CN1563407A - Preparation technique for producing natural carotenoid by using radioresistant coccus - Google Patents
Preparation technique for producing natural carotenoid by using radioresistant coccus Download PDFInfo
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- CN1563407A CN1563407A CN 200410017087 CN200410017087A CN1563407A CN 1563407 A CN1563407 A CN 1563407A CN 200410017087 CN200410017087 CN 200410017087 CN 200410017087 A CN200410017087 A CN 200410017087A CN 1563407 A CN1563407 A CN 1563407A
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- 150000001747 carotenoids Chemical class 0.000 title claims description 37
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
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- UPYKUZBSLRQECL-UKMVMLAPSA-N Lycopene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1C(=C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=C)CCCC2(C)C UPYKUZBSLRQECL-UKMVMLAPSA-N 0.000 abstract 2
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- 241001003009 Deinococcus radiodurans R1 Species 0.000 description 1
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Abstract
This invention discloses a production technology for producing natural carotene utilizing the anti-radiation cocci with the strain classification of deinococcus radiodurans to be inoculated in a TGY solid culture medium to be activated, passaged, the activated liquid seeds are inoculated into a bean cake powder culture medium matched with beam cake powder for fermentation and the fermentated liquid is centrifuged washed, extracted to become natural kind carotene.
Description
Technical field
The invention belongs to zymotechnique, relate generally to the zymotechnique of producing natural carotenoid, especially about utilizing radioresistant cocci Deinococcus radiodurans to produce the production technique of natural carotenoid.
Background technology
Carotenoid is the important plant pigments of a class, at occurring in nature, extensively is present in each kind of plant, and discovery is also arranged in animal and microorganism.A large amount of studies confirm that, except the medical functions to illness in eye, carotenoid is to cardiovascular disorder, some tumour, aging, photosensitive diseases, and cataractous generation development has certain prevention, delays and therapeutic action.Particularly it can also improve people's immunologic function.The class Radix Dauci Sativae has been expedited the emergence of the suitability for industrialized production of carotenoid in the application of aspects such as clinical medicine, clinical pharmacology, foodstuffs industry and cosmetic industry and the fast development of research in recent years.Carotenoid is as natural pigment, and fodder additives has suitable pharmaceutical use.
At present, the preparation of natural carotenoid mainly contains two kinds of sources: the 1. 2. microbe-derived natural carotenoid of natural carotenoid in plant (as Radix Dauci Sativae, salt algae, Dunaliella salina) source.Owing to utilize plant extract natural carotenoid complex process, the production that utilizes algae to carry out is not easy to control, and cost is all very high, so utilize growth cycle short, the microorganism that production technique is simpler and cost is lower carries out large-scale natural carotenoid production and has significant advantage.The major microorganisms of having found at present to synthesize carotenoid has lactobacterium helveticus, rhodotorula glutinis, phycomyces blakesleeanus, three spore cloth Laplaces mould etc.
Summary of the invention
The objective of the invention is to overcome the deficiency that prior art exists, a kind of production technique of utilizing radioresistant cocci to produce natural carotenoid is provided, the strain classification of the radioresistant cocci that the present invention is used is Deinococcusradiodurans, preserving number is: ATCC NO.13939, bacterium numbering is R1, and the step of production technique of the present invention is:
(1) activation of bacterial classification, go down to posterity: with the radioresistant cocci streak inoculation on peptone yeast extract glucose (TGY) solid medium, cultivate 45-48h for 30-32 ℃, picking list colony inoculation is cultured to logarithmic phase in triangular flask, the TGY solid medium consists of peptone 5g/l, yeast extract 3g/l, glucose 1g/l.
(2) fermentation culture: activatory liquid seeds in (1) is inserted the soybean cake powder substratum that is mixed with soybean cake powder carry out fermentation culture, pH6.0～8.0, cultivate 32-36h for 30 ℃, the prescription of soybean cake powder substratum is mainly soybean cake powder, adds carbon source and/or adds nitrogenous source, the preferred 30g/l of soybean cake powder, add carbon source and be mainly glucose, sucrose, preferred glucose 1～3g/l, glucose 3g/l most preferably, sucrose 1～3g/l, add the preferred SODIUMNITRATE 0.5～1.5g/l of nitrogenous source, most preferably SODIUMNITRATE 1.5g/l.
(3) extraction of natural carotenoid: take out cultured thalline fermented liquid in (2), centrifugal, the washing wet thallus adds a certain amount of methyl alcohol, vibrates under the room temperature, centrifugal 10 minutes of 5000rpm, get supernatant, add isopyknic chloroform and water, carry out reextraction, collect lower floor's organic phase, vacuum-drying is the carotenoid dry product.The methyl alcohol add-on is a bacterium liquid: methyl alcohol (V/V)=5: 3, in supernatant liquor, add isopyknic chloroform and water, and ratio is a chloroform: water (V/V)=3: 1.
