CN1377956A - Ocean bacteria capable of producing haematochrome and method for producing haematochrome using said bacteria - Google Patents
Ocean bacteria capable of producing haematochrome and method for producing haematochrome using said bacteria Download PDFInfo
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- CN1377956A CN1377956A CN 01110272 CN01110272A CN1377956A CN 1377956 A CN1377956 A CN 1377956A CN 01110272 CN01110272 CN 01110272 CN 01110272 A CN01110272 A CN 01110272A CN 1377956 A CN1377956 A CN 1377956A
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Abstract
The present invention provides a kind of ocean bacteria capable of producing haematochrome in the preservation number of CGMCC No.0527. The present invention also provides the method of producing natural haemotochrome, including culturing the ocean bacteria of claim 1; and extracting haematochrome from the culture. The present invention also provides a kind of haemtochrome.
Description
The present invention relates to a kind of method of producing the marine bacteria bacterial strain of haematochrome and utilizing this bacterium production haematochrome.The invention still further relates to a kind of haematochrome.
Pigment is divided into synthetic colour and natural pigment, synthetic colour does not only have nutritive value and also has in various degree toxicity, some in addition can be carcinogenic, that therefore uses in foods and cosmetics industry is fewer and feweri, it is good to the substitute is security, and have the natural pigment of certain nutritive value and pharmacological function, but the natural pigment kind of exploitation far can not satisfy the needs in market at present.
Existing natural pigment is mostly by plant and lower animal tissue extraction, because the animals and plants wild resource is limited, it is long to cultivate or culture the needed cycle, and raw-material cost is high and under-supply, has limited the scale of production.Microbial reproduction is fast, can obtain in a large number by artificial culture, is the Biological resources that a class has higher potentiality to be exploited, and particularly the uniqueness of marine microorganism meta-bolites and novelty have obtained affirming of domestic and international scientific and technological circle.
Some related microorganisms chromatogenous report that ferments is all arranged at present both at home and abroad, but in industrial production, use seldom.Land fungi monascus, the pigment of production are pigments of present unique usefulness microorganisms producing of using on market as foodstuff additive.Monascus cultivate to need ripe rice as solid medium, and the growth and breeding cycle is longer, and single natural red colouring matter is difficult to satisfy market demand, and the haematochrome that present domestic makeup use mostly is the chemosynthesis pigment greatly.Utilize the marine microorganism artificial culture to extract pigment, all do not report both at home and abroad.Many marine microorganisms can both chromogenesis, but general pigment generation is less, and the visual inspection colony colour is shallow, and the generation of pigment is extremely unstable, and temperature, illumination, nutrition all can influence the generation of its pigment, can not be as the bacterial strain of producing pigment.
The purpose of this invention is to provide a kind of marine bacteria that produces haematochrome.
Another object of the present invention provides a kind of method of utilizing bacterium of the present invention to produce haematochrome.
A further object of the present invention provides a kind of natural red colouring matter.
The invention provides a kind of marine bacteria that produces haematochrome, bacterial strain code name S-9801.Its form is shaft-like or arc is shaft-like, 0.5-1.2 μ m, no gemma, peritrichous, sports type, Gram-negative is cultivated 24h, the bacterium colony Jinyuan Garden shape of formation for 28 ℃ at protein culture medium (every 1L seawater contains nitrogenous source 0.5%, carbon source 0.2%), more smooth, bright red, pigment is insoluble to substratum.This bacterial strain has peptonization to litmus milk; The glucose oxidase reacting positive; Oxidase negative; The catalase feminine gender.Preliminary evaluation is Flavobacterium (Flavobacterium sp.)
Bacterial strain all can be grown under 15 ℃-35 ℃ culture temperature, and 25 ℃-28 ℃ is optimum growth temperature, and under this temperature, strain liquid is cultivated 12 hours beginning chromogenesises, cultivates 36 hours, and the pigment that bacterial strain produces can reach maximum value.
