CN102492644A - Bacterial strain capable of producing alternan and method of applying same in fermentation production of alternan - Google Patents
Bacterial strain capable of producing alternan and method of applying same in fermentation production of alternan Download PDFInfo
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- CN102492644A CN102492644A CN2011104464069A CN201110446406A CN102492644A CN 102492644 A CN102492644 A CN 102492644A CN 2011104464069 A CN2011104464069 A CN 2011104464069A CN 201110446406 A CN201110446406 A CN 201110446406A CN 102492644 A CN102492644 A CN 102492644A
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Abstract
The invention provides a bacterial strain capable of producing alternan and a method of applying the same in fermentation production of alternan, which belongs to the field of food biotechnology. The invention provides a bacterial strain of Leuconostoc citreum SK24.002 with an accession number of CCTCC NO: M 2011398; the bacterial strain is a novel bacterial strain for high yield of glucan-alternan and is obtained through plate colony feature selection of Chinese traditional fermented pickled vegetables, primary fermentation separate shake-flask culture and contrast test of products by using NMR and IR technologies. Alternan can be obtained through liquid submerged fermentation, and yield of the alternan is more than 25g/L. The alternan easily dissolves in water, has low viscosity and good processing stability and can be applied as prebiotics, a thickening agent, a filling agent and the like in a plurality of fields like food, health products, daily chemicals, medicines, high-molecular materials, etc.
Description
Technical field
The present invention relates to a strain and replace sugared high yield bacterium and produce alternately sugar, belong to technical field of food biotechnology with this strain fermentation method.
Background technology
Along with the development of society, people live very big variation have taken place, like rhythm of life quickening, social competition's fierceness, dietary structure change, environmental pollution etc.These change the health that is threatening people invariably, and most crowd is in sub-health state.At present, the public has increasing choose to use food to change this situation to the self health status growing interest, is that the master is advanced to " preventiveing treatment of disease " and health care from " treating the disease affected ", turns to " initiatively healthy " from " passive medical treatment ".For the direction of complying with the modern food development in science and technology and the health demand that satisfies the human consumer, hypoglycemia, functional carbohydrate low in calories have become the trend of 21 century heath food.
VISOSE is to be the polymeric carbohydrate that monomer forms with α-D-Glucopyranose, is the most important polysaccharide of occurring in nature.Because the difference of VISOSE link mode, the polymerization degree, space structure etc. forms character and the very big broad variety of difference in appearance, like common starch, Mierocrystalline cellulose, DEXTRAN 500.000 etc.The research of Chinese scholars shows that VISOSE has the immunity anticancer effect, and preventing hypertension, arteriosclerosis help fat-reducing; And can be used as plasma substitute, biogel, high-quality gel, edible low-heat VISOSE additive, makeup, petroleum assistant, B.T. insecticide synergist etc., and be with a wide range of applications, there are many investigators to be devoted to VISOSE research.Wherein alternately sugar (alternan) contains the glycosidic link of (1,6) and (1,3) two types, and it is as a kind of novel VISOSE, has good physiological function and processing characteristics thereby by extensive concern.At present, abroad some scholars report that the production by biological Sucrose:glucan alpha1 utilizes small molecular sugar to carry out receptor response and generates alternately sugar, but alternately candy output is very low for the most of microbe product, does not far reach industrialization demands.
The inventor investigates and has studied prior art further and studies replacing sugared bacterial classification and working method, and finally we find that a strain replaces sugared high yield bacterium and produces alternately sugar with this strain fermentation method.Based on above-mentioned discovery the present invention has been proposed.
Summary of the invention
The object of the present invention is to provide and a kind ofly replace sugared high yield bacterium and with this strain fermentation production method of sugar alternately, gained replaces sugar and can be applicable to a plurality of fields such as food, healthcare products, daily use chemicals, medicine, macromolecular material.
Technical scheme of the present invention: from China's traditional zymotic pickles, separate obtaining a strain and produce and replace sugared bacterial strain, classification called after lemon leukonid (
Leuconostoc citreum) SK 24.002, being preserved in Chinese typical culture collection center, deposit number is CCTCC M 2011398.
Adopting lemon leukonid CCTCC NO:M 2011398 is starting strain, alternately sugared through seed culture, liquid submerged fermentation production.Production stage is:
(1) seed culture: seed culture medium (g/L): glucose 5 ~ 10, yeast powder 10 ~ 20, Calcium Chloride Powder Anhydrous 0.02 ~ 0.025, potassium hydrogenphosphate 10 ~ 15, bitter salt 0.2 ~ 0.5, Manganous sulfate monohydrate 0.01 ~ 0.015, pH6.8 ~ 7.0.Culture condition: 8 ~ 12h, 30 ~ 37 ℃, 160 ~ 200 rpm.
(2) liquid submerged fermentation: fermention medium (g/L): sucrose 100 ~ 120, yeast extract paste 15 ~ 20, Calcium Chloride Powder Anhydrous 0.02 ~ 0.03, potassium hydrogenphosphate 15 ~ 20, bitter salt 0.2 ~ 0.5, tween 80 5 ~ 10 mL/L substratum, pH 6.8 ~ 7.0.Fermentation condition: with the seed culture medium of step (1) after seed culture by volume per-cent 6% ~ 10% insert in the fermention medium fermentation time 24 ~ 48h, 30 ~ 37 ℃ of temperature, 160 ~ 200 rpm.
