sterilization, cooling.
7. The method of preparation according to claim 6, characterized in that the consumption by 29-31% hydrochloric acid or an edible alkaline pH adjusted.
8. A production method according to claim 6, characterized in that the high pressure saturated vapor pressure of 0. 09-0. LlMpa.
9. A production method according to claim 6, characterized in that the sterilization temperature of 118-121
V, sterilization time of 25-35 minutes.
10, prepared according to the method as claimed in claim 6, characterized in that the cooling water is indirectly cooled to 25- 35 ° C through the cooling coils.
II, a process for producing nisin, wherein - the above medium access food grade lactic acid bacteria, the internal pressure of 0. 1- 0. 5 MPa, temperature
PCT/CN2006/000637 2005-05-26 2006-04-11 A culture medium, preparation method thereof and method for culturing nisin WO2006125362A1 (en)

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JP5931064B2 (en) * 2011-06-29 2016-06-08 雪印種苗株式会社 Process for the preparation of new lactic acid bacteria and silage or fermented feed using the same
CN103642709B (en) * 2013-08-28 2015-10-28 浙江工业大学 Nisin and its application in a microbial fermentation in the preparation of feed additives

Citations (2)

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Publication number Priority date Publication date Assignee Title
US6207411B1 (en) * 1995-04-12 2001-03-27 Teagasc Bacteriocins
CN1400304A (en) * 2002-07-16 2003-03-05 李盛学 Liquid-solid combined fermentation process of probiotics by using bifidobacteria as main component

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EP1273237A1 (en) * 2001-07-02 2003-01-08 CSK Food Enrichment B.V. Nisin-producing starter cultures for fermented food products

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6207411B1 (en) * 1995-04-12 2001-03-27 Teagasc Bacteriocins
CN1400304A (en) * 2002-07-16 2003-03-05 李盛学 Liquid-solid combined fermentation process of probiotics by using bifidobacteria as main component

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LU W. ET AL.: "Effects of sucrose, lactate and KH2PO4 on nisin production by L. Lactis sp. Lactis ATCC 11454", THE CHINESE JOURNAL OF PROCESS ENGINEERING, vol. 4, no. 1, February 2004 (2004-02-01), ISSN 1009-606X, pages 28 - 31, XP008077165 *

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