The S-9801 bacterial strain all can be grown in the scope of the contained NaCl concentration 0-5.0% of nutrient solution, but the NaCl concentration of nutrient solution is 0 o'clock, and strain growth is very poor, can not chromogenesis.NaCl concentration is 5.0% o'clock, and strain growth is normal, and the amount of pigment of generation is less.Bacterial strain is well-grown in the scope of NaCl concentration 1.0-3.0%, and the amount of pigment of generation is big, and NaCl concentration changes in this scope, and is not obvious to the influence of strain growth and pigment production.
Bacterial strain all can be grown in the scope of the pH of nutrient solution 3-9, and produces haematochrome.The pH value is 9 o'clock, and it is little that strain growth and other are handled difference, but the amount of pigment that produces is less, suitable growth pH 4-8.
After measured, the artificial medium that the S-9801 bacterial strain is fit to is peptone or Tryptones 5-7g yeast powder 1-3g, glucose 2-4g, NaCl 20-30g, MgCl.6H2O1.1-2.3g, KCl 0.15-0.3g, agar 17g, water 1000ml.Cultivate temperature required 25 ℃-28 ℃, pH 4-8.
The S-9801 bacterial strain is fit to the liquid growth, can pass through the fermentor tank enlarged culturing, and it is little to have occupation space, and the more manageable characteristics of industrial scale help scale operation.Marine bacteria S-9801 bacterial strain, vitality is strong, and is simple to nutritional requirement, and growth scope is wide, and the haematochrome of generation is strong to the tolerance of acid, alkali, heat, light, and the animal toxicity test of being done through Chinese medical courses in general institute drug screening center shows that this pigment toxicity is very low.Bacterial strain can obtain in a large number by the fermentor tank liquid fermenting simultaneously, and the more existing natural pigment Biological resources of its growth and breeding speed are fast.Haematochrome is bright in luster, and the natural red colouring matter as makeup use has good development prospect.
Marine bacteria S-9801 of the present invention has carried out preservation on December 27th, 2000 at Chinese culture presevation management committee common micro-organisms center, and the preservation centre address is: Beijing, Zhong Guan-cun, postcode: 100080.Preserving number is: CGMCC NO.0527
The present invention also provides a kind of method of producing natural red colouring matter, comprising: cultivate marine bacteria of the present invention and extract haematochrome from culture.
In aforesaid method, the cultivation of bacterial strain of the present invention is a nitrogenous source at Tryptones (preferred 0.7% weight)+yeast powder (preferred 0.3% weight), glucose (preferred 0.3% weight) is carbon source, NaCl concentration is 1.8%-2.1% (weight), pH6-7, the condition liquid culture that temperature is 28 ℃, the pigment of generation can reach 1.5g/L.
From culture, fermented liquid pH can be adjusted to 2-4 during the separate red pigment, be preferably about 3.At this moment pigment sedimentation is the most thorough.When extracting, preferably use alcoholic solvent, preferably make solvent with ethanol, can make pigment safer like this.
The present invention also provides a kind of haematochrome of being produced by marine bacteria of the present invention.The dry crude product of pigment is a rose, and pure product are scarlet, and external environment is red below pH12, and the pH value is that 12 or 12 above tones become yellow.Pigment has stability preferably to light, heat.
The pigmentary colours that the S-9801 bacterial strain produces are bright-coloured, and pigment production is big, and the chromatogenous character of bacterial strain is more stable.The S-9801 wild strain is from seawater, and the low nutrient environment of seawater is brought up it to the undemanding characteristic of nutritional requirement.
China is populous nation, and the needed amount of pigment of foods and cosmetics industry is very big, and the pigment that uses mostly is synthetic colour greatly at present, natural pigment quantity not sufficient 10%.Wherein the natural pigment kind is few, and industrial scale is little, and price is expensive to be major cause.And natural pigment yields poorly mainly because Biological resources lack or the cost height.The discovery of S-9801 bacterial strain not only increases new variety for China's natural pigment, the more important thing is that this bacterial strain can pass through the liquid fermentation tank suitability for industrialized production, therefore, this invention will produce positive pushing effect to the natural pigment industry of China, simultaneously, this discovery also will be opened up a new way for the utilization of Living marine resources.