The product alternately fermentation yield of sugar reaches 25-40g/L, and the glycosidic link mode of connection is that α (1,3) alternately connects with α (1,6), and both ratios are 0.6:1-1:1; Molecular mass is 1.0 * 10
6~ 5.0 * 10
7
The product alternately postprandial blood sugar of sugar generates index less than 55%, belongs to hypoglycemia type VISOSE.
Adopt IR to press KBr pressed disc method and alternately sugared functional group and the glycosidic link mode of connection of nuclear magnetic resonance nmr technical measurement; Wherein chemical shift is that the peak value of 99.95ppm and 98.67ppm is represented Glucopyranose α-1 respectively on 13C NMR collection of illustrative plates; 3 with α-1,6 glycosidic link in the quantity of connected anomeric carbon.
Alternately the mensuration of the molecular weight of sugar adopts the method for high performance size exclusion chromatography (HPSEC) to measure.
Alternately the postprandial blood sugar of sugar generates assessment of indices and calculates and press the glucose oxidase method that FAO/WHO recommends.
Beneficial effect of the present invention: the alternately superior strain of sugar of a strain has been proposed, the lemon leukonid (
Leuconostoc citreum) SK 24.001 microorganism strains are the new bacterial strain of a strain, make alternately sugar with this bacterial strain through seed culture, liquid submerged fermentation, output reaches 25-40g/L.This bacterium is suitable for liquid continuously ferments and cultivates and high-cell-density cultivation, and fermentation time is short, cost is low, output is high, is suitable for scale operation.The product of the present invention's preparation replaces sugar and can be applicable to a plurality of fields such as food, medicine, household chemicals, and market outlook are very good, and economic benefit is wide.
The biological material specimens preservation: the lemon leukonid (
Leuconostoc citreum) SK 24.002, depositary institution's title: Chinese typical culture collection center, be called for short CCTCC, address: Chinese Wuhan Wuhan University, deposit number is CCTCC NO:M 2011398, preservation date on November 18th, 2011.
Description of drawings
Fig. 1 replaces the glycan molecule structure iron.
Fig. 2
13C NMR figure.
Embodiment
Further illustrate content of the present invention below in conjunction with instance, but the content that the present invention protected not only is confined to following instance.
The fermentation culture of embodiment 1 lemon leukonid SK24.002
Seed culture: seed culture medium, glucose 10 g, yeast powder 10g, Calcium Chloride Powder Anhydrous 0.02g, potassium hydrogenphosphate 15g, bitter salt 0.5g, Manganous sulfate monohydrate 0.01g, zero(ppm) water is settled to 1000mL, regulates pH to 6.8.Cultivate 12h at 30 ℃ with 200 rpm shaking tables.
Liquid submerged fermentation is cultivated: fermention medium, and sucrose 100g, yeast extract paste 20g, Calcium Chloride Powder Anhydrous 0.03g, potassium hydrogenphosphate 20g, bitter salt 0.2g, tween 80 5mL, zero(ppm) water is settled to 1000mL, regulates pH to 7.0; Be inoculated in the fermention medium by 6%, cultivate 24h with the 160rpm shaking table under 37 ℃, finally replace sugared content and reach 30.5g/L.
The fermentation culture of embodiment 2 lemon leukonid SK24.002
Seed culture medium: glucose 5 g, yeast powder 20g, Calcium Chloride Powder Anhydrous 0.025g, potassium hydrogenphosphate 10g, bitter salt 0.2g, Manganous sulfate monohydrate 0.01g, zero(ppm) water is settled to 1000mL, regulates pH to 7.0.Under 37 ℃, cultivate 8h with 160 rpm shaking tables.
Liquid submerged fermentation substratum: sucrose 120g, yeast extract paste 15g, Calcium Chloride Powder Anhydrous 0.02g; Potassium hydrogenphosphate 15g, bitter salt 0.5g, tween 80 10mL; Zero(ppm) water is settled to 1000mL, regulates pH to 6.9, is inoculated in the fermention medium by 10%; Cultivate 48h with the 200rpm shaking table under 30 ℃, finally replace sugared content and reach 38.4g/L.
Practical implementation case described herein only illustrates as the present invention's spirit is tested to do with part.The technician in the related field of the present invention can make various modifications or replenishes or adopt similar mode to substitute described practical implementation case, but can't depart from spirit of the present invention or surmount the defined scope of appended claims.
Claims (4)
1. a strain is produced and is replaced sugared bacterial strain, classification called after lemon leukonid (
Leuconostoc citreum) SK 24.002, being preserved in Chinese typical culture collection center, deposit number is CCTCC NO:M 2011398.