Microorganism of the present invention is radioresistant cocci bacterial classification Deinococcus radiodurans and mutation thereof.
The invention has the advantages that:
(1) in by preferred crude liquid substratum provided by the invention, can get natural carotenoid dry product 2.27g/l through extracting;
(2) the general microbial fermentation extraction of this technical process is more simple, and cost is lower;
(3) radioresistant cocci belongs to prokaryotic organism, and the bacterial strain breeding is fast, and the production cycle is shorter, is suitable for large-scale production.
Description of drawings
Fig. 1 is a process flow sheet of the present invention.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiments, and these examples should be understood that only to illustrate, but not limit the scope of the invention by any way.
Used microorganism is radioresistant cocci (Deinococcus radiodurans R1) among the present invention, one class can be stood the prokaryotic micro-organisms of extreme environments such as ultraviolet ray, radioactive rays, arid, the biological property of this bacterium is, compare with other species, it has superpower radiation hardness ability and DNA repair ability.Have the scholar with the resistance of oxidation of this bacterium owing to wherein carotenoid material, think that simultaneously it is the color source of this red bacterium.The present invention utilizes radioresistant cocci can produce natural carotenoid 2.27g/l.
Embodiment 1: the substratum (PH=7.0) that is mixed with soybean cake powder 30g/l carries out fermentation culture and is extracted as example
(1) with the radioresistant cocci streak inoculation on the TGY solid medium, substratum consists of peptone 5g/l, yeast extract 3g/l, glucose 1g/l cultivates 48h for 32 ℃, picking list colony inoculation is cultured to logarithmic phase in triangular flask;
(2) aforesaid liquid seed 2% inoculum size is by volume inserted the big right substratum (PH=7.0) that contains soybean cake powder 30g/l,, carry out fermentation culture in 30 ℃ of cultivation 36h;
(3) get a certain amount of cultured thalline fermented liquid, through 5000rpm centrifugal 10 minutes, wash twice then, in fermented liquid: the ratio of methyl alcohol (V/V)=5: 3, add a certain amount of methyl alcohol, the 0.5h that vibrates under the room temperature, centrifugal 10 minutes of 5000rpm gets supernatant; In chloroform: the ratio of water (V/V)=3: 1, in supernatant liquor, add isopyknic chloroform and water, carry out reextraction, leave standstill for some time behind the mixing, collect lower floor's organic phase, vacuum-drying gets carotenoid dry product 1.25g/l, and control group (through the TGY fermentation culture) produces carotenoid 0.80g/l, is 1.56 times of control group.
Embodiment 2: with by soybean cake powder 30g/l, it is example that the substratum that glucose 1g/l is configured to carries out fermentation culture
(1) with the radioresistant cocci streak inoculation on the TGY solid medium, substratum consists of peptone 5g/l, yeast extract 3g/l, glucose 1g/l cultivates 48h for 32 ℃, picking list colony inoculation is cultured to logarithmic phase in triangular flask;
(2) aforesaid liquid seed 2% inoculum size is by volume inserted by soybean cake powder 30g/l, the substratum that glucose 1g/l is mixed with (PH=7.0) in 30 ℃ of cultivation 36h, carries out fermentation culture;
(3) get a certain amount of cultured thalline fermented liquid, through 5000rpm centrifugal 10 minutes, wash twice then, in fermented liquid: the ratio of methyl alcohol (V/V)=5: 3, add a certain amount of methyl alcohol, the 0.5h that vibrates under the room temperature, centrifugal 10 minutes of 5000rpm gets supernatant; In chloroform: the ratio of water (V/V)=3: 1, in supernatant liquor, add isopyknic chloroform and water, carry out reextraction, leave standstill for some time behind the mixing, collect lower floor's organic phase, vacuum-drying gets carotenoid dry product 1.89g/l, and control group (cultivating through the soybean cake powder natural medium) produces carotenoid 1.25g/l, is 1.51 times of control group.
Embodiment 3: with soybean cake powder 30g/l, the substratum that sucrose 1g/l is mixed with carries out fermentation culture and is extracted as example
(1) with the radioresistant cocci streak inoculation on the TGY solid medium, substratum consists of peptone 5g/l, yeast extract 3g/l, glucose 1g/l cultivates 48h for 32 ℃, picking list colony inoculation is cultured to logarithmic phase in triangular flask;
(2) aforesaid liquid seed 2% inoculum size is by volume inserted by soybean cake powder 30g/l, the substratum that sucrose 1g/l is mixed with (PH=7.0) in 30 ℃ of cultivation 36h, carries out fermentation culture;
(3) get a certain amount of cultured thalline fermented liquid, through 5000rpm centrifugal 10 minutes, wash twice then, in fermented liquid: the ratio of methyl alcohol (V/V)=5: 3, add a certain amount of methyl alcohol, the 0.5h that vibrates under the room temperature, centrifugal 10 minutes of 5000rpm gets supernatant; In chloroform: the ratio of water (V/V)=3: 1, in supernatant liquor, add isopyknic chloroform and water, carry out reextraction, leave standstill for some time behind the mixing, collect lower floor's organic phase, vacuum-drying gets carotenoid dry product 1.74g/l, and control group (cultivating through the soybean cake powder natural medium) produces carotenoid 1.25g/l, is 1.39 times of control group.