The mensuration of the NaCl concentration of embodiment 1 strain growth, pH scope and culture temperature
The S-9801 bacterial strain is a basic culture solution with the peptone nutrient solution, and the 100ml nutrient solution inserts 5ml seed liquor (as follows), and NaCl concentration is established 0,1.5%, 2.0%, 3.0%, 5.0%5 concentration level; The pH value is established 3,5,7,94 potential of hydrogen, 25 ℃ of cultivations, respectively 12,24h takes a sample once, surveys OD
538Value.
Bacterial strain is a basic culture solution with the peptone nutrient solution, under 15 ℃, 20 ℃, 25 ℃, 28 ℃, 30 ℃, 35 ℃, condition, cultivate respectively, observe the time that produces haematochrome, put then under the suitable temperature and cultivate, respectively at 12,24,36,48,60h takes a sample once, with 752 type ultraviolet spectrophotometers, survey OD
538Value is analyzed the yeast culture pigment production and is reached the maximum time.
Test-results shows that the S-9801 bacterial strain all can be grown in the scope of the contained NaCl concentration 0-5.0% of nutrient solution, but the NaCl concentration of nutrient solution is 0 o'clock, and strain growth is very poor, can not chromogenesis.NaCl concentration is 5.0% o'clock, and strain growth is normal, and the amount of pigment of generation is less.Bacterial strain is well-grown in the scope of NaCl concentration 1.0-3.0%, and the amount of pigment of generation is big, and NaCl concentration changes in this scope, and is not obvious to the influence of strain growth and pigment production.
The S-9801 bacterial strain all can be grown in the scope of the pH of nutrient solution 3-9, and produces haematochrome.The pH value is 9 o'clock, and it is little that strain growth and other are handled difference, but the amount of pigment that produces is less.
Bacterial strain all can be grown under 15 ℃-35 ℃ culture temperature, and 25-28 ℃ is optimum growth temperature, under this temperature, strain liquid is cultivated and can be reached logarithmic phase in 12 hours, and the beginning chromogenesis, to cultivate 36 hours, the pigment that bacterial strain produces can reach maximum value.
Table 1 S-9801 bacterial strain nutrient solution NaCl, pH, difference and culture temperature,
Chromogenesis information slip during asynchronism(-nization)
Embodiment 2
| ????NaCl(％) ????OD 538 | ????0??????1.5?????2.0??????3.0??????5.0 ????0.17???0.99????1.01?????0.99?????0.48 |
| ????PH ????OD 538 | ????????3???????5???????7????????9 ????????0.98????0.97????0.95?????0.57 |
| The temperature ℃ product pigment time (h) | ????15????20??????25??????28??????30????35 ????38????24??????12??????12??????17????20 |
| Incubation time (h) OD 538 | ????????12?????24?????36??????48??????60 ????????0.50???0.89???1.20????1.20????1.11 |
The testing laboratory's cultivation of S-9801 bacterial strain and the acquisition of pigment crude product
Peptone 5g yeast powder 2g, glucose 2g, NaCl 21g, MgCl.6H
2O 1.8g, KCl0.2g, agar 17g, water 1000ml.25 ℃-28 ℃ of temperature are regulated nutrient solution pH6-7, and plate is poured in sterilization into, and the S-9801 bacterial strain is inserted in the cooling back, cultivates the fresh lawn of 24h, inserts Tryptones 7g yeast powder 3g again, glucose 3g, NaCl 21g, MgCl.6H2O1.8g, KCl 0.2g, water 1000ml.In the aseptic culture fluid of pH6-7, sample-loading amount 120ml/250ml, 150rpm, 28 ℃, about shaking culture 30 minutes, bacterium begins breeding, and bacterial strain breeding in 12 hours reaches exponential phase of growth, and beginning chromogenesis, cultivate after 24-36 hour and take out, regulate fermented liquid to pH3, centrifugal (5000rpm) 10 minutes, abandoning supernatant, throw out adds dehydrated alcohol (throw out of every 1000ml fermented liquid adds 200ml ethanol) and fully keeps ethanolic soln in the dissolving back, repeats 3 times, merges ethanolic soln, ethanol through the rotary evaporation evaporate to dryness, can be obtained the haematochrome crude extract.