2. one kind with the said bacterial strain CCTCC of the claim 1 NO:M 2011398 fermentative prodn method of sugar alternately, it is characterized in that adopting lemon leukonid CCTCC NO:M 2011398 to be starting strain, through seed culture, liquid submerged fermentation production sugar alternately;
(1) seed culture: seed culture medium is in g/L: glucose 5 ~ 10, yeast powder 10 ~ 20, Calcium Chloride Powder Anhydrous 0.02 ~ 0.025, potassium hydrogenphosphate 10 ~ 15, bitter salt 0.2 ~ 0.5, Manganous sulfate monohydrate 0.01 ~ 0.015, pH6.8 ~ 7.0; Culture condition: 8 ~ 12h, 30 ~ 37 ℃, 160 ~ 200 rpm;
(2) liquid submerged fermentation: fermention medium is in g/L: sucrose 100 ~ 120, and yeast extract paste 15 ~ 20, Calcium Chloride Powder Anhydrous 0.02 ~ 0.03, potassium hydrogenphosphate 15 ~ 20, bitter salt 0.2 ~ 0.5, tween 80 5 ~ 10mL/L, pH 6.8 ~ 7.0; Fermentation condition: with the seed culture medium of step (1) after seed culture by volume per-cent 6% ~ 10% insert in the fermention medium fermentation time 24 ~ 48h, 30 ~ 37 ℃ of temperature, 160 ~ 200 rpm; Alternately the fermentation yield of sugar reaches 25-40g/L.
3. method according to claim 2 is characterized in that the alternately molecular chain structure of sugar of product: the glycosidic link mode of connection is that α (1,3) alternately connects with α (1,6), and both ratios are 0.6:1-1:1; Molecular mass is 1.0 * 10
6~ 5.0 * 10
7
4. method according to claim 2 is characterized in that the alternately sugared postprandial blood sugar of product generates index less than 55%, belongs to hypoglycemia type VISOSE.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732489A (en) * | 2012-07-16 | 2012-10-17 | 江南大学 | Controlling method for producing alternansucrase in high yield by promoting fermentation of leuconostoc citreum |
| CN105076406A (en) * | 2015-08-27 | 2015-11-25 | 光明乳业股份有限公司 | Fermented milk drink rich in fructosan and production method thereof |
| CN105285096A (en) * | 2015-08-27 | 2016-02-03 | 光明乳业股份有限公司 | Fermented milk beverage rich in levan and preparation method of fermented milk beverage |
| CN111254102A (en) * | 2020-04-29 | 2020-06-09 | 中国农业大学 | Leuconostoc citreum and application thereof in precipitated starch slurry |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101665794A (en) * | 1999-02-08 | 2010-03-10 | 拜尔生物科学有限公司 | Nucleic acid molecules encoding alternansucrase |
| CN102186885A (en) * | 2008-10-17 | 2011-09-14 | 拜尔作物科学股份公司 | Alternan derivatives |
-
2011
- 2011-12-28 CN CN 201110446406 patent/CN102492644B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101665794A (en) * | 1999-02-08 | 2010-03-10 | 拜尔生物科学有限公司 | Nucleic acid molecules encoding alternansucrase |
| CN102186885A (en) * | 2008-10-17 | 2011-09-14 | 拜尔作物科学股份公司 | Alternan derivatives |
Non-Patent Citations (4)
| Title |
|---|
| H.A.WYCKOFF ET AL.: "Isolation and Characterization of Microorganisms with Alternan Hydrolytic Activity", 《CURRENT MICROBIOLOGY》 * |
| PETER BIELY ET AL.: "Purification and Properties of Alternanase, a Novel Endo--1,3--1,6-d-Glucanase", 《EUROPEAN JOURNAL OF BIOCHEMISTRY》 * |
| TD LEATHERS ET AL.: "Modification of alternan by novel Penicillium spp.", 《JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY》 * |
| 黄锡全等: "L.sp.HXQ001菌株16SrRNA基因序列及聚类分析", 《中国微生态学杂志》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732489A (en) * | 2012-07-16 | 2012-10-17 | 江南大学 | Controlling method for producing alternansucrase in high yield by promoting fermentation of leuconostoc citreum |
| CN105076406A (en) * | 2015-08-27 | 2015-11-25 | 光明乳业股份有限公司 | Fermented milk drink rich in fructosan and production method thereof |
| CN105285096A (en) * | 2015-08-27 | 2016-02-03 | 光明乳业股份有限公司 | Fermented milk beverage rich in levan and preparation method of fermented milk beverage |
| CN105076406B (en) * | 2015-08-27 | 2019-01-08 | 光明乳业股份有限公司 | A kind of leben and preparation method thereof rich in levulan |
| CN105285096B (en) * | 2015-08-27 | 2019-01-29 | 光明乳业股份有限公司 | A kind of leben and preparation method thereof rich in levulan levan |
| CN111254102A (en) * | 2020-04-29 | 2020-06-09 | 中国农业大学 | Leuconostoc citreum and application thereof in precipitated starch slurry |
| CN111254102B (en) * | 2020-04-29 | 2020-09-08 | 中国农业大学 | Leuconostoc citreum and application thereof in precipitated starch slurry |
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| CN102492644B (en) | 2012-12-05 |
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