Embodiment 4: with by soybean cake powder 30g/l, it is example that the substratum that SODIUMNITRATE 0.5g/l is configured to (PH=6.0) carries out fermentation culture
(1) with the radioresistant cocci streak inoculation on the TGY solid medium, substratum consists of peptone 5g/l, yeast extract 3g/l, glucose 1g/l cultivates 48h for 32 ℃, picking list colony inoculation is cultured to logarithmic phase in triangular flask;
(2) aforesaid liquid seed 2% inoculum size is by volume inserted by soybean cake powder 30g/l, the substratum that SODIUMNITRATE 0.5g/l is mixed with (PH=7.0) in 30 ℃ of cultivation 36h, carries out fermentation culture;
(3) get a certain amount of cultured thalline fermented liquid, through 5000rpm centrifugal 10 minutes, wash twice then, in fermented liquid: the ratio of methyl alcohol (V/V)=5: 3, add a certain amount of methyl alcohol, the 0.5h that vibrates under the room temperature, centrifugal 10 minutes of 5000rpm gets supernatant; In chloroform: the ratio of water (V/V)=3: 1, in supernatant liquor, add isopyknic chloroform and water, carry out reextraction, leave standstill for some time behind the mixing, collect lower floor's organic phase, vacuum-drying gets carotenoid dry product 1.30mg/ml, and control group (cultivating through the soybean cake powder natural medium) produces carotenoid 1.25g/l, is 1.04 times of control group.
Embodiment 5: with soybean cake powder, the substratum that glucose 3g/l, SODIUMNITRATE 1.5g/l are configured to (PH=6.0) carries out fermentation culture and is extracted as example
(1) with the radioresistant cocci streak inoculation on the TGY solid medium, substratum consists of peptone 5g/l, yeast extract 3g/l, glucose 1g/l cultivates 48h for 32 ℃, picking list colony inoculation is cultured to logarithmic phase in triangular flask;
(2) aforesaid liquid seed 2% inoculum size is by volume inserted by soybean cake powder, the substratum that glucose 3g/l, SODIUMNITRATE 1.5g/l are configured to (PH=6.0) in 30 ℃ of cultivation 36h, carries out fermentation culture;
(3) get a certain amount of cultured thalline fermented liquid, through 5000rpm centrifugal 10 minutes, wash twice then, in fermented liquid: the ratio of methyl alcohol (V/V)=5: 3, add a certain amount of methyl alcohol, the 0.5h that vibrates under the room temperature, centrifugal 10 minutes of 5000rpm gets supernatant; In chloroform: the ratio of water (V/V)=3: 1, in supernatant liquor, add isopyknic chloroform and water, carry out reextraction, leave standstill for some time behind the mixing, take off a layer organic phase, vacuum-drying gets carotenoid dry product 2.27g/l, and control group (cultivating through the soybean cake powder natural medium) produces carotenoid 1.25g/l, is 1.82 times of control group.
The present invention is illustrated by above description and embodiment, more than is described as nonrestrictively, does not limit claim scope of the present invention.
Claims (10)
1. production technique of utilizing radioresistant cocci to produce natural carotenoid, the strain classification of used radioresistant cocci is Deinococcus radiodurans, preserving number is ATCC NO.13939, and bacterium numbering is R1, it is characterized in that: the step of this production technique is:
(1) activation of bacterial classification, go down to posterity: the radioresistant cocci streak inoculation on peptone yeast extract glucose (TGY) solid medium, is cultivated 45-48h for 30-32 ℃, and picking list colony inoculation is cultured to logarithmic phase in triangular flask;
(2) fermentation culture: activatory liquid seeds in (1) is inserted the substratum that is mixed with soybean cake powder carry out fermentation culture, pH6.0～8.0,30 ℃ cultivation 32-36h;
(3) extraction of natural carotenoid: take out cultured thalline fermented liquid in (2), centrifugal, the washing wet thallus adds a certain amount of methyl alcohol, vibrates under the room temperature, centrifugal 10 minutes of 5000rpm, get supernatant, add isopyknic chloroform and water, carry out reextraction, collect lower floor's organic phase, vacuum-drying is the carotenoid dry product.
2. the production technique of utilizing radioresistant cocci to produce natural carotenoid as claimed in claim 1, the described culture medium prescription that is mixed with soybean cake powder are soybean cake powder, add carbon source and/or add nitrogenous source.