The different pH values of table 2 are to the effect of settling of thalline
| ?PH | ?1 | ?2 | ?3 | ?4 | ?5 | ?6 | ?7 | ?8 | ?9 | ?10 | ?11 | ?12 |
| ?OD 538 | ?1.45 | ?1.45 | ?1.49 | ?1.46 | ?1.45 | ?1.29 | ?0.78 | ?0.44 | ?0.34 | ?0.18 | ?0.10 | ?0.056 |
As can be seen from the table, the pH value was at 3～4 o'clock, and the effect of settling of thalline is best.
Adopt chloroform direx process and adjust pH to make the thalline sedimentation use ethanol extraction method again, all can obtain the pigment crude product, preceding a kind of method is simple to operate, fermented liquid directly adds chloroform and can extract, the highest pigment crude product 1.3g that obtains of every 1000ml fermented liquid, but the chloroform consumption is big, and the toxicity of chloroform is also bigger simultaneously; It is centrifugal that a kind of method in back need transfer fermented liquid proper pH value to make after the thalline sedimentation, use extraction using alcohol again, operation is complicated slightly, but saving solvent, alcohol toxicity is less, extract after the thalline sedimentation more complete, the highest pigment crude product 1.5g that obtains of every 1000ml fermented liquid, therefore more suitable with ethanol extraction method.
The haematochrome crude extract is through 100 ℃ of wet heat treatment 1h, drying treatment 2h, and its color and 40 ℃ of drying treatment differences are little, and the absorbancy rate of loss is respectively 0 and 7.1%.Slightly carry pigment and put 10d under the natural light, or treatment with ultraviolet light is no more than 1h, absorbance does not see and changes that treatment with ultraviolet light surpasses 1 hour, and the absorbancy rate of loss is 6.7%.Pigment is through the institute of Materia Medica,Chinese Academy of Medical Sciences acute toxic test, abdominal injection LD
50Be 670.04mg/kg, the toxicity of gastric infusion significantly is lower than abdominal injection, and minimum lethal dose is greater than 2000mg/kg, and toxicity is very low.The dissolving of embodiment 3 pigments in different solvents and the influence of different pH to the pigmentary colour accent:
Haematochrome through extraction using alcohol is got 0.2g, and is with ethanol, methyl alcohol, acetone, ether, the chloroform dissolving of 20ml, fixed molten to 20ml with ethanol respectively after 40 ℃ of oven dry of lysate respectively, surveys OD
538Value is observed its dissolved complexity.An amount of pigment dissolve with ethanol is regulated pH with NaOH and HCl respectively and is respectively 3,5,9,11,12, with pH7 in contrast, observes the variation of pigment tone.
Table 3 haematochrome is with the tonal variation under the OD value after the different solvents dissolving and the different pH values
| The pH tone | 3579 10 11 12 red red Huangs |
| Solvent OD 538 | Methanol acetone ether chloroform 0.57 0.50 0.48 0.39 0.37 |
The result shows that in the scope of pH 2-11, haematochrome is very stable.Haematochrome all can dissolve in ethanol, methyl alcohol, acetone, chloroform, ether, 5 kinds of solvents, but the dissolution degree difference, according to the OD after the different solvents dissolving
538Measure, its solubleness order from big to small is methyl alcohol, ethanol, acetone, ether, chloroform.