3. as the arbitrary described substratum that is mixed with soybean cake powder of claim 1-2, described interpolation carbon source is mainly glucose, sucrose.
4. as the arbitrary described substratum that is mixed with soybean cake powder of claim 1-2, the preferred SODIUMNITRATE of described interpolation nitrogenous source.
5. as claim 1 or the 3 described substratum that are mixed with soybean cake powder, the preferred 1～3g/l glucose of described interpolation carbon source, 1～3g/l sucrose.
6. as claim 1 or the 4 described substratum that are mixed with soybean cake powder, the preferred 0.5～1.5g/l SODIUMNITRATE of described interpolation nitrogenous source.
7. as the arbitrary described substratum that is mixed with soybean cake powder of claim 1-6, the preferred 30g/l of described soybean cake powder.
8. the extraction of natural carotenoid as claimed in claim 1, described methyl alcohol add-on preferred proportion is a fermented liquid: methyl alcohol (V/V)=5: 3.
9. the extraction of natural carotenoid as claimed in claim 1, described isopyknic chloroform and the water of in supernatant liquor, adding, preferred proportion is a chloroform: water (V/V)=3: 1.
10. as the arbitrary described production technique of utilizing radioresistant cocci to produce natural carotenoid of claim 1-9, described microorganism is radioresistant cocci bacterial classification Deinococcus radiodurans and mutation thereof.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101326960B (en) * | 2008-07-10 | 2010-12-15 | 浙江大学 | Use of radioresistant cocci in preparing feedstuff additive for improving fertility of sow |
| CN101402987B (en) * | 2008-10-21 | 2011-10-26 | 浙江大学 | Method for improving production volume of radioresistant deinococcus carotenoid |
| US20140017724A1 (en) * | 2009-01-19 | 2014-01-16 | Centre National De La Recherche Scientifique | Methods for isolating bacteria |
| CN104152389A (en) * | 2014-08-29 | 2014-11-19 | 厦门大学 | High-efficiency algal-inhibition active compound Deinoxanthin and preparation method and application thereof |
| CN108103134A (en) * | 2017-12-23 | 2018-06-01 | 安徽工程大学 | A kind of water-soluble carotenoid and its fermentation preparation |
| CN108372307A (en) * | 2018-02-02 | 2018-08-07 | 浙江大学 | A kind of preparation method of nanogold, nanogold and application |
| CN113025525A (en) * | 2021-03-22 | 2021-06-25 | 浙江大学 | Anti-oxidation protective agent based on deinococcus radiodurans and preparation method thereof |
| CN115029274A (en) * | 2021-11-25 | 2022-09-09 | 宜宾五粮液股份有限公司 | Novel carotenoid-producing deinococcus strains and use thereof |
-
2004
- 2004-03-16 CN CN 200410017087 patent/CN1281761C/en not_active Expired - Fee Related
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101326960B (en) * | 2008-07-10 | 2010-12-15 | 浙江大学 | Use of radioresistant cocci in preparing feedstuff additive for improving fertility of sow |
| CN101402987B (en) * | 2008-10-21 | 2011-10-26 | 浙江大学 | Method for improving production volume of radioresistant deinococcus carotenoid |
| US20140017724A1 (en) * | 2009-01-19 | 2014-01-16 | Centre National De La Recherche Scientifique | Methods for isolating bacteria |
| US9279134B2 (en) * | 2009-01-19 | 2016-03-08 | Deinove | Methods for isolating bacteria |
| CN104152389A (en) * | 2014-08-29 | 2014-11-19 | 厦门大学 | High-efficiency algal-inhibition active compound Deinoxanthin and preparation method and application thereof |
| CN108103134A (en) * | 2017-12-23 | 2018-06-01 | 安徽工程大学 | A kind of water-soluble carotenoid and its fermentation preparation |
| CN108103134B (en) * | 2017-12-23 | 2021-06-04 | 安徽工程大学 | Water-soluble carotenoid and fermentation preparation method thereof |
| CN108372307A (en) * | 2018-02-02 | 2018-08-07 | 浙江大学 | A kind of preparation method of nanogold, nanogold and application |
| CN113025525A (en) * | 2021-03-22 | 2021-06-25 | 浙江大学 | Anti-oxidation protective agent based on deinococcus radiodurans and preparation method thereof |
| CN115029274A (en) * | 2021-11-25 | 2022-09-09 | 宜宾五粮液股份有限公司 | Novel carotenoid-producing deinococcus strains and use thereof |
| CN115029274B (en) * | 2021-11-25 | 2024-01-23 | 宜宾五粮液股份有限公司 | New strain of genus Sphaerococcus for producing carotenoid and its use |
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| CN1281761C (en) | 2006-10-25 |
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