Embodiment 4
The separation and purification of pigment
(1) with a certain amount of pigment crude extract through silica gel column chromatography, normal hexane wet method dress post, sample on the dry method.With normal hexane and chloroform: methyl alcohol gradient elution (50: 1,40: 1,30: 1,20: 1,10: 0), collect pigment part elutriant, solvent evaporated;
(2) further sample is dissolved in a little chloroform: methyl alcohol (1: 1) is gone up Sephadex LH-2 post, chloroform: methyl alcohol (1: 1) wash-out, and flow velocity 1ml/min collects the pigment elutriant, concentrates;
(3) preparation thin layer, the silica gel H bed board, chloroform: normal hexane: methyl alcohol (5: 5: 1) is as developping agent.Launch the back and excise red stripes, grind the silica gel that soaks under the excision, carry out repeatedly 3 times, merge after-filtration, concentrate, weigh with 60 ℃ of methyl alcohol with blade.The thin layer point sample is checked purity;
(4) recrystallization concentrates the pigment extract and is dissolved in the 5ml methyl alcohol, slowly adds 2ml water, 4 ℃ of refrigerator overnight, suction filtration in the time of 60 ℃.Obtain the pigment crystallization, 60 ℃, 10h vacuum-drying is weighed.
(5) HPLC analyzes: the C18 post, and methyl alcohol: water (2: 1) wash-out, detect wavelength 534nm, 254nm.Flow velocity 1ml/min.With test sample purity.
Through ultraviolet, infrared and nuclear magnetic resonance measuring analysis pigment structure, think that tentatively this pigment is a conjugation pyroles pigment.
Claims (6)
1. marine bacteria that produces haematochrome, its preserving number is CGMCC NO.0527.
2. a method of producing natural red colouring matter comprises: cultivate the described marine bacteria of claim 1; With from culture, extract haematochrome.
3. in accordance with the method for claim 2, wherein, described marine bacteria is to be nitrogenous source at Tryptones, and glucose is carbon source, and NaCl concentration is to carry out under the condition of 1.8%-2.1% (weight).
4. in accordance with the method for claim 2, wherein, extracting haematochrome from culture is by at first fermented liquid pH being adjusted to 2-4, using alcoholic solvent to extract then.
5. in accordance with the method for claim 2, wherein, extracting haematochrome from culture is by at first fermented liquid pH being adjusted to 3, using ethanol to extract as solvent then.
6. haematochrome that produces by the described marine bacteria of claim 1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434466A (en) * | 2016-10-14 | 2017-02-22 | 天津科技大学 | Rhodococcus ruber for generating natural haematochrome and preparation method and application thereof |
| CN108004150A (en) * | 2017-12-23 | 2018-05-08 | 安徽工程大学 | A kind of epicoccum nigrum LS10H and its application |
| CN112574588A (en) * | 2020-12-29 | 2021-03-30 | 泉州师范学院(石狮)生态智能织物工程技术研究院 | Microbial pyrrole red pigment dye liquor and method for dyeing textiles by using same as dye |
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2001
- 2001-04-05 CN CN 01110272 patent/CN1132931C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434466A (en) * | 2016-10-14 | 2017-02-22 | 天津科技大学 | Rhodococcus ruber for generating natural haematochrome and preparation method and application thereof |
| CN106434466B (en) * | 2016-10-14 | 2019-10-18 | 天津科技大学 | A kind of Rhodococcus ruber and the preparation method and application thereof generating natural red colouring matter |
| CN108004150A (en) * | 2017-12-23 | 2018-05-08 | 安徽工程大学 | A kind of epicoccum nigrum LS10H and its application |
| CN112574588A (en) * | 2020-12-29 | 2021-03-30 | 泉州师范学院(石狮)生态智能织物工程技术研究院 | Microbial pyrrole red pigment dye liquor and method for dyeing textiles by using same as dye